The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

PubMed Central

German, M S; Moss, L G; Wang, J; Rutter, W J

1992-01-01

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators. Images PMID:1549125

Value of the intravenous and oral glucose tolerance tests for detecting subtle impairments in insulin sensitivity and beta-cell function in former gestational diabetes.

PubMed

Tura, A; Mari, A; Prikoszovich, T; Pacini, G; Kautzky-Willer, A

2008-08-01

Women with former gestational diabetes mellitus (fGDM) often show defects in both insulin sensitivity and beta-cell function but it is not clear which defect plays the major role or which appears first. This might be because fGDM women are often studied as a unique group and not divided according to their glucose tolerance. Different findings might also be the result of using different tests. Our aim was to study insulin sensitivity and beta-cell function with two independent glucose tolerance tests in fGDM women divided according to their glucose tolerance. A total of 108 fGDM women divided into normal glucose tolerance (IGT; N = 82), impaired glucose metabolism (IGM; N = 20) and overt type 2 diabetes (T2DM; N = 6) groups, and 38 healthy control women (CNT) underwent intravenous (IVGTT) and oral glucose tolerance tests (OGTT). Measurements Insulin sensitivity and beta-cell function were assessed by both the IVGTT and the OGTT. Both tests revealed impaired insulin sensitivity in the normotolerant group compared to controls (IVGTT: 4.2 +/- 0.3 vs. 5.4 +/- 0.4 10(-4) min(-1) (microU/ml)(-1); OGTT: 440 +/- 7 vs. 472 +/- 9 ml min(-1) m(-2)). Conversely, no difference was found in beta-cell function from the IVGTT. However, some parameters of beta-cell function by OGTT modelling analysis were found to be impaired: glucose sensitivity (106 +/- 5 vs. 124 +/- 7 pmol min(-1) m(-2) mm(-1), P = 0.0407) and insulin secretion at 5 mm glucose (168 +/- 9 vs. 206 +/- 10 pmol min(-1) m(-2), P = 0.003). Both insulin sensitivity and beta-cell function are impaired in normotolerant fGDM but the subtle defect in beta-cell function is disclosed only by OGTT modelling analysis.

Arsenic Exposure and Calpain-10 Polymorphisms Impair the Function of Pancreatic Beta-Cells in Humans: A Pilot Study of Risk Factors for T2DM

PubMed Central

Díaz-Villaseñor, Andrea; Cruz, Laura; Cebrián, Arturo; Hernández-Ramírez, Raúl U.; Hiriart, Marcia; García-Vargas, Gonzálo; Bassol, Susana; Sordo, Monserrat; Gandolfi, A. Jay; Klimecki, Walter T.; López-Carillo, Lizbeth; Cebrián, Mariano E.; Ostrosky-Wegman, Patricia

2013-01-01

The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function. PMID:23349674

GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells.

PubMed

Tran, Dat Q; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M

2009-08-11

TGF-beta family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-beta is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFbeta-binding protein (LTBP) to produce a large latent form. Latent TGF-beta is also found on the surface of activated FOXP3(+) regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-beta to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-beta and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-beta expression on activated Tregs and recombinant latent TGF-beta1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-beta on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism.

Deficiency of bone marrow beta3-integrin enhances non-functional neovascularization.

PubMed

Watson, Alan R; Pitchford, Simon C; Reynolds, Louise E; Direkze, Natalie; Brittan, Mairi; Alison, Malcolm R; Rankin, Sara; Wright, Nicholas A; Hodivala-Dilke, Kairbaan M

2010-03-01

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

Beta-lactam antibiotics modulate T-cell functions and gene expression via covalent binding to cellular albumin.

PubMed

Mor, Felix; Cohen, Irun R

2013-02-19

Recent work has suggested that beta-lactam antibiotics might directly affect eukaryotic cellular functions. Here, we studied the effects of commonly used beta-lactam antibiotics on rodent and human T cells in vitro and in vivo on T-cell-mediated experimental autoimmune diseases. We now report that experimental autoimmune encephalomyelitis and adjuvant arthritis were significantly more severe in rats treated with cefuroxime and other beta-lactams. T cells appeared to mediate the effect: an anti-myelin basic protein T-cell line treated with cefuroxime or penicillin was more encephalitogenic in adoptive transfer experiments. The beta-lactam ampicillin, in contrast to cefuroxime and penicillin, did not enhance encephalomyelitis, but did inhibit the autoimmune diabetes developing spontaneously in nonobese diabetic mice. Gene expression analysis of human peripheral blood T cells showed that numerous genes associated with T helper 2 (Th2) and T regulatory (Treg) differentiation were down-regulated in T cells stimulated in the presence of cefuroxime; these genes were up-regulated in the presence of ampicillin. The T-cell protein that covalently bound beta-lactam antibiotics was found to be albumin. Human and rodent T cells expressed albumin mRNA and protein, and penicillin-modified albumin was taken up by rat T cells, leading to enhanced encephalitogenicity. Thus, beta-lactam antibiotics in wide clinical use have marked effects on T-cell behavior; beta-lactam antibiotics can function as immunomodulators, apparently through covalent binding to albumin.

Neurotensin protects pancreatic beta cells from apoptosis.

PubMed

Coppola, Thierry; Béraud-Dufour, Sophie; Antoine, Aurélie; Vincent, Jean-Pierre; Mazella, Jean

2008-01-01

The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both beta-TC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal.

Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming human beta cell function

PubMed Central

Rodriguez-Diaz, Rayner; Dando, Robin; Jacques-Silva, M. Caroline; Fachado, Alberto; Molina, Judith; Abdulreda, Midhat; Ricordi, Camillo; Roper, Stephen D.; Berggren, Per-Olof; Caicedo, Alejandro

2011-01-01

Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development. PMID:21685896

Regulation of pancreatic islet beta-cell mass by growth factor and hormone signaling.

PubMed

Huang, Yao; Chang, Yongchang

2014-01-01

Dysfunction and destruction of pancreatic islet beta cells is a hallmark of diabetes. Better understanding of cellular signals in beta cells will allow development of therapeutic strategies for diabetes, such as preservation and expansion of beta-cell mass and improvement of beta-cell function. During the past several decades, the number of studies analyzing the molecular mechanisms, including growth factor/hormone signaling pathways that impact islet beta-cell mass and function, has increased exponentially. Notably, somatolactogenic hormones including growth hormone (GH), prolactin (PRL), and insulin-like growth factor-1 (IGF-1) and their receptors (GHR, PRLR, and IGF-1R) are critically involved in beta-cell growth, survival, differentiation, and insulin secretion. In this chapter, we focus more narrowly on GH, PRL, and IGF-1 signaling, and GH-IGF-1 cross talk. We also discuss how these signaling aspects contribute to the regulation of beta-cell proliferation and apoptosis. In particular, our novel findings of GH-induced formation of GHR-JAK2-IGF-1R protein complex and synergistic effects of GH and IGF-1 on beta-cell signaling, proliferation, and antiapoptosis lead to a new concept that IGF-1R may serve as a proximal component of GH/GHR signaling. © 2014 Elsevier Inc. All rights reserved.

Incretin hormone receptors are required for normal beta cell development and function in female mice.

PubMed

Omar, Bilal; Ahlkvist, Linda; Yamada, Yuchiro; Seino, Yutaka; Ahrén, Bo

2016-05-01

The incretin hormones, glucose dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), potentiate insulin secretion and are responsible for the majority of insulin secretion that occurs after a meal. They may also, however, have a fundamental role in pancreatic beta cell development and function, independently of their role in potentiating insulin secretion after a meal. This has led to observations that a loss of GIP or GLP-1 action affects normal beta cell function, however each one of the incretin hormones may compensate when the action of the other is lost and therefore the overall impact of the incretin hormones on beta cell function is not known. We therefore utilized a mouse line deficient in both the GLP-1 and GIP receptor genes, the double incretin receptor knockout (DIRKO), to determine the consequences of a lifelong, complete lack of incretin hormone action on beta cell function, in vivo, in intact animals. We found that DIRKO mice displayed impaired glucose tolerance and insulin secretion in response to both oral glucose and mixed meal tolerance tests compared to wild-type mice. Assessment of beta cell function using the hyperglycemic clamp technique revealed an 80% decrease in first phase insulin response in DIRKO mice, but a normal second phase insulin secretion. A similar decline was seen when wild-type mice were given acute intravenous injection of glucose together with the GLP-1 receptor antagonist Ex9-39. Ex vivo assessments of the pancreas revealed significantly fewer islets in the pancreata of DIRKO mice despite no differences in total pancreatic mass. Insulin secretion from isolated islets of DIRKO mice was impaired to a similar extent to that seen during the hyperglycemic clamp. Insulin secretion in wild-type islets was impaired by acute treatment with Ex9-39 to a similar extent as the in vivo intravenous glucose tolerance tests. In conclusion, a loss of the action of both incretin hormones results in direct impairment of beta cell function both in vivo and in vitro in a process that appears to be independent of the intestinally secreted incretin hormones. We therefore conclude that the incretin hormones together significantly impact both beta-cell function and beta-cell development. Copyright © 2016. Published by Elsevier Inc.

Clinical characteristics and beta cell function in Chinese patients with newly diagnosed type 2 diabetes mellitus with different levels of serum triglyceride.

PubMed

Zheng, Shuang; Zhou, Huan; Han, Tingting; Li, Yangxue; Zhang, Yao; Liu, Wei; Hu, Yaomin

2015-04-29

To explore clinical characteristics and beta cell function in Chinese patients with newly diagnosed drug naive type 2 diabetes mellitus (T2DM) with different levels of serum triglyceride (TG). Patients with newly diagnosed T2DM (n = 624) were enrolled and divided into different groups according to levels of serum TG. All patients underwent oral glucose tolerance tests and insulin releasing tests. Demographic data, lipid profiles, glucose levels, and insulin profiles were compared between different groups. Basic insulin secretion function index (homeostasis model assessment for beta cell function index, HOMA-β), modified beta cell function index (MBCI), glucose disposition indices (DI), and early insulin secretion function index (insulinogenic index, IGI) were used to evaluate the beta cell function. Patients of newly diagnosed T2DM with hypertriglyceridemia were younger, fatter and had worse lipid profiles, glucose profiles, and high insulin levels than those with normal TG. There is no difference in early phase insulin secretion among groups of newly diagnosed T2DM patients with different TG levels. The basal beta cell function (HOMA-β and MBCI) initially increased along rising TG levels and then decreased as the TG levels rose further. The insulin sensitivity was relatively high in patients with a low level of TG and low with a high level of TG. Hypertriglyceridemia influences clinical characteristics and β cell function of Chinese patients with newly diagnosed T2DM. A better management of dyslipidemia may, to some extent, reduce the effect of lipotoxicity, thereby improving glucose homeostasis in patients with newly diagnosed T2DM.

Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

PubMed

Jansson, Keith H; Castillo, Deborah G; Morris, Joseph W; Boggs, Mary E; Czymmek, Kirk J; Adams, Elizabeth L; Schramm, Lawrence P; Sikes, Robert A

2014-01-01

Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

NASA Technical Reports Server (NTRS)

Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

2003-01-01

CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function

PubMed Central

Pi, Jingbo; Zhang, Qiang; Fu, Jingqi; Woods, Courtney G.; Hou, Yongyong; Corkey, Barbara E; Collins, Sheila; Andersen, Melvin E.

2009-01-01

This review focuses on the emerging evidence that reactive oxygen species (ROS) derived from glucose metabolism, such as H2O2, act as metabolic signaling molecules for glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Particular emphasis is placed on the potential inhibitory role of endogenous antioxidants, which rise in response to oxidative stress, in glucose-triggered ROS and GSIS. We propose that cellular adaptive response to oxidative stress challenge, such as nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant induction, plays paradoxical roles in pancreatic beta-cell function. On the one hand, induction of antioxidant enzymes protects beta-cells from oxidative damage and possible cell death, thus minimizing oxidative damage-related impairment of insulin secretion. On the other hand, the induction of antioxidant enzymes by Nrf2 activation blunts glucose-triggered ROS signaling, thus resulting in reduced GSIS. These two premises are potentially relevant to impairment of beta-cells occurring in the late and early stage of Type 2 diabetes, respectively. In addition, we summarized our recent findings that persistent oxidative stress due to absence of uncoupling protein 2 activates cellular adaptive response which is associated with impaired pancreatic beta-cell function. PMID:19501608

Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

PubMed Central

Tondeleir, Davina; Noelanders, Rivka; Bakkali, Karima; Ampe, Christophe

2014-01-01

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11. PMID:24409333

cGMP may have trophic effects on beta cell function comparable to those of cAMP, implying a role for high-dose biotin in prevention/treatment of diabetes.

PubMed

McCarty, Mark F

2006-01-01

Incretin hormones have trophic effects on beta cell function that can aid prevention and treatment of diabetes. cAMP is the primary mediator of these effects, and has been shown to potentiate glucose-stimulated insulin secretion, promote proper beta cells differentiation by increasing expression of the crucial transcription factor PDX-1, and prevent beta cell apoptosis. cGMP's role in beta cell function has received far less scrutiny, but there is emerging evidence that it may have a trophic impact on beta cell function analogous to that of cAMP. An increase in plasma glucose boosts beta cell production of cGMP, which acts as a feed-forward mediator to enhance glucose-stimulated insulin secretion. cGMP also has an anti-apoptotic effect in beta cells, and there is now indirect evidence that it promotes expression of PDX-1. Supraphysiological concentrations of biotin can directly activate guanylate cyclase, and there is limited evidence that high intakes of this vitamin can be therapeutically beneficial in diabetics and in rodent models of diabetes. Beneficial effects of cGMP on muscle insulin sensitivity and on control of hepatic glucose output may contribute to biotin's utility in diabetes. The fact that nitric oxide/cGMP exert a range of favorable effects on vascular health should further encourage exploration of biotin's preventive and therapeutic potential. If an appropriate high-dose biotin regimen could achieve a modest systemic increase in guanylate cyclase activity, without entailing unacceptable side effects or risks, such a regimen might have considerable potential for promoting vascular health and preventing or managing diabetes.

Topologically heterogeneous beta cell adaptation in response to high-fat diet in mice.

PubMed

Ellenbroek, Johanne H; Töns, Hendrica A; de Graaf, Natascha; Loomans, Cindy J; Engelse, Marten A; Vrolijk, Hans; Voshol, Peter J; Rabelink, Ton J; Carlotti, Françoise; de Koning, Eelco J

2013-01-01

Beta cells adapt to an increased insulin demand by enhancing insulin secretion via increased beta cell function and/or increased beta cell number. While morphological and functional heterogeneity between individual islets exists, it is unknown whether regional differences in beta cell adaptation occur. Therefore we investigated beta cell adaptation throughout the pancreas in a model of high-fat diet (HFD)-induced insulin resistance in mice. C57BL/6J mice were fed a HFD to induce insulin resistance, or control diet for 6 weeks. The pancreas was divided in a duodenal (DR), gastric (GR) and splenic (SR) region and taken for either histology or islet isolation. The capacity of untreated islets from the three regions to adapt in an extrapancreatic location was assessed by transplantation under the kidney capsule of streptozotocin-treated mice. SR islets showed 70% increased beta cell proliferation after HFD, whereas no significant increase was found in DR and GR islets. Furthermore, isolated SR islets showed twofold enhanced glucose-induced insulin secretion after HFD, as compared with DR and GR islets. In contrast, transplantation of islets isolated from the three regions to an extrapancreatic location in diabetic mice led to a similar decrease in hyperglycemia and no difference in beta cell proliferation. HFD-induced insulin resistance leads to topologically heterogeneous beta cell adaptation and is most prominent in the splenic region of the pancreas. This topological heterogeneity in beta cell adaptation appears to result from extrinsic factors present in the islet microenvironment.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

PubMed

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

EPConDB: a web resource for gene expression related to pancreatic development, beta-cell function and diabetes.

PubMed

Mazzarelli, Joan M; Brestelli, John; Gorski, Regina K; Liu, Junmin; Manduchi, Elisabetta; Pinney, Deborah F; Schug, Jonathan; White, Peter; Kaestner, Klaus H; Stoeckert, Christian J

2007-01-01

EPConDB (http://www.cbil.upenn.edu/EPConDB) is a public web site that supports research in diabetes, pancreatic development and beta-cell function by providing information about genes expressed in cells of the pancreas. EPConDB displays expression profiles for individual genes and information about transcripts, promoter elements and transcription factor binding sites. Gene expression results are obtained from studies examining tissue expression, pancreatic development and growth, differentiation of insulin-producing cells, islet or beta-cell injury, and genetic models of impaired beta-cell function. The expression datasets are derived using different microarray platforms, including the BCBC PancChips and Affymetrix gene expression arrays. Other datasets include semi-quantitative RT-PCR and MPSS expression studies. For selected microarray studies, lists of differentially expressed genes, derived from PaGE analysis, are displayed on the site. EPConDB provides database queries and tools to examine the relationship between a gene, its transcriptional regulation, protein function and expression in pancreatic tissues.

Effects of 18beta-glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells.

PubMed

Huang, Shih-Horng; Wu, Jiahn-Chun; Hwang, Ra-Der; Yeo, Hui-Lin; Wang, Seu-Mei

2003-09-01

Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to alpha-, beta-, and gamma-catenin, which were seen as continuous, linear staining of cell-cell adherens junction. Treatment with 18beta-glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time- and dose-dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell-cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18beta-glycyrrhetinic acid stimulated a two- to three-fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18beta-glycyrrhetinic acid was partially mediated by extracellular signal-related kinase and calcium/calmodulin-dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells. Copyright 2003 Wiley-Liss, Inc.

Treatment of prediabetes

PubMed Central

Kanat, Mustafa; DeFronzo, Ralph A; Abdul-Ghani, Muhammad A

2015-01-01

Progression of normal glucose tolerance (NGT) to overt diabetes is mediated by a transition state called impaired glucose tolerance (IGT). Beta cell dysfunction and insulin resistance are the main defects in type 2 diabetes mellitus (type 2 DM) and even normoglycemic IGT patients manifest these defects. Beta cell dysfunction and insulin resistance also contribute to the progression of IGT to type 2 DM. Improving insulin sensitivity and/or preserving functions of beta-cells can be a rational way to normalize the GT and to control transition of IGT to type 2 DM. Loosing weight, for example, improves whole body insulin sensitivity and preserves beta-cell function and its inhibitory effect on progression of IGT to type 2 DM had been proven. But interventions aiming weight loss usually not applicable in real life. Pharmacotherapy is another option to gain better insulin sensitivity and to maintain beta-cell function. In this review, two potential treatment options (lifestyle modification and pharmacologic agents) that limits the IGT-type 2 DM conversion in prediabetic subjects are discussed. PMID:26464759

Heterocellular interaction enhances recruitment of {alpha} and {beta}-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Talhouk, Rabih S.; Mroue, Rana; Mokalled, Mayssa

2008-11-01

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation ({beta}-casein expression) was evaluated. Heterocellular interaction is critical for {beta}-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complexmore » components ({alpha}-catenin, {beta}-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although {beta}-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and {beta}-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear {beta}-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of {beta}-catenin in GJ complexes.« less

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

PubMed

Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Increased androgen levels in rats impair glucose-stimulated insulin secretion through disruption of pancreatic beta cell mitochondrial function.

PubMed

Wang, Hongdong; Wang, Xiaping; Zhu, Yunxia; Chen, Fang; Sun, Yujie; Han, Xiao

2015-11-01

Although insulin resistance is recognized to contribute to the reproductive and metabolic phenotypes of polycystic ovary syndrome (PCOS), pancreatic beta cell dysfunction plays an essential role in the progression from PCOS to the development of type 2 diabetes. However, the role of insulin secretory abnormalities in PCOS has received little attention. In addition, the precise changes in beta cells and the underlying mechanisms remain unclear. In this study, we therefore attempted to elucidate potential mechanisms involved in beta cell alterations in a rat model of PCOS. Glucose-induced insulin secretion was measured in islets isolated from DHT-treated and control rats. Oxygen consumption rate (OCR), ATP production, and mitochondrial copy number were assayed to evaluate mitochondrial function. Glucose-stimulated insulin secretion is significantly decreased in islets from DHT-treated rats. On the other hand, significant reductions are observed in the expression levels of several key genes involved in mitochondrial biogenesis and in mitochondrial OCR and ATP production in DHT-treated rat islets. Meanwhile, we found that androgens can directly impair beta cell function by inducing mitochondrial dysfunction in vitro in an androgen receptor dependent manner. For the first time, our study demonstrates that increased androgens in female rats can impair glucose-stimulated insulin secretion partly through disruption of pancreatic beta cell mitochondrial function. This work has significance for hyperandrogenic women with PCOS: excess activation of the androgen receptor by androgens may provoke beta cell dysfunction via mitochondrial dysfunction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Requirement for Pdx1 in specification of latent endocrine progenitors in zebrafish

PubMed Central

2011-01-01

Background Insulin-producing beta cells emerge during pancreas development in two sequential waves. Recently described later-forming beta cells in zebrafish show high similarity to second wave mammalian beta cells in developmental capacity. Loss-of-function studies in mouse and zebrafish demonstrated that the homeobox transcription factors Pdx1 and Hb9 are both critical for pancreas and beta cell development and discrete stage-specific requirements for these genes have been uncovered. Previously, exocrine and endocrine cell recovery was shown to follow loss of pdx1 in zebrafish, but the progenitor cells and molecular mechanisms responsible have not been clearly defined. In addition, interactions of pdx1 and hb9 in beta cell formation have not been addressed. Results To learn more about endocrine progenitor specification, we examined beta cell formation following morpholino-mediated depletion of pdx1 and hb9. We find that after early beta cell reduction, recovery occurs following loss of either pdx1 or hb9 function. Unexpectedly, simultaneous knockdown of both hb9 and pdx1 leads to virtually complete and persistent beta cell deficiency. We used a NeuroD:EGFP transgenic line to examine endocrine cell behavior in vivo and developed a novel live-imaging technique to document emergence and migration of late-forming endocrine precursors in real time. Our data show that Notch-responsive progenitors for late-arising endocrine cells are predominantly post mitotic and depend on pdx1. By contrast, early-arising endocrine cells are specified and differentiate independent of pdx1. Conclusions The nearly complete beta cell deficiency after combined loss of hb9 and pdx1 suggests functional cooperation, which we clarify as distinct roles in early and late endocrine cell formation. A novel imaging approach permitted visualization of the emergence of late endocrine cells within developing embryos for the first time. We demonstrate a pdx1-dependent progenitor population essential for the formation of duct-associated, second wave endocrine cells. We further reveal an unexpectedly low mitotic activity in these progenitor cells, indicating that they are set aside early in development. PMID:22034951

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

PubMed Central

1995-01-01

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

A Synopsis of Factors Regulating Beta Cell Development and Beta Cell Mass

PubMed Central

Prasadan, Krishna; Shiota, Chiyo; Xiangwei, Xiao; Ricks, David; Fusco, Joseph; Gittes, George

2016-01-01

The insulin-secreting beta cells in the endocrine pancreas regulate blood glucose levels, and loss of functional beta cells leads to insulin deficiency, hyperglycemia (high blood glucose) and diabetes mellitus. Current treatment strategies for type-1 (autoimmune) diabetes are islet transplantation, which has significant risks and limitations, or normalization of blood glucose with insulin injections, which is clearly not ideal. The type-1 patients can lack insulin counter-regulatory mechanism; therefore, hypoglycemia is a potential risk. Hence, a cell-based therapy offers a better alternative for the treatment of diabetes. Past research was focused on attempting to generate replacement beta cells from stem cells, however, recently there has been an increasing interest in identifying mechanisms that will lead to the conversion of pre-existing differentiated endocrine cells into beta cells. The goal of this review is to provide an overview of several of the key factors that regulate new beta cell formation (neogenesis) and beta cell proliferation. PMID:27105622

Impaired compensatory beta-cell function and growth in response to high-fat diet in LDL receptor knockout mice

PubMed Central

Oliveira, Ricardo B d; Carvalho, Carolina P d F; Polo, Carla C; Dorighello, Gabriel d G; Boschero, Antônio C; Oliveira, Helena C F d; Collares-Buzato, Carla B

2014-01-01

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr−/− mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr−/− mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr−/− mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr−/− mice showed no significant changes in beta-cell mass, but lower islet–duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr−/− mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion. PMID:24853046

Functional characterization of the beta-adrenergic receptor subtypes expressed by CA1 pyramidal cells in the rat hippocampus.

PubMed

Hillman, Kristin L; Doze, Van A; Porter, James E

2005-08-01

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists indicates that beta2-AR activation is mediating the increased AP frequency. Knowledge of functional AR expression in CA1 pyramidal neurons will aid future long-term potentiation studies by allowing selective manipulation of specific beta-AR subtypes.

A critical role for transcription factor Smad4 in T cell function independent of transforming growth factor beta receptor signaling

PubMed Central

Gu, Ai-Di; Zhang, Song; Wang, Yunqi; Xiong, Hui; Curtis, Thomas A.; Wan, Yisong Y.

2014-01-01

Summary Transforming growth factor-beta (TGF-β) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-β signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-βR) deletion and compromised T-cell-mediated tumor rejection. While Smad4 was dispensable for T cell generation, homeostasis and effector function, it was essential for T cell proliferation following activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-βR-independent role for Smad4 in promoting T cell function, autoimmunity and anti-tumor immunity. PMID:25577439

Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.

2005-03-03

Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha}more » co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.« less

Different downstream signalling of CCK1 receptors regulates distinct functions of CCK in pancreatic beta cells.

PubMed

Ning, Shang-lei; Zheng, Wen-shuai; Su, Jing; Liang, Nan; Li, Hui; Zhang, Dao-lai; Liu, Chun-hua; Dong, Jun-hong; Zhang, Zheng-kui; Cui, Min; Hu, Qiao-Xia; Chen, Chao-chao; Liu, Chang-hong; Wang, Chuan; Pang, Qi; Chen, Yu-xin; Yu, Xiao; Sun, Jin-peng

2015-11-01

Cholecystokinin (CCK) is secreted by intestinal I cells and regulates important metabolic functions. In pancreatic islets, CCK controls beta cell functions primarily through CCK1 receptors, but the signalling pathways downstream of these receptors in pancreatic beta cells are not well defined. Apoptosis in pancreatic beta cell apoptosis was evaluated using Hoechst-33342 staining, TUNEL assays and Annexin-V-FITC/PI staining. Insulin secretion and second messenger production were monitored using ELISAs. Protein and phospho-protein levels were determined by Western blotting. A glucose tolerance test was carried out to examine the functions of CCK-8s in streptozotocin-induced diabetic mice. The sulfated carboxy-terminal octapeptide CCK26-33 amide (CCK-8s) activated CCK1 receptors and induced accumulation of both IP3 and cAMP. Whereas Gq -PLC-IP3 signalling was required for the CCK-8s-induced insulin secretion under low-glucose conditions, Gs -PKA/Epac signalling contributed more strongly to the CCK-8s-mediated insulin secretion in high-glucose conditions. CCK-8s also promoted formation of the CCK1 receptor/β-arrestin-1 complex in pancreatic beta cells. Using β-arrestin-1 knockout mice, we demonstrated that β-arrestin-1 is a key mediator of both CCK-8s-mediated insulin secretion and of its the protective effect against apoptosis in pancreatic beta cells. The anti-apoptotic effects of β-arrestin-1 occurred through cytoplasmic late-phase ERK activation, which activates the 90-kDa ribosomal S6 kinase-phospho-Bcl-2-family protein pathway. Knowledge of different CCK1 receptor-activated downstream signalling pathways in the regulation of distinct functions of pancreatic beta cells could be used to identify biased CCK1 receptor ligands for the development of new anti-diabetic drugs. © 2015 The British Pharmacological Society.

β-Arrestin2 plays a key role in the modulation of the pancreatic beta cell mass in mice.

PubMed

Ravier, Magalie A; Leduc, Michele; Richard, Joy; Linck, Nathalie; Varrault, Annie; Pirot, Nelly; Roussel, Morgane M; Bockaert, Joël; Dalle, Stéphane; Bertrand, Gyslaine

2014-03-01

Beta cell failure due to progressive secretory dysfunction and limited expansion of beta cell mass is a key feature of type 2 diabetes. Beta cell function and mass are controlled by glucose and hormones/neurotransmitters that activate G protein-coupled receptors or receptor tyrosine kinases. We have investigated the role of β-arrestin (ARRB)2, a scaffold protein known to modulate such receptor signalling, in the modulation of beta cell function and mass, with a specific interest in glucagon-like peptide-1 (GLP-1), muscarinic and insulin receptors. β-arrestin2-knockout mice and their wild-type littermates were fed a normal or a high-fat diet (HFD). Glucose tolerance, insulin sensitivity and insulin secretion were assessed in vivo. Beta cell mass was evaluated in pancreatic sections. Free cytosolic [Ca(2+)] and insulin secretion were determined using perifused islets. The insulin signalling pathway was evaluated by western blotting. Arrb2-knockout mice exhibited impaired glucose tolerance and insulin secretion in vivo, but normal insulin sensitivity compared with wild type. Surprisingly, the absence of ARRB2 did not affect glucose-stimulated insulin secretion or GLP-1- and acetylcholine-mediated amplifications from perifused islets, but it decreased the islet insulin content and beta cell mass. Additionally, there was no compensatory beta cell mass expansion through proliferation in response to the HFD. Furthermore, Arrb2 deletion altered the islet insulin signalling pathway. ARRB2 is unlikely to be involved in the regulation of insulin secretion, but it is required for beta cell mass plasticity. Additionally, we provide new insights into the mechanisms involved in insulin signalling in beta cells.

Neurotensin receptor-2 and -3 are crucial for the anti-apoptotic effect of neurotensin on pancreatic beta-TC3 cells.

PubMed

Béraud-Dufour, Sophie; Coppola, Thierry; Massa, Fabienne; Mazella, Jean

2009-12-01

The neuropeptide neurotensin (NT) has been recently shown to protect pancreatic beta cells from toxic agents-induced apoptosis through interaction with the NT receptor-2 (NTSR2) and activation of the phosphatidylinositol-3 kinase pathway. However, expression of the NT receptor-3/sortilin (NTSR3) in the mouse pancreatic beta cell line -TC3 led us to investigate its possible functional role in these cells. By using siRNA, immunoprecipitation, co-localization and caspase-3 assays,we provide evidence for a functional endogenous interaction between NTSR2 and NTSR3. Expression of both receptors is necessary for the protective action of NT on staurosporine-induced caspase-3 activity in -TC3 cells. Moreover, NTSR2 and NTSR3 co-immunoprecipitate and are co-localized at the plasma membrane. Thus, the NT response in beta cells is controlled by the formation of a functional complex between NTSR2 and NTSR3.

TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

PubMed

Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

2010-01-01

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

TGF-beta1 inhibits Cx43 expression and formation of functional syncytia in cultured smooth muscle cells from human detrusor.

PubMed

Neuhaus, Jochen; Heinrich, Marco; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

2009-02-01

Human detrusor smooth muscle cells (hBSMCs) are coupled by connexin 43 (Cx43)-positive gap junctions to form functional syncytia. Gap junctional communication likely is necessary for synchronised detrusor contractions and is supposed to be altered in voiding disturbances. Other authors have shown that the pleiotropic cytokine TGF-beta1 upregulates Cx43 expression in human aortic smooth muscle cells. In this study, we examined the TGF-beta1 effects on Cx43 expression in cultured hBSMCs. hBSMC cultures, established from patients undergoing cystectomy, were treated with recombinant human TGF-beta1. Cx43 expression was then examined by Western blotting, real-time PCR, and immunocytochemistry. Dye-injection experiments were used to study the size of functional syncytia. Dye-coupling experiments revealed stable formation of functional syncytia in passaged cell cultures (P1-P4). Stimulation with TGF-beta1 led to significant reduction of Cx43 immunoreactivity and coupling. Cx43 protein expression was significantly downregulated and Cx43 mRNA was only 30% of the control level. Interestingly, low phosphorylation species of Cx43 were particularly affected. Our experiments demonstrated a significant down regulation of connexin 43 by TGF-beta1 in cultured hBSMCs. These findings support the view that TGF-beta1 is involved in the pathophysiology of urinary bladder dysfunction.

Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

PubMed

Green, Alastair D; Vasu, Srividya; Moffett, R Charlotte; Flatt, Peter R

2016-06-01

We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Beta cell function after weight loss: a clinical trial comparing gastric bypass surgery and intensive lifestyle intervention

PubMed Central

Hofsø, D; Jenssen, T; Bollerslev, J; Ueland, T; Godang, K; Stumvoll, M; Sandbu, R; Røislien, J; Hjelmesæth, J

2011-01-01

Objective The effects of various weight loss strategies on pancreatic beta cell function remain unclear. We aimed to compare the effect of intensive lifestyle intervention (ILI) and Roux-en-Y gastric bypass surgery (RYGB) on beta cell function. Design One year controlled clinical trial (ClinicalTrials.gov identifier NCT00273104). Methods One hundred and nineteen morbidly obese participants without known diabetes from the MOBIL study (mean (s.d.) age 43.6 (10.8) years, body mass index (BMI) 45.5 (5.6) kg/m2, 84 women) were allocated to RYGB (n=64) or ILI (n=55). The patients underwent repeated oral glucose tolerance tests (OGTTs) and were categorised as having either normal (NGT) or abnormal glucose tolerance (AGT). Twenty-nine normal-weight subjects with NGT (age 42.6 (8.7) years, BMI 22.6 (1.5) kg/m2, 19 women) served as controls. OGTT-based indices of beta cell function were calculated. Results One year weight reduction was 30 % (8) after RYGB and 9 % (10) after ILI (P<0.001). Disposition index (DI) increased in all treatment groups (all P<0.05), although more in the surgery groups (both P<0.001). Stimulated proinsulin-to-insulin (PI/I) ratio decreased in both surgery groups (both P<0.001), but to a greater extent in the surgery group with AGT at baseline (P<0.001). Post surgery, patients with NGT at baseline had higher DI and lower stimulated PI/I ratio than controls (both P<0.027). Conclusions Gastric bypass surgery improved beta cell function to a significantly greater extent than ILI. Supra-physiological insulin secretion and proinsulin processing may indicate excessive beta cell function after gastric bypass surgery. PMID:21078684

Generation of Beta Cells from Human Pluripotent Stem Cells: Potential for Regenerative Medicine

PubMed Central

Nostro, Maria Cristina; Keller, Gordon

2015-01-01

The loss of beta cells in Type I Diabetes ultimately leads to insulin dependence and major complications that are difficult to manage by insulin injections. Given the complications associated with long-term administration of insulin, cell-replacement therapy is now under consideration as an alternative treatment that may someday provide a cure for this disease. Over the past 10 years, islet transplantation trials have demonstrated that it is possible to replenish beta cell function in Type I Diabetes patients and, at least temporarily, eliminate their dependency on insulin. While not yet optimal, the success of these trials has provided proof-of-principle that cell replacement therapy is a viable option for treating diabetes. Limited access to donor islets has launched a search for alternative source of beta cells for cell therapy purposes and focused the efforts of many investigators on the challenge of deriving such cells from human embryonic and induced pluripotent stem cells. Over the past five years, significant advances have been made in understanding the signaling pathways that control lineage development from hPSCs and as a consequence, it is now possible to routinely generate human insulin producing cells from both hESCs and hiPSCs. While these achievements are impressive, significant challenges do still exist, as the majority of insulin producing cells generated under these conditions are polyhormonal and non functional, likely reflecting the emergence of the polyhormonal population that is known to arise in the early embryo during the phase of pancreatic development known as the ‘first transition’. Functional beta cells, which arise during the second phase or transition of pancreatic development have been generated from hPSCs, however they are detected only following transplantation of progenitor stage cells into immunocompromised mice. With this success, our challenge now is to define the pathways that control the development and maturation of this second transition population from hPSCs, and establish conditions for the generation of functional beta cells in vitro. PMID:22750147

Chronology of endocrine differentiation and beta-cell neogenesis.

PubMed

Miyatsuka, Takeshi

2016-01-01

Diabetes is a chronic and incurable disease, which results from absolute or relative insulin insufficiency. Therefore, pancreatic beta cells, which are the only type of cell that expresses insulin, is considered to be a potential target for the cure of diabetes. Although the findings regarding beta-cell neogenesis during pancreas development have been exploited to induce insulin-producing cells from non-beta cells, there are still many hurdles towards generating fully functional beta cells that can produce high levels of insulin and respond to physiological signals. To overcome these problems, a solid understanding of pancreas development and beta-cell formation is required, and several mouse models have been developed to reveal the unique features of each endocrine cell type at distinct developmental time points. Here I review our understanding of pancreas development and endocrine differentiation focusing on recent progresses in improving temporal cell labeling in vivo.

Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma.

PubMed

Laouar, Yasmina; Sutterwala, Fayyaz S; Gorelik, Leonid; Flavell, Richard A

2005-06-01

Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

Differential expression of E-cadherin at the surface of rat beta-cells as a marker of functional heterogeneity.

PubMed

Bosco, Domenico; Rouiller, Dominique G; Halban, Philippe A

2007-07-01

The aim of this study was to assess whether the expression of E-cadherin at the surface of rat beta-cells is regulated by insulin secretagogues and correlates with insulin secretion. When cultured under standard conditions, virtually all beta-cells expressed E-cadherin observed by immunofluorescence, but heterogeneous staining was observed. Using fluorescence-activated cell sorting (FACS), two beta-cell sub-populations were sorted: one that was poorly labeled ('ECad-low') and another that was highly labeled ('ECad-high'). After 1-h stimulation with 16.7 mM glucose, insulin secretion (reverse hemolytic plaque assay) from individual ECad-high beta-cells was higher than that from ECad-low beta-cells. Ca2+-dependent beta-cell aggregation was increased at 16.7 mM glucose when compared with 2.8 mM glucose. E-cadherin at the surface of beta-cells was increased after 18 h at 11.1 and 22.2 mM glucose when compared with 2.8 mM glucose, with the greatest increase at 22.2 mM glucose + 0.5 mM isobutylmethylxanthine (IBMX). While no labeling was detected on freshly trypsinized cells, the proportion of stained cells increased in a time-dependent manner during culture for 1, 3, and 24 h. This recovery was faster when cells were incubated at 16.7 vs 2.8 mM glucose. Cycloheximide inhibited expression of E-cadherin at 2.8 mM glucose, but not at 16.7 mM, while depolymerization of actin by either cytochalasin B or latrunculin B increased surface E-cadherin at low glucose. In conclusion, these results show that expression of E-cadherin at the surface of islet beta-cells is controlled by secretagogues including glucose, correlates with insulin secretion, and can serve as a surface marker of beta-cell function.

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion

PubMed Central

1995-01-01

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein. PMID:7699339

Different susceptibility of rat pancreatic alpha and beta cells to hypoxia.

PubMed

Bloch, Konstantin; Vennäng, Julia; Lazard, Daniel; Vardi, Pnina

2012-06-01

Insulin-producing beta cells are known to be highly susceptible to hypoxia, which is a major factor in their destruction after pancreatic islet transplantation. However, whether the glucagon-producing pancreatic islet alpha cells are sensitive to hypoxia is not known. Our objective was to compare the sensitivity of alpha and beta cells to hypoxia. Isolated rat pancreatic islets were exposed to hypoxia (1% oxygen, 94% N(2), 5% CO(2)) for 3 days. The viability of the alpha and beta cells, as well as the stimulus-specific secretion of glucagon and insulin, was evaluated. A quantitative analysis of the proportion of beta to alpha cells indicated that, under normoxic conditions, islet cells were composed mainly of beta cells (87 ± 3%) with only 13 ± 3% alpha cells. Instead, hypoxia treatment significantly increased the proportion of alpha cells (40 ± 13%) and decreased the proportion of beta cells to 60 ± 13%. Using the fluorescent TUNEL assay we found that only a few percent of beta cells and alpha cells were apoptotic in normoxia. In contrast, hypoxia induced an abundance of apoptotic beta cells (61 ± 22%) and had no effect on the level of apoptosis in alpha cells. In conclusion, this study demonstrates that hypoxia results in severe functional abnormality in both beta and alpha cells while alpha cells display significantly decreased rate of apoptosis compared to intensive apoptotic injury of beta cells. These findings have implications for the understanding of the possible role of hypoxia in the pathophysiology of diabetes.

A thymosin beta15-like peptide promotes intersegmental myotome extension in the chicken embryo.

PubMed

Chankiewitz, Verena; Morosan-Puopolo, Gabriela; Yusuf, Faisal; Rudloff, Stefan; Pröls, Felicitas; Kleff, Veronika; Hofmann, Dietrich Kurt; Brand-Saberi, Beate

2014-03-01

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15(avian) in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15(avian) expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15(avian) has a function in normal myotome development. Ectopic over-expression of thymosin beta15(avian) leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15(avian) has a still undescribed function in promoting myocyte elongation.

Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process

PubMed Central

Ghosal, Abhisek; Sekar, Thillai V.

2014-01-01

Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na+-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na+-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078

Arsenic-gene interactions and beta-cell function in the Strong Heart Family Study.

PubMed

Balakrishnan, Poojitha; Navas-Acien, Ana; Haack, Karin; Vaidya, Dhananjay; Umans, Jason G; Best, Lyle G; Goessler, Walter; Francesconi, Kevin A; Franceschini, Nora; North, Kari E; Cole, Shelley A; Voruganti, V Saroja; Gribble, Matthew O

2018-06-01

We explored arsenic-gene interactions influencing pancreatic beta-cell activity in the Strong Heart Family Study (SHFS). We considered 42 variants selected for associations with either beta-cell function (31 variants) or arsenic metabolism (11 variants) in the SHFS. Beta-cell function was calculated as homeostatic model - beta corrected for insulin resistance (cHOMA-B) by regressing homeostatic model - insulin resistance (HOMA-IR) on HOMA-B and adding mean HOMA-B. Arsenic exposure was dichotomized at the median of the sum of creatinine-corrected inorganic and organic arsenic species measured by high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). Additive GxE models for cHOMA-B were adjusted for age and ancestry, and accounted for family relationships. Models were stratified by center (Arizona, Oklahoma, North Dakota and South Dakota) and meta-analyzed. The two interactions between higher vs. lower arsenic and SNPs for cHOMA-B that were nominally significant at P < 0.05 were with rs10738708 (SNP overall effect -3.91, P = 0.56; interaction effect with arsenic -31.14, P = 0.02) and rs4607517 (SNP overall effect +16.61, P = 0.03; interaction effect with arsenic +27.02, P = 0.03). The corresponding genes GCK and TUSC1 suggest oxidative stress and apoptosis as possible mechanisms for arsenic impacts on beta-cell function. No interactions were Bonferroni-significant (1.16 × 10 -3 ). Our findings are suggestive of oligogenic moderation of arsenic impacts on pancreatic β-cell endocrine function, but were not Bonferroni-significant. Copyright © 2018 Elsevier Inc. All rights reserved.

Metabolomic profiling of amino acids and beta-cell function relative to insulin sensitivity in youth

USDA-ARS?s Scientific Manuscript database

In longitudinal studies of adults, elevated amino acid (AA) concentrations predicted future type 2 diabetes mellitus (T2DM). The aim of the present investigation was to examine whether increased plasma AA concentrations are associated with impaired beta-cell function relative to insulin sensitivity ...

In vitro differentiation of rat bone marrow mesenchymal stem cells into hepatocytes.

PubMed

Feng, Zhihui; Li, Changying; Jiao, Shuxian; Hu, Bin; Zhao, Lin

2011-01-01

To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (BMSCs) into hepatocytes and to find a new source for therapies of hepatic diseases. We isolated BMSCs for subsequent differentiation in the presence of hepatocyte growth factor (HGF) or beta-nerve growth factor (beta-NGF). Cell morphology was observed and cell surface phenotypings were detected by flow cytometry. a1-antitrypsin (AAT) expression of the hepatocytes was confirmed by immunocytochemistry and albumin expression was validated by real time PCR and western blotting. The expression of high-affinity nerve growth factor receptor (TrkA) and the activation of Erk pathway were detected by western blotting. Hepatocyte functional activity was confirmed by uptake of indocyanine green (ICG) assay. Small round cells appeared in the presence of HGF on day 10 or beta-NGF on day 12. Differentiated cells expressed albumin and had functional characteristics of hepatocytes, such as uptake of ICG. BMSCs were positive for TrkA. HGF and beta-NGF significantly upregulated the protein levels of phospho-Erk. BMSCs could differentiate into hepatocytes in the differentiation media including HGF or beta-NGF. Combination of HGF and beta-NGF significantly increased the efficiency of hepatic differentiation.

Lubrol-RAFTs in melanoma cells: a molecular platform for tumor-promoting ephrin-B2-integrin-beta1 interaction.

PubMed

Meyer, Stefanie; Orsó, Evelyn; Schmitz, Gerd; Landthaler, Michael; Vogt, Thomas

2007-07-01

Ephrins control cell motility and matrix adhesion. These functions play a pivotal role in cancer progression, for example, in malignant melanomas. We have previously shown that the ephrin-B2-tumor-promoting action is partly mediated by integrin-beta1 interaction. However, the subcellular prerequisites for molecular interaction like molecular proximity and co-compartmentalization have not been elucidated yet. Specific cholesterol-rich microdomains, termed lipid rafts (RAFTs), are known to be essential for functional ephrin-B2 signalling and integrin-mediated effects. Therefore, we addressed the question whether RAFT co-compartmentalization of both molecules could provide the molecular platform for their tumor-promoting interaction. In this study, we show that overexpressed ephrin-B2 is not only compartmentalized to classical Triton X-100 RAFTs in B16 melanoma cells, but also to the recently defined Lubrol-RAFTs. Interestingly, in the melanoma cells investigated, integrin-beta1 is also preferentially detected in such Lubrol-RAFTs. Accordingly, the presence of ephrin-B2 and integrin-beta1 in RAFTs and their function in cell migration and matrix attachment are highly sensitive to RAFT disruption by cholesterol depletion. Confocal fluorescence microscopy analyses also support the concept of a close molecular proximity and functional interplay of ephrin-B2 and integrin-beta1 in the plasma membrane. We conclude that Lubrol-RAFTs probably represent the platform for tumor-promoting ephrin-B2-integrin-beta1 interaction, which could become an interesting target for future antitumoral therapies.

Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arakawa, Masayuki; Ebato, Chie; Mita, Tomoya

2009-12-18

Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4more » daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.« less

A human beta cell line with drug inducible excision of immortalizing transgenes

PubMed Central

Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

2015-01-01

Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of transforming growth factor beta in a mouse model of breast cancer.

PubMed

Nam, Jeong-Seok; Suchar, Adam M; Kang, Mi-Jin; Stuelten, Christina H; Tang, Binwu; Michalowska, Aleksandra M; Fisher, Larry W; Fedarko, Neal S; Jain, Alka; Pinkas, Jan; Lonning, Scott; Wakefield, Lalage M

2006-06-15

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors.

PubMed

Uberti, Michelle A; Hague, Chris; Oller, Heide; Minneman, Kenneth P; Hall, Randy A

2005-04-01

The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

Transforming growth factor-beta inhibits human antigen-specific CD4+ T cell proliferation without modulating the cytokine response.

PubMed

Tiemessen, Machteld M; Kunzmann, Steffen; Schmidt-Weber, Carsten B; Garssen, Johan; Bruijnzeel-Koomen, Carla A F M; Knol, Edward F; van Hoffen, Els

2003-12-01

Transforming growth factor (TGF)-beta has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-beta on antigen-specific established memory T cells has not been investigated yet. In this study antigen-specific CD4(+) T cell clones (TCC) were used to determine the effect of TGF-beta on antigen-specific proliferation, the activation status of the T cells and their cytokine production. This study demonstrates that TGF-beta is an adequate suppressor of antigen-specific T cell proliferation, by reducing the cell-cycle rate rather than induction of apoptosis. Addition of TGF-beta resulted in increased CD69 expression and decreased CD25 expression on T cells, indicating that TGF-beta is able to modulate the activation status of in vivo differentiated T cells. On the contrary, the antigen-specific cytokine production was not affected by TGF-beta. Although TGF-beta was suppressive towards the majority of the T cells, insensitivity of a few TCC towards TGF-beta was also observed. This could not be correlated to differential expression of TGF-beta signaling molecules such as Smad3, Smad7, SARA (Smad anchor for receptor activation) and Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate). In summary, TGF-beta has a pronounced inhibitory effect on antigen-specific T cell proliferation without modulating their cytokine production.

Inhibition of glycogen synthase kinase 3beta during heart failure is protective.

PubMed

Hirotani, Shinichi; Zhai, Peiyong; Tomita, Hideharu; Galeotti, Jonathan; Marquez, Juan Pablo; Gao, Shumin; Hong, Chull; Yatani, Atsuko; Avila, Jesús; Sadoshima, Junichi

2007-11-26

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3beta during heart failure could be compensatory.

Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer

2009-04-24

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline,more » the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.« less

Elliptical-P cells in the avian perilymphatic interface of the Tegmentum vasculosum

NASA Technical Reports Server (NTRS)

Fermin, C. D.; Lee, D. H.; Martin, D. S.

1995-01-01

Elliptical cells (E-P) are present at the perilymphatic interface lumen (PIL) of the lagena. The E-P cells often separate from the tegmentum vasculosum (TV) and have touching processes that form a monolayer between the K+ rich perilymph and the Na+ rich endolymph, similar to the mammalian Reissner's membrane. We examined the TV of chicks (Gallus domesticus) and quantitated the expression of anti-S100 alphaalphabetabeta and S100 beta. There was a 30% increase of S100 beta saturation in the light cells facing the PIL when compared to other TV light cells. We show that: (1) the dimer anti- S100 alphaalphabetabeta and the monomer anti-S100 beta are expressed preferentially in the light cells and the E-P cells of TV; (2) expression of S100 beta is higher in light cells facing the PIL than in adjacent cells; (3) the expression of the dimer S100 alphaalphabetabeta and monomer S100 beta overlaps in most inner ear cell types, including the cells of the TV, most S100 alphaalphabetabeta positive cells express S 100 beta, but S100 beta positive cells do not always express S100 alphaalphabetabeta; and (4) the S100 beta expression in light cells, the abundant Na+-K+ ATPase on dark cells of the TV, and previously demonstrated co-localization of S100 beta/GABA in sensory cells suggest that S100 beta could have, in the inner ear, a dual neurotrophic-ionic modulating function.

Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

PubMed

Pinho, Andreia V; Bensellam, Mohammed; Wauters, Elke; Rees, Maxine; Giry-Laterriere, Marc; Mawson, Amanda; Ly, Le Quan; Biankin, Andrew V; Wu, Jianmin; Laybutt, D Ross; Rooman, Ilse

2015-01-01

Sirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear. This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas. We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas. This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

Hypothyroidism in utero stimulates pancreatic beta cell proliferation and hyperinsulinaemia in the ovine fetus during late gestation.

PubMed

Harris, Shelley E; De Blasio, Miles J; Davis, Melissa A; Kelly, Amy C; Davenport, Hailey M; Wooding, F B Peter; Blache, Dominique; Meredith, David; Anderson, Miranda; Fowden, Abigail L; Limesand, Sean W; Forhead, Alison J

2017-06-01

Thyroid hormones are important regulators of growth and maturation before birth, although the extent to which their actions are mediated by insulin and the development of pancreatic beta cell mass is unknown. Hypothyroidism in fetal sheep induced by removal of the thyroid gland caused asymmetric organ growth, increased pancreatic beta cell mass and proliferation, and was associated with increased circulating concentrations of insulin and leptin. In isolated fetal sheep islets studied in vitro, thyroid hormones inhibited beta cell proliferation in a dose-dependent manner, while high concentrations of insulin and leptin stimulated proliferation. The developing pancreatic beta cell is therefore sensitive to thyroid hormone, insulin and leptin before birth, with possible consequences for pancreatic function in fetal and later life. The findings of this study highlight the importance of thyroid hormones during pregnancy for normal development of the fetal pancreas. Development of pancreatic beta cell mass before birth is essential for normal growth of the fetus and for long-term control of carbohydrate metabolism in postnatal life. Thyroid hormones are also important regulators of fetal growth, and the present study tested the hypotheses that thyroid hormones promote beta cell proliferation in the fetal ovine pancreatic islets, and that growth retardation in hypothyroid fetal sheep is associated with reductions in pancreatic beta cell mass and circulating insulin concentration in utero. Organ growth and pancreatic islet cell proliferation and mass were examined in sheep fetuses following removal of the thyroid gland in utero. The effects of triiodothyronine (T 3 ), insulin and leptin on beta cell proliferation rates were determined in isolated fetal ovine pancreatic islets in vitro. Hypothyroidism in the sheep fetus resulted in an asymmetric pattern of organ growth, pancreatic beta cell hyperplasia, and elevated plasma insulin and leptin concentrations. In pancreatic islets isolated from intact fetal sheep, beta cell proliferation in vitro was reduced by T 3 in a dose-dependent manner and increased by insulin at high concentrations only. Leptin induced a bimodal response whereby beta cell proliferation was suppressed at the lowest, and increased at the highest, concentrations. Therefore, proliferation of beta cells isolated from the ovine fetal pancreas is sensitive to physiological concentrations of T 3 , insulin and leptin. Alterations in these hormones may be responsible for the increased beta cell proliferation and mass observed in the hypothyroid sheep fetus and may have consequences for pancreatic function in later life. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Conversion of immortal liver progenitor cells into pancreatic endocrine progenitor cells by persistent expression of Pdx-1.

PubMed

Jin, Cai-Xia; Li, Wen-Lin; Xu, Fang; Geng, Zhen H; He, Zhi-Ying; Su, Juan; Tao, Xin-Rong; Ding, Xiao-Yan; Wang, Xin; Hu, Yi-Ping

2008-05-01

The conversion of expandable liver progenitor cells into pancreatic beta cells would provide a renewable cell source for diabetes cell therapy. Previously, we reported the establishment of liver epithelial progenitor cells (LEPCs). In this work, LEPCs were modified into EGFP/Pdx-1 LEPCs, cells with stable expression of both Pdx-1 and EGFP. Unlike previous work, with persistent expression of Pdx-1, EGFP/Pdx-1 LEPCs acquired the phenotype of pancreatic endocrine progenitor cells rather than giving rise to insulin-producing cells directly. EGFP/Pdx-1 LEPCs proliferated vigorously and expressed the crucial transcription factors involved in beta cell development, including Ngn3, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6, Isl1, MafA and endogenous Pdx-1, but did not secrete insulin. When cultured in high glucose/low serum medium supplemented with cytokines, EGFP/Pdx-1 LEPCs stopped proliferating and gave rise to functional beta cells without any evidence of exocrine or other islet cell lineage differentiation. When transplanted into diabetic SCID mice, EGFP/Pdx-1 LEPCs ameliorated hyperglycemia by secreting insulin in a glucose regulated manner. Considering the limited availability of beta cells, we propose that our experiments will provide a framework for utilizing the immortal liver progenitor cells as a renewable cell source for the generation of functional pancreatic beta cells.

Beneficial effect of 17{beta}-estradiol on hyperglycemia and islet {beta}-cell functions in a streptozotocin-induced diabetic rat model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamabe, Noriko; Kang, Ki Sung; Zhu Baoting, E-mail: BTZhu@kumc.ed

2010-11-15

The modulating effect of estrogen on glucose homeostasis remains a controversial issue at present. In this study, we sought to determine the beneficial effect of 17{beta}-estradiol (E{sub 2}) on hyperglycemia and islet {beta}-cell functions in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were injected i.p. with STZ to induce a relatively mild diabetic condition. The rats were then treated with E{sub 2} orally at 500 {mu}g/kg body weight/day for 15 days to evaluate the modulating effect on hyperglycemia, insulin secretion, and islet {beta}-cell proliferation. E{sub 2} administration for 10 days significantly lowered plasma glucose levels, increased plasma insulin levels, andmore » improved glucose tolerance by attenuating insulin response to oral glucose loading. These beneficial effects of E{sub 2} were accompanied by increases in islet number and volume, rate of islet cell proliferation, and the amount of insulin secreted. The growth-stimulatory effect of E{sub 2} on islet cells was linked to the functions of the estrogen receptor {alpha}. Notably, these protective effects of E{sub 2} on diabetic conditions were basically not observed when the STZ-treated rats had a more severe degree of islet damage and hyperglycemia. Taken together, we conclude that E{sub 2} can promote the regeneration of damaged pancreatic islets by stimulating {beta}-cell proliferation in diabetic rats, and this effect is accompanied by improvements in glucose tolerance and a decrease in plasma glucose levels. These findings suggest that oral administration of E{sub 2} may be beneficial in diabetic patients with an accelerated loss of islet {beta}-cells.« less

Measuring beta-cell function relative to insulin sensitivity in youth: Does the hyperglycemic clamp suffice?

USDA-ARS?s Scientific Manuscript database

To compare beta-cell function relative to insulin sensitivity, disposition index (DI), calculated from two clamps (2cDI, insulin sensitivity from the hyperinsulinemic-euglycemic clamp and first-phase insulin from the hyperglycemic clamp) with the DI calculated from the hyperglycemic clamp alone (hcD...

Role for the TRPV1 channel in insulin secretion from pancreatic beta cells.

PubMed

Diaz-Garcia, Carlos Manlio; Morales-Lázaro, Sara L; Sánchez-Soto, Carmen; Velasco, Myrian; Rosenbaum, Tamara; Hiriart, Marcia

2014-06-01

Transient receptor potential channels have been put forward as regulators of insulin secretion. A role for the TRPV1 ion channel in insulin secretion has been suggested in pancreatic beta cell lines. We explored whether TRPV1 is functionally expressed in RINm5F and primary beta cells from neonate and adult rats. We examined if capsaicin could activate cationic non-selective currents. Our results show that TRPV1 channels are not functional in insulin-secreting cells, since capsaicin did not produce current activation, not even under culture conditions known to induce the expression of other ion channels in these cells. Although TRPV1 channels seem to be irrelevant for the physiology of isolated beta cells, they may play a role in glucose homeostasis acting through the nerve fibers that regulate islet function. At the physiological level, we observed that Trpv1 (-/-) mice presented lower fasting insulin levels than their wild-type littermates, however, we did not find differences between these experimental groups nor in the glucose tolerance test or in the insulin secretion. However, we did find that the Trpv1 (-/-) mice exhibited a higher insulin sensitivity compared to their wild-type counterparts. Our results demonstrate that TRPV1 does not contribute to glucose-induced insulin secretion in beta cells as was previously thought, but it is possible that it may control insulin sensitivity.

Expression of S100 beta in sensory and secretory cells of the vertebrate inner ear

NASA Technical Reports Server (NTRS)

Fermin, C. D.; Martin, D. S.

1995-01-01

We evaluated anti-S100 beta expression in the chick (Gallus domesticus) inner ear and determined that: 1) the monomer anti-S100 beta is expressed differentially in the vestibular and auditory perikarya; 2) expression of S100 beta in the afferent nerve terminals is time-related to synapse and myelin formation; 3) the expression of the dimer anti-S100 alpha alpha beta beta and monomer anti-S100 beta overlaps in most inner ear cell types. Most S100 alpha alpha beta beta positive cells express S100 beta, but S100 beta positive cells do not always express S100 alpha alpha beta beta. 4) the expression of S100 beta is diffused over the perikaryal cytoplasm and nuclei of the acoustic ganglia but is concentrated over the nuclei of the vestibular perikarya. 6) S100 beta is expressed in secretory cells, and it is co-localized with GABA in sensory cells. 7) Color thresholding objective quantitation indicates that the amount of S100 beta was higher (mean 22, SD +/- 4) at E19 than at E9 (mean 34, SD +/- 3) in afferent axons. 8) Moreover, S100 beta was unchanged between E11-E19 in the perikaryal cytoplasm, but did change over the nuclei. At E9, 74%, and at E21, 5% of vestibular perikarya were positive. The data suggest that S100 beta may be physically associated with neuronal and ionic controlling cells of the vertebrate inner ear, where it could provide a dual ionic and neurotrophic modulatory function.

Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma.

PubMed Central

Altomonte, M.; Montagner, R.; Fonsatti, E.; Colizzi, F.; Cattarossi, I.; Brasoveanu, L. I.; Nicotra, M. R.; Cattelan, A.; Natali, P. G.; Maio, M.

1996-01-01

Human endoglin (CD105) is a member of the transforming growth factor beta (TGF-beta) receptor family that binds TGF-beta1 and -beta3, but not TGF-beta2, on human endothelial cells. Immunohistochemical analyses demonstrated that CD105 is expressed on normal and neoplastic cells of the melanocytic lineage. The anti-CD105 MAb, MAEND3, stained 50, 25 and 34% of intradermal naevi, primary and metastatic melanomas investigated, respectively, and nine out of 12 melanoma cell lines. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that CD105 expressed by melanoma cells consists of a homodimeric protein with an apparent molecular weight of 180 and 95 kDa under non-reducing and reducing conditions. Cross-linking of 125I-labelled TGF-beta1 to melanoma cells, Mel 97, by disuccinimidyl suberate (DSS) demonstrated that CD105 expressed on pigmented cells binds TGF-beta1; the pattern of binding of TGF-beta1 to melanoma cells was found to be similar to that of human umbilical vein endothelial cells. The addition of exogenous, bioactive TGF-beta1 significantly (P<0.05) inhibited the growth of CD105-positive melanoma cells, Mel 97, but did not affect that of CD105-negative melanoma cells, F0-1. These data, altogether, demonstrate that CD105 is expressed on pigmented cells and might play a functionally relevant role in the biology of human melanoma cells by regulating their sensitivity to TGF-betas. Images Figure 1 Figure 3 Figure 4 PMID:8932339

Insulin-producing cells could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells

PubMed Central

2013-01-01

Objective The aim of this study was to compare the difference between insulin-producing cells (IPCs) and normal human pancreatic beta cells both in physiological function and morphological features in cellular level. Methods The levels of insulin secretion were measured by enzyme-linked immunosorbent assay. The insulin gene expression was determined by real-time quantitative polymerase chain reaction. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy. Results IPCs and normal human pancreatic beta cells were similar to each other under the observation in AFM with the porous structure features in the cytoplasm. Both number of membrane particle size and average roughness of normal human beta cells were higher than those of IPCs. Conclusions Our results firstly revealed that the cellular ultrastructure of IPCs was closer to that of normal human pancreatic beta cells, but they still could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. PMID:23421382

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

PubMed

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

2009-08-07

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

Diverse roles of integrin receptors in articular cartilage.

PubMed

Shakibaei, M; Csaki, C; Mobasheri, A

2008-01-01

Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

Functionalization of DNA Nanostructures for Cell Signaling Applications

NASA Astrophysics Data System (ADS)

Pedersen, Ronnie O.

Transforming growth factor beta (TGF-beta) is an important cytokine responsible for a wide range of different cellular functions including extracellular matrix formation, angiogenesis and epithelial-mesenchymal transition. We have sought to use self-assembling DNA nanostructures to influence TGF-beta signaling. The predictable Watson Crick base pairing allows for designing self-assembling nanoscale structures using oligonucleotides. We have used the method of DNA origami to assemble structures functionalized with multiple peptides that bind TGF-beta receptors outside the ligand binding domain. This allows the nanostructures to cluster TGF-beta receptors and lower the energy barrier of ligand binding thus sensitizing the cells to TGF-beta stimulation. To prove efficacy of our nanostructures we have utilized immunofluorescent staining of Smad2/4 in order to monitor TGF-beta mediated translocation of Smad2/4 to the cell nucleus. We have also utilized Smad2/4 responsive luminescence constructs that allows us to quantify TGF-beta stimulation with and without nanostructures. To functionalize our nanostructures we relied on biotin-streptavidin linkages. This introduces a multivalency that is not necessarily desirable in all designs. Therefore we have investigated alternative means of functionalization. The first approach is based on targeting DNA nanostructure by using zinc finger binding proteins. Efficacy of zinc finger binding proteins was assayed by the use of enzyme-linked immunosorbent (ELISA) assay and atomic force microscopy (AFM). While ELISA indicated a relative specificity of zinc finger proteins for target DNA sequences AFM showed a high degree of non-specific binding and insufficient affinity. The second approach is based on using peptide nucleic acid (PNA) incorporated in the nanostructure through base pairing. PNA is a synthetic DNA analog consisting of a backbone of repeating N-(2-aminoethyl)-glycine units to which purine and pyrimidine bases are linked by amide bonds. The solid phase synthesis of PNA allows for convenient extension of the backbone into a peptide segment enabling peptide functionalization of DNA nanostructures. We have investigated how the neutral character of PNA alters the incorporation in DNA based nanostructures compared to a DNA control using biotinylation and AFM. Results indicate that PNA can successfully be used as a way of functionalizing DNA nanostructures. Additionally we have shown that functionalized nanostructures are capable of sensitizing cells to TGF-beta stimulation.

Function of beta 2-adrenergic receptors in chronic localized myalgia.

PubMed

Maekawa, Kenji; Kuboki, Takuo; Inoue, Eitoku; Inoue-Minakuchi, Mami; Suzuki, Koji; Yatani, Hirofumi; Clark, Glenn T

2003-01-01

To investigate alteration of beta 2-adrenergic receptor (beta 2 AR) function in chronic localized myalgia subjects by evaluating levels of the beta 2 AR second messenger, cyclic adenosine monophosphate (cAMP), in mononuclear cells after beta AR-agonist stimulation. Eleven chronic localized myalgia subjects and 21 matched healthy controls participated in this study. Peripheral blood (30 cc) was drawn from the subjects' anterocubital vein. Mononuclear cells were isolated from the total blood by using the Ficoll-Hypaque gradient technique. Basal and stimulated intracellular cAMP levels were determined by enzyme immunoassay using a commercially available kit. Aliquots of 5 x 10(6) cells were incubated with or without stimulation of the beta AR-agonist isoproterenol for 5 minutes. Five different concentrations of isoproterenol (10(-3) M to 10(-7) M) were utilized. cAMP levels in both groups were tested statistically by a 2-way repeated-measures ANOVA with 2 predictors, group difference and isoproterenol concentration difference. As with isoproterenol stimulation, the cAMP responses to forskolin, which activates adenylyl cyclase directly and produces cAMP, bypassing the cell surface receptors were also measured. The basal cAMP levels in both groups (myalgia: 0.33 +/- 0.02 pmol/5 x 10(6) cells; control: 0.43 +/- 0.10 pmol/5 x 10(6) cells) were almost identical, and isoproterenol-produced cAMP levels increased dose-dependently in both groups. No significant differences in the mean cAMP levels were observed between the groups (P = .909). Significant increases were observed according to the isoproterenol concentration increase (P < .0001). The cAMP responses to forskolin stimulation also showed no significant group difference (P = .971). These results suggest that beta 2 AR function is not different between localized myalgia subjects and healthy individuals.

Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice.

PubMed

Rui, Jinxiu; Deng, Songyan; Lebastchi, Jasmin; Clark, Pamela L; Usmani-Brown, Sahar; Herold, Kevan C

2016-05-01

Type 1 diabetes is caused by the immunological destruction of pancreatic beta cells. Preclinical and clinical data indicate that there are changes in beta cell function at different stages of the disease, but the fate of beta cells has not been closely studied. We studied how immune factors affect the function and epigenetics of beta cells during disease progression and identified possible triggers of these changes. We studied FACS sorted beta cells and infiltrating lymphocytes from NOD mouse and human islets. Gene expression was measured by quantitative real-time RT-PCR (qRT-PCR) and methylation of the insulin genes was investigated by high-throughput and Sanger sequencing. To understand the role of DNA methyltransferases, Dnmt3a was knocked down with small interfering RNA (siRNA). The effects of cytokines on methylation and expression of the insulin gene were studied in humans and mice. During disease progression in NOD mice, there was an inverse relationship between the proportion of infiltrating lymphocytes and the beta cell mass. In beta cells, methylation marks in the Ins1 and Ins2 genes changed over time. Insulin gene expression appears to be most closely regulated by the methylation of Ins1 exon 2 and Ins2 exon 1. Cytokine transcription increased with age in NOD mice, and these cytokines could induce methylation marks in the insulin DNA by inducing methyltransferases. Similar changes were induced by cytokines in human beta cells in vitro. Epigenetic modification of DNA by methylation in response to immunological stressors may be a mechanism that affects insulin gene expression during the progression of type 1 diabetes.

Etiopathogenesis of type 1 diabetes mellitus: prognostic factors for the evolution of residual β cell function

PubMed Central

2009-01-01

Type 1A diabetes mellitus (T1ADM) is a progressive autoimmune disease mediated by T lymphocytes with destruction of beta cells. Up to now, we do not have precise methods to assess the beta cell mass, "in vivo" or "ex-vivo". The studies about its genetic susceptibility show strong association with class II antigens of the HLA system (particularly DQ). Others genetics associations are weaker and depend on the population studied. A combination of precipitating events may occur at the beginning of the disease. There is a silent loss of immune-mediated beta cells mass which velocity has an inverse relation with the age, but it is influenced by genetic and metabolic factors. We can predict the development of the disease primarily through the determination of four biochemically islet auto antibodies against antigens like insulin, GAD65, IA2 and Znt8. Beta cell destruction is chronically progressive but at clinical diagnosis of the disease a reserve of these cells still functioning. The goal of secondary disease prevention is halt the autoimmune attack on beta cells by redirecting or dampening the immune system. It is remains one of the foremost therapeutic goals in the T1ADM. Glycemic intensive control and immunotherapeutic agents may preserve beta-cell function in newly diagnosed patients with T1ADM. It may be assessed through C-peptide values, which are important for glycemic stability and for the prevention of chronic complications of this disease. This article will summarize the etiopathogenesis mechanisms of this disease and the factors can influence on residual C-peptide and the strategies to it preservation. PMID:19961609

MicroRNA-320a suppresses human colon cancer cell proliferation by directly targeting {beta}-catenin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jian-Yong; State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 710032 Xi'an; Huang, Yi

2012-04-20

Highlights: Black-Right-Pointing-Pointer miR-320a is downregulated in human colorectal carcinoma. Black-Right-Pointing-Pointer Overexpression of miR-320a inhibits colon cancer cell proliferation. Black-Right-Pointing-Pointer {beta}-Catenin is a direct target of miR-320a in colon cancer cells. Black-Right-Pointing-Pointer miR-320a expression inversely correlates with mRNA expression of {beta}-catenin's target genes in human colon carcinoma. -- Abstract: Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-320a) in human colorectal carcinoma. However, its expression pattern and underlying mechanisms in the development and progression of colorectal carcinoma has not been elucidated clearly. Here, we performed real-time PCR to examine the expression levels of miR-320a in colonmore » cancer cell lines and tumor tissues. And then, we investigated its biological functions in colon cancer cells by a gain of functional strategy. Further more, by the combinational approaches of bioinformatics and experimental validation, we confirmed target associations of miR-320a in colorectal carcinoma. Our results showed that miR-320a was frequently downregulated in cancer cell lines and colon cancer tissues. And we demonstrated that miR-320a restoration inhibited colon cancer cell proliferation and {beta}-catenin, a functionally oncogenic molecule was a direct target gene of miR-320a. Finally, the data of real-time PCR showed the reciprocal relationship between miR-320a and {beta}-catenin's downstream genes in colon cancer tissues. These findings indicate that miR-320a suppresses the growth of colon cancer cells by directly targeting {beta}-catenin, suggesting its application in prognosis prediction and cancer treatment.« less

N-terminal tyrosine phosphorylation of caveolin-2 negates anti-proliferative effect of transforming growth factor beta in endothelial cells

PubMed Central

Abel, Britain; Willoughby, Cara; Jang, Sungchan; Cooper, Laura; Xie, Leike; Vo-Ransdell, Chi; Sowa, Grzegorz

2012-01-01

Here we show that tyrosine phosphorylation of caveolin-2 (Cav-2) negatively regulates the anti-proliferative function of transforming growth factor beta (TGF-beta) in endothelial cells. In contrast to wild-type-Cav-2, retroviral re-expression of Y19/27F-Cav-2 in Cav-2 knockout endothelial cells did not affect anti-proliferative effect of TGF-beta compared to empty vector. Conversely, although less effective than wild-type, re-expression of S23/36A-Cav-2 reduced the effect of TGF-beta compared to empty vector. This differential effect of tyrosine and serine phosphorylation mutants of Cav-2 correlated with TGF-beta-induced Smad3 phosphorylation and transcriptional activation of plasminogen activator inhibitor-1. Thus tyrosine-phosphorylated Cav-2 counteracts anti-proliferative effect of TGF-beta in endothelial cells. PMID:22819829

Loss of c-myc repression coincides with ovarian cancer resistance to transforming growth factor beta growth arrest independent of transforming growth factor beta/Smad signaling.

PubMed

Baldwin, Rae Lynn; Tran, Hang; Karlan, Beth Y

2003-03-15

Many epithelial carcinomas, including ovarian, are refractory to the antiproliferative effects of transforming growth factor (TGF) beta. In some cancers, TGF-beta resistance has been linked to TGF-beta receptor II (TbetaR-II) and Smad4 mutations; however, in ovarian cancer, the mechanism of resistance remains unclear. Primary ovarian epithelial cell cultures were used as a model system to determine the mechanisms of TGF-beta resistance. To simulate in vivo responses to TGF-beta, primary cultures derived from normal human ovarian surface epithelium (HOSE) and from ovarian carcinomas (CSOC) were grown on collagen I gel, the predominant matrix molecule in the ovarian tumor milieu. When treated with 5 ng/ml TGF-beta for 72 h, HOSE (n = 11) proliferation was inhibited by 20 +/- 21% on average. In contrast, CSOC (n = 10) proliferation was stimulated 5 +/- 10% in response to TGF-beta (a statistically significant difference in response when compared with HOSE; P = 0.001). To dissect the TGF-beta/Smad signaling pathway we used a quantitative RNase protection assay (RPA) for measuring mRNA levels of TGF-beta pathway components in 20 HOSE and 20 CSOC cultures. Basal mRNA levels of TGF-beta receptors I and II, downstream signaling components Smad2, 3, 4, 6, 7, and the transcriptional corepressors Ski and SnoN did not show a statistically significant difference between HOSE and CSOC, and cannot explain their differential susceptibility to TGF-beta-induced cell cycle arrest. To assess functional differences of the TGF-beta pathway in TGF-beta-sensitive HOSE and TGF-beta-resistant CSOC, we measured Smad2/4 and 3/4 complex induction after TGF-beta treatment. HOSE and CSOC showed equivalent Smad2/4 and 3/4 complex induction after TGF-beta exposure for 0, 0.5, 2, and 4 h. It has been proposed that SnoN and Ski are corepressors of the TGF-beta/Smad pathway and undergo TGF-beta-induced degradation followed by reinduction of SnoN mRNA. However, our data show equivalent SnoN degradation in HOSE and CSOC, and equivalent SnoN mRNA induction after TGF-beta treatment. Surprising, TGF-beta-induced Ski degradation was not observed in HOSE or CSOC, suggesting that Ski may not function as a TGF-beta/Smad corepressor in ovarian epithelial cells. These data implied that the TGF-beta/Smad pathway remains functional in CSOC, although CSOC cells are resistant to antimitogenic TGF-beta effects. CSOC resistance to TGF-beta coincided with the loss of c-myc down-regulation. These data suggest that TGF-beta/Smad signaling is blocked downstream of Smad complex formation or that an alternate signaling pathway other than TGF-beta/Smad may transmit TGF-beta-induced cell cycle arrest in the ovarian epithelium.

Beta-lactamase induction and cell wall metabolism in Gram-negative bacteria

PubMed Central

Zeng, Ximin; Lin, Jun

2013-01-01

Production of beta-lactamases, the enzymes that degrade beta-lactam antibiotics, is the most widespread and threatening mechanism of antibiotic resistance. In the past, extensive research has focused on the structure, function, and ecology of beta-lactamases while limited efforts were placed on the regulatory mechanisms of beta-lactamases. Recently, increasing evidence demonstrate a direct link between beta-lactamase induction and cell wall metabolism in Gram-negative bacteria. Specifically, expression of beta-lactamase could be induced by the liberated murein fragments, such as muropeptides. This article summarizes current knowledge on cell wall metabolism, beta-lactam antibiotics, and beta-lactamases. In particular, we comprehensively reviewed recent studies on the beta-lactamase induction by muropeptides via two major molecular mechanisms (the AmpG–AmpR–AmpC pathway and BlrAB-like two-component regulatory system) in Gram-negative bacteria. The signaling pathways for beta-lactamase induction offer a broad array of promising targets for the discovery of new antibacterial drugs used for combination therapies. Therefore, to develop effective mitigation strategies against the widespread beta-lactam resistance, examination of the molecular basis of beta-lactamase induction by cell wall fragment is highly warranted. PMID:23734147

Expression and function of CD8 alpha/beta chains on rat and human mast cells.

PubMed

Kim, Mi-Sun; Kim, Sung-Hoon; Lee, Hye-Jung; Kim, Hyung-Min

2004-03-01

The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. RT-PCR and Western blot analysis identified the presence of CD8 alpha/beta chains on the mast cells, and immunohistochemistry confirmed CD8alpha expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 alpha/beta chains on rat mast cells induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.

Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human A{beta}{sub 1-42} and tau

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakravarthy, Balu, E-mail: Balu.Chakravarthy@nrc-cnrc.gc.ca; Gaudet, Chantal; Menard, Michel

2012-10-12

Highlights: Black-Right-Pointing-Pointer A{beta} and tau-induced neurofibrillary tangles play a key role in Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}{sub 1-42} and mutant tau protein together reduce the primary cilium length. Black-Right-Pointing-Pointer This shortening likely reduces cilium-dependent neurogenesis and memory function. Black-Right-Pointing-Pointer This provides a model of an A{beta}/tau targeting of a neuronal signaling organelle. -- Abstract: The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatinmore » receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75{sup NTR}). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both A{beta}{sub 1-42} and the mutant human tau protein tau{sub P301L,} the dentate granule cells still had immunostainable SSTR3- and p75{sup NTR}-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of A{beta}{sub 1-42} or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of A{beta}{sub 1-42} and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.« less

Synthetic alleles at position 121 define a functional domain of human interleukin-1 beta.

PubMed

Ambrosetti, D C; Palla, E; Mirtella, A; Galeotti, C; Solito, E; Navarra, P; Parente, L; Melli, M

1996-06-01

The non-conservative substitution of the tyrosine residue at position 121 of human interleukin-1 beta (IL-1 beta) generates protein mutants showing strong reduction of the capacity to induce (a) prostaglandin E2 (PGE2) release from fibroblasts and smooth muscle cells, (b) murine T-cells proliferation and (c) activation of interleukin-6 (IL-6) gene expression. It is generally accepted that these functions are mediated by the type-I interleukin-1 receptor (IL-1RI). However, the mutant proteins maintain the binding affinity to the types-I and II IL-1 receptors, which is the same as the control IL-1 beta, suggesting that this amino acid substitution does not alter the structure of the molecule, except locally. Thus we have identified a new functional site of IL-1 beta different from the known receptor binding region, responsible for fundamental IL-1 beta functions. Moreover, we show that the same mutants maintain at least two hypothalamic functions, that is, the in vitro short-term PGE2 release from rat hypothalamus and the induction of fever in rabbits. This result suggests that there is yet another site of the molecule responsible for the hypothalamic functions, implying that multiple active sites on the IL-1 beta molecule, possibly binding to more than one receptor chain, trigger different signals.

Lack of the central nervous system- and neural crest-expressed forkhead gene Foxs1 affects motor function and body weight.

PubMed

Heglind, Mikael; Cederberg, Anna; Aquino, Jorge; Lucas, Guilherme; Ernfors, Patrik; Enerbäck, Sven

2005-07-01

To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-beta-galactosidase reporter gene "knock-in" (Foxs1beta-gal/beta-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a beta-galactosidase reporter gene. Staining for beta-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1beta-gal/beta-gal mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1beta-gal/lbeta-gal males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1beta-gal/beta-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.

[Associations of insulin resistance and pancreatic beta-cell function with plasma glucose level in type 2 diabetes].

PubMed

Nian, Xiaoping; Sun, Gaisheng; Dou, Chunmei; Hou, Hongbo; Fan, Xiuping; Yu, Hongmei; Ma, Ling; He, Bingxian

2002-06-10

To investigate the influence of insulin resistance and pancreatic beta-cell function on plasma glucose level in type 2 diabetes so as to provide theoretical basis for reasonable selection of hypoglycemic agents. The plasma non-specific insulin (NSINS), true insulin (TI) and glucose in eight-one type 2 diabetics, 38 males and 43 females, with a mean age of 53 years, were examined 0, 30, 60 and 120 minutes after they had 75 grams of instant noodles. The patients were divided into two groups according to their fasting plasma glucose (FPG): group A (FPG < 8.89 mmol/L) and group B (FPG> = 8.89 mmol/L). The insulin resistance was evaluated by HOMA-IR, the beta-cell function was evaluated by HOMA-beta formula and the formula deltaI(30)/deltaG(30) = (deltaI(30)-deltaI(0))/(deltaG(30)-deltaG(0)). The insulin area under curve (INSAUC) was evaluated by the formula INSAUC=FINS/2+INS(30)+INS(60)+INS(120)/2. The mean FPG was 6.23 mmol/L in group A and 12.6 mmol/L in group B. PG2H was 11.7 mmol/L in group A and 19.2 mmol/L in group B. The TI levels in group B at 0, 30, 60, 120 min during standard meal test were significantly higher than those in group A: 6.15 +/- 1.06 vs 4.77 +/- 1.06, 9.76 +/- 1.1 vs 5.88 +/- 1.1,14.68 +/- 1.11 vs 6.87 +/- 1.1 and 17.13 +/- 1.12 vs 8.0 +/- 1.1 microU/dl (all P< 0.01). The NSINS showed the same trend. The insulin resistance in group B was 1.5 times that in group A. With the insulin resistance adjusted, the beta cell function in group A was 5 to 6 times that in group B. The INSAUC in group A was 1.66 times larger than that in group B, especially the INSAUC for true insulin (2 times larger). The contribution of insulin resistance and beta cell function to PG2H was half by half in group A and 1:8 in group B. beta cell function calculated by insulin (Homa-beta) explained 41% of the plasma glucose changes in group A and 54% of the plasma glucose changes in group B. The contribution of insulin deficiency to plasma glocose was 3.3.times that of insulin resistance in group A and was 9.5 times that of insulin resistance in group B. Insulin sensitivity explained 12% of the plasma glucose changes in group A, and only 5.7% of the plasma glucose changes in group B. Diabetics with FPG greater than 8.89 mmol/L have both higher insulin resistance and poorer beta-cell function, their hyperglycemia being caused mainly by beta-cell failure, The combined use of insulin sensitizer and insulin or insulintropic agents during the initial stage of treatment is effective.

Differences in beta-cell function and insulin secretion in Black vs. White obese adolescents: Do incretin hormones play a role?

USDA-ARS?s Scientific Manuscript database

Black youth are at higher risk for type 2 diabetes (T2D) than their White peers. Previously we demonstrated that for the same degree of insulin sensitivity, Black youth have an upregulated beta-cell function and insulin hypersecretion, in response to intravenous (IV) glucose, compared with Whites. T...

Beta cell function and BMI in ethnically diverse children with newly diagnosed autoimmune type 1 diabetes

USDA-ARS?s Scientific Manuscript database

The objective of our study was to examine the relationship between BMI and beta-cell function at diagnosis of autoimmune type 1 diabetes (T1D) in a large group of ethnically diverse children. Cross-sectional analysis of 524 children (60.8% White, 19.5% Hispanic, 14.5% African-American, 5.2% other n...

Evidences that beta1 integrin and Rac1 are involved in the overriding effect of laminin on myelin-associated glycoprotein inhibitory activity on neuronal cells.

PubMed

Laforest, Sullivan; Milanini, Julie; Parat, Fabrice; Thimonier, Jean; Lehmann, Maxime

2005-11-01

During neurite elongation, migrating growth cones encounter both permissive and inhibitory substrates, such as laminin and MAG (myelin-associated glycoprotein), respectively. Here, we demonstrated on two neuronal cell lines (PC12 and N1E-115), that laminin and collagen hampered, in a dose-dependent manner, MAG inhibitory activity on several integrin functions, i.e., neurite growth, cell adhesion and cell spreading. Using a function blocking antibody, in PC12 cells, we showed that alpha1beta1 integrin is required in these phenomena. In parallel, we observed that MAG perturbs actin dynamics and lamellipodia formation during early steps of cell spreading. This seemed to be independent of RhoA activation, but dependent of Rac-1 inhibition by MAG. Laminin overrode MAG activity on actin and prevented MAG inhibition NGF-induced Rac1 activation. In conclusion, we evidenced antagonistic signaling between MAG receptors and beta1 integrins, in which Rac-1 may have a central function.

Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

PubMed

Liu, Haiyan; Javaheri, Ali; Godar, Rebecca J; Murphy, John; Ma, Xiucui; Rohatgi, Nidhi; Mahadevan, Jana; Hyrc, Krzysztof; Saftig, Paul; Marshall, Connie; McDaniel, Michael L; Remedi, Maria S; Razani, Babak; Urano, Fumihiko; Diwan, Abhinav

2017-01-01

Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells

PubMed Central

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W.; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S.; Troncoso, Patricia; Maity, Sankar N.; Navone, Nora M.

2012-01-01

Purpose To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction. Experimental Design We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs. Results Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling. Conclusion We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients. PMID:22298898

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

PubMed Central

Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh; Batchuluun, Battsetseg; Nagy, Kristina; Neely, Eric; Gull, Rida; Nagy, Andras; Wheeler, Michael B.

2016-01-01

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. PMID:27755557

Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells.

PubMed

Tóth, Beáta; Garabuczi, Eva; Sarang, Zsolt; Vereb, György; Vámosi, György; Aeschlimann, Daniel; Blaskó, Bernadett; Bécsi, Bálint; Erdõdi, Ferenc; Lacy-Hulbert, Adam; Zhang, Ailiang; Falasca, Laura; Birge, Raymond B; Balajthy, Zoltán; Melino, Gerry; Fésüs, László; Szondy, Zsuzsa

2009-02-15

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3) to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3) cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance.

PubMed

Tamoi, Masahiro; Kurotaki, Hideki; Fukamizo, Tamo

2007-07-01

In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

PubMed

Belkin, A M; Zhidkova, N I; Balzac, F; Altruda, F; Tomatis, D; Maier, A; Tarone, G; Koteliansky, V E; Burridge, K

1996-01-01

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

Inhibition of Transforming Growth Factor-Beta1 SignalingAttenuates Ataxia Telangiectasia Mutated Activity in Response toGenotoxic Stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirshner, Julia; Jobling, Michael F.; Pajares, Maria Jose

2006-01-01

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor {beta} (TGF{beta})-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF{beta} inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf{beta}I null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF{beta} type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced H2AX radiation-induced foci; and increased radiosensitivity compared with TGF{beta} competent cells.more » We determined that loss of TGF{beta} signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF{beta} restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf{beta}I, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF{beta} may be used to advantage in cancer therapy.« less

Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function.

PubMed

Rosenberg, I M; Göke, M; Kanai, M; Reinecker, H C; Podolsky, D K

1997-10-01

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

Granulocyte-macrophage and macrophage colony-stimulating factors differentially regulate alpha v integrin expression on cultured human macrophages.

PubMed

De Nichilo, M O; Burns, G F

1993-03-15

The colony-stimulating factors (CSFs) greatly influence mature macrophage function in vitro: macrophage (M)-CSF induces maturation of monocytes and enhances differentiated cell function; granulocyte-macrophage (GM)-CSF stimulates a variety of antimicrobial functions. In vivo M-CSF is thought to promote differentiation, and GM-CSF is thought to potentiate the inflammatory response. One mechanism by which these differential effects may be achieved is through the receptor-mediated interaction of macrophages with their extracellular matrix. Here we show that M-CSF induces specifically the expression of the alpha v beta 5 integrin receptor, whereas GM-CSF rapidly induces mRNA and surface expression of the alpha v beta 3 integrin. The M-CSF-treated cells acquire a flattened epitheloid phenotype, and on vitronectin the alpha v beta 5 is located in adhesion plaques. These cells do not bind collagen or laminin. In contrast, cells treated with GM-CSF adopt an elongated phenotype on a number of substrates, including collagen and laminin, and express alpha v beta 3 at the leading edge of cells on vitronectin. These results suggest that a primary means by which the CSFs exert their individual effects on mature cells may be through regulating integrin expression.

Biological functions of hCG and hCG-related molecules

PubMed Central

2010-01-01

Background hCG is a term referring to 4 independent molecules, each produced by separate cells and each having completely separate functions. These are hCG produced by villous syncytiotrophoblast cells, hyperglycosylated hCG produced by cytotrophoblast cells, free beta-subunit made by multiple primary non-trophoblastic malignancies, and pituitary hCG made by the gonadotrope cells of the anterior pituitary. Results and discussion hCG has numerous functions. hCG promotes progesterone production by corpus luteal cells; promotes angiogenesis in uterine vasculature; promoted the fusion of cytotrophoblast cell and differentiation to make syncytiotrophoblast cells; causes the blockage of any immune or macrophage action by mother on foreign invading placental cells; causes uterine growth parallel to fetal growth; suppresses any myometrial contractions during the course of pregnancy; causes growth and differentiation of the umbilical cord; signals the endometrium about forthcoming implantation; acts on receptor in mother's brain causing hyperemesis gravidarum, and seemingly promotes growth of fetal organs during pregnancy. Hyperglycosylated hCG functions to promote growth of cytotrophoblast cells and invasion by these cells, as occurs in implantation of pregnancy, and growth and invasion by choriocarcinoma cells. hCG free beta-subunit is produced by numerous non-trophoblastic malignancies of different primaries. The detection of free beta-subunit in these malignancies is generally considered a sign of poor prognosis. The free beta-subunit blocks apoptosis in cancer cells and promotes the growth and malignancy of the cancer. Pituitary hCG is a sulfated variant of hCG produced at low levels during the menstrual cycle. Pituitary hCG seems to mimic luteinizing hormone actions during the menstrual cycle. PMID:20735820

Toward beta cell replacement for diabetes

PubMed Central

Johannesson, Bjarki; Sui, Lina; Freytes, Donald O; Creusot, Remi J; Egli, Dieter

2015-01-01

The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes. PMID:25733347

Preoperative Beta Cell Function Is Predictive of Diabetes Remission After Bariatric Surgery.

PubMed

Souteiro, Pedro; Belo, Sandra; Neves, João Sérgio; Magalhães, Daniela; Silva, Rita Bettencourt; Oliveira, Sofia Castro; Costa, Maria Manuel; Saavedra, Ana; Oliveira, Joana; Cunha, Filipe; Lau, Eva; Esteves, César; Freitas, Paula; Varela, Ana; Queirós, Joana; Carvalho, Davide

2017-02-01

Bariatric surgery can improve glucose metabolism in obese patients with diabetes, but the factors that can predict diabetes remission are still under discussion. The present study aims to examine the impact of preoperative beta cell function on diabetes remission following surgery. We investigated a cohort of 363 obese diabetic patients who underwent bariatric surgery. The impact of several preoperative beta cell function indexes on diabetes remission was explored through bivariate logistic regression models. Postoperative diabetes remission was achieved in 39.9 % of patients. Younger patients (p < 0.001) and those with lower HbA1c (p = 0.001) at the baseline evaluation had higher odds of diabetes remission. Use of oral anti-diabetics and insulin therapy did not reach statistical significance when they were adjusted for age and HbA1c. Among the evaluated indexes of beta cell function, higher values of insulinogenix index, Stumvoll first- and second-phase indexes, fasting C-peptide, C-peptide area under the curve (AUC), C-peptide/glucose AUC, ISR (insulin secretion rate) AUC, and ISR/glucose AUC predicted diabetes remission even after adjustment for age and HbA1c. Among them, C-peptide AUC had the higher discriminative power (AUC 0.76; p < 0.001). Patients' age and preoperative HbA1c can forecast diabetes remission following surgery. Unlike other studies, our group found that the use of oral anti-diabetics and insulin therapy were not independent predictors of postoperative diabetes status. Preoperative beta cell function, mainly C-peptide AUC, is useful in predicting diabetes remission, and it should be assessed in all obese diabetic patients before bariatric or metabolic surgery.

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

PubMed Central

De Groef, Sofie; Leuckx, Gunter; Van Gassen, Naomi; Staels, Willem; Cai, Ying; Yuchi, Yixing; Coppens, Violette; De Leu, Nico; Heremans, Yves; Baeyens, Luc; Van de Casteele, Mark; Heimberg, Harry

2015-01-01

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories. PMID:26273954

Sequential Coating of Insulin Secreting Beta Cells within Multilayers of Polysaccharide Nanogels.

PubMed

Bal, Tugba; Oran, Dilem Ceren; Sasaki, Yoshihiro; Akiyoshi, Kazunari; Kizilel, Seda

2018-05-01

Pancreatic islet transplantation has emerged as a promising treatment for type-1 diabetes (T1D); however, its clinical application is still limited by the life-long use of immunosuppressive drugs, insufficient number of islets to achieve normoglycemia, and large transplantation volume. This paper reports a unique approach for nanothin coating of insulin secreting beta cell aggregates. The coating is based on hydrophobic and covalent interactions between natural acrylate modified cholesterol bearing pullulan (CHPOA) nanogels and MIN6 beta cell aggregates. Beta cell aggregates are prepared as spheroids through hanging drop method, which is optimized with respect to hanging drop volume and initial number of beta cells. These aggregates, defined as pseudoislets, are coated with sequential layers of nanogels and are evaluated as viable and functional for insulin secretion. Coating experiments are carried out using physiologically compatible medium, where pseudoislets are not brought in contact with toxic prepolymer solutions used in existing approaches. This study offers new opportunities through coating of islets with advanced functional materials under completely physiological conditions for clinical translation of cell transplantation technology. The technique developed here will establish a new paradigm for creating tolerable grafts for other chronic diseases such as anemia, cancer, central nervous system (CNS) diseases. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heat shock protein 90{beta}: A novel mediator of vitamin D action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angelo, Giana; Mineral Bioavailability Laboratory, 711 Washington Street, Boston, MA 02111; Lamon-Fava, Stefania

2008-03-14

We investigated the role of Heat shock protein 90 (Hsp90) in vitamin D action in Caco-2 cells using geldanamycin (GA) to block Hsp90 function and RNA interference to reduce Hsp90{beta} expression. When cells were exposed to GA, vitamin D-mediated gene expression and transcriptional activity were inhibited by 69% and 54%, respectively. Gel shift analysis indicated that GA reduced vitamin D-mediated DNA binding activity of the vitamin D receptor (VDR). We tested the specific role of Hsp90{beta} by knocking down its expression with stably expressed short hairpin RNA. Vitamin D-induced gene expression and transcriptional activity were reduced by 90% and 80%,more » respectively, in Hsp90{beta}-deficient cells. Nuclear protein for VDR and RXR{alpha}, its heterodimer partner, were not reduced in Hsp90{beta}-deficient cells. These findings indicate that Hsp90{beta} is needed for optimal vitamin D responsiveness in the enterocyte and demonstrate a specific role for Hsp90{beta} in VDR signaling.« less

Expansion of natural (NK1+) T cells that express alpha beta T cell receptors in transporters associated with antigen presentation-1 null and thymus leukemia antigen positive mice

PubMed Central

1996-01-01

Thymic selection of natural killer-1+ natural T cells that express alpha beta T cell receptors requires a conserved beta 2-microglobulin- associated molecule, presumably CD1d, displayed by CD4+8+ thymocytes. Here we demonstrate that positive selection of natural T cells occurs independent of transporters associated with antigen presentation-1 (TAP- 1) function. Moreover, natural T cells in TAP-1o/o mice are numerically expanded. Several H-2 class Ib molecules function in a TAP-independent manner, suggesting that if expressed in TAP-1o/o thymocytes, they could play a role in natural T cell development. Of these class Ib molecules, H-2TL is expressed by TAP-1o/o thymocytes. Moreover, we find that thymi of TL+ mice congenic or transgenic for H-2T18 also have a numerically expanded natural T cell repertoire compared with TL- mice. This expansion, as in TAP-1o/o thymi, is evident in each of the limited T cell receptor V beta chains expressed by natural T cells, suggesting that TL and CD1d impact similar repertoires. Thus TL, in addition to CD1d, plays a role in natural T cell development. PMID:8879233

17-Beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells via activation of extracellular signal-regulated kinase through the G protein-coupled receptor 30.

PubMed

Kleuser, Burkhard; Malek, Daniela; Gust, Ronald; Pertz, Heinz H; Potteck, Henrik

2008-12-01

Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.

Log (TG)/HDL-C ratio as a predictor of decreased islet beta cell function in patients with type 2 diabetes: 6-year cohort study.

PubMed

Zhou, Meicen; Li, Zengyi; Min, Rui; Dong, Yaxiu; Sun, Qi; Li, Yuxiu

2015-09-01

The aim of the present study was to explore whether the triglyceride to high density lipoprotein cholesterol ratio [log (TG)/HDL-C] and peripheral blood leukocytes DNA telomere length could predict future islet beta cell function decreased in Chinese type 2 diabetes mellitus (T2DM) during a 6-year cohort. Sixty T2DM patients (without insulin treatment at baseline) were included in the 6-year cohort study. Peripheral blood leukocytes DNA telomere length, HbA1c, blood lipid profile, fatty fat acid, glucose, insulin and C peptide (3 h after a mixed meal) were determined. Delta C peptide area under curve (Delta CP AUC) was used to reflect change in beta cell secretion function (Delta CP AUC = baseline CP AUC - CP AUC after 6 years). Subjects were divided into slow decrease of beta cell function group (Delta CP AUCslow group) and fast decrease group (Delta CP AUCfast group) according to median of Delta CP AUC. Baseline demographic characteristics, clinical variables between two groups were compared. Correlations between baseline data and Delta CP AUC were analyzed. Baseline log (TG)/HDL-C was positively correlated with Delta CP AUC (r = 0.306, P = 0.027); log (TG)/HDL-C in Delta CP AUCfast group was higher than that in Delta CP AUCslow group (0.103 ± 0.033 vs 0.083 ± 0.030, P = 0.027). There was no significant difference in DNA telomere length between the two groups. Change in DNA telomere length over 6 years was not significantly correlated with baseline blood lipid. In Chinese T2DM patients, high baseline log (TG)/HDL-C ratio predicts fast progression of islet beta cell dysfunction. It may be a simple index to predict progression speed of islet beta cell dysfunction. © 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

King, K.; Caron, M.G.; Lefkowitz, R.J.

1990-10-05

To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR andmore » a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.« less

Predominant Improvement of Alpha Cell Function after Steroid Therapy in a Patient with Autoimmune Pancreatitis: Case Report.

PubMed

Takeshima, Ken; Ariyasu, Hiroyuki; Iwakura, Hiroshi; Kawai, Shintaro; Uraki, Shinsuke; Inaba, Hidefumi; Furuta, Machi; Warigaya, Kenji; Murata, Shin-Ichi; Akamizu, Takashi

2018-06-01

Autoimmune pancreatitis (AIP) is a subset of inflammatory pancreatic disease, responsive to corticosteroid therapy. It is prone to being affected by diabetes mellitus, but the effectiveness of steroid therapy on pancreatic endocrine function is still controversial. We present a case of AIP, focusing on pancreatic endocrine function after steroid therapy. The patient was referred to our hospital with exacerbation of diabetic control and pancreatic swelling. By admission, the insulin secretory capacity was severely impaired. The patient was diagnosed with AIP and treated with prednisolone, resulting in marked improvement of the pancreatic swelling. Glycemic control worsened transiently after initiation of steroid therapy, but insulin requirements decreased along with tapering prednisolone dosage. Pancreatic cytology showed that the acinar structure had been destroyed, and the islets had disappeared. Insulin and glucagon immunostaining revealed slightly scattered alpha and beta cells within the fibrotic stroma. The patient notably showed improved pancreatic alpha cell function predominantly after steroid therapy, despite partial improvement of beta cell function. An imbalance between alpha and beta cell function may contribute to insufficient diabetic control in some patients with AIP. The pancreatic endocrine function test in combination with pancreatic cytology could be helpful when considering the treatment strategy for diabetic control in patients with AIP.

Unraveling the role of the ghrelin gene peptides in the endocrine pancreas.

PubMed

Granata, Riccarda; Baragli, Alessandra; Settanni, Fabio; Scarlatti, Francesca; Ghigo, Ezio

2010-09-01

The ghrelin gene peptides include acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob). AG, mainly produced by the stomach, exerts its central and peripheral effects through the GH secretagogue receptor type 1a (GHS-R1a). UAG, although devoid of GHS-R1a-binding affinity, is an active peptide, sharing with AG many effects through an unknown receptor. Ob was discovered as the G-protein-coupled receptor 39 (GPR39) ligand; however, its physiological actions remain unclear. The endocrine pancreas is necessary for glucose homeostasis maintenance. AG, UAG, and Ob are expressed in both human and rodent pancreatic islets from fetal to adult life, and the pancreas is the major source of ghrelin in the perinatal period. GHS-R1a and GPR39 expression has been shown in beta-cells and islets, as well as specific binding sites for AG, UAG, and Ob. Ghrelin colocalizes with glucagon in alpha-islet cells, but is also uniquely expressed in epsilon-islet cells, suggesting a role in islet function and development. Indeed, AG, UAG, and Ob regulate insulin secretion in beta-cells and isolated islets, promote beta-cell proliferation and survival, inhibit beta-cell and human islet cell apoptosis, and modulate the expression of genes that are essential in pancreatic islet cell biology. They even induce beta-cell regeneration and prevent diabetes in streptozotocin-treated neonatal rats. The receptor(s) mediating their effects are not fully characterized, and a signaling crosstalk has been suggested. The present review summarizes the newest findings on AG, UAG, and Ob expression in pancreatic islets and the role of these peptides on beta-cell development, survival, and function.

Drp1 guarding of the mitochondrial network is important for glucose-stimulated insulin secretion in pancreatic beta cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reinhardt, Florian; Schultz, Julia; Waterstradt, Rica

Mitochondria form a tubular network in mammalian cells, and the mitochondrial life cycle is determined by fission, fusion and autophagy. Dynamin-related protein 1 (Drp1) has a pivotal role in these processes because it alone is able to constrict mitochondria. However, the regulation and function of Drp1 have been shown to vary between cell types. Mitochondrial morphology affects mitochondrial metabolism and function. In pancreatic beta cells mitochondrial metabolism is a key component of the glucose-induced cascade of insulin secretion. The goal of the present study was to investigate the action of Drp1 in pancreatic beta cells. For this purpose Drp1 wasmore » down-regulated by means of shDrp1 in insulin-secreting INS1 cells and mouse pancreatic islets. In INS1 cells reduced Drp1 expression resulted in diminished expression of proteins regulating mitochondrial fusion, namely mitofusin 1 and 2, and optic atrophy protein 1. Diminished mitochondrial dynamics can therefore be assumed. After down-regulation of Drp1 in INS1 cells and spread mouse islets the initially homogenous mitochondrial network characterised by a moderate level of interconnections shifted towards high heterogeneity with elongated, clustered and looped mitochondria. These morphological changes were found to correlate directly with functional alterations. Mitochondrial membrane potential and ATP generation were significantly reduced in INS1 cells after Drp1down-regulation. Finally, a significant loss of glucose-stimulated insulin secretion was demonstrated in INS1 cells and mouse pancreatic islets. In conclusion, Drp1 expression is important in pancreatic beta cells to maintain the regulation of insulin secretion. -- Highlights: •Down-regulation of Drp1 in INS1 cells reduces mitochondrial fusion protein expression. •Mitochondrial membrane potential in INS1 cells is diminished after Drp1 down-regulation. •Mitochondria become elongated after down-regulation of Drp1 in beta cells. •Down-regulation of Drp1 in islets evokes loss of glucose-stimulated insulin secretion.« less

Central infusion of leptin improves insulin resistance and suppresses beta-cell function, but not beta-cell mass, primarily through the sympathetic nervous system in a type 2 diabetic rat model.

PubMed

Park, Sunmin; Ahn, Il Sung; Kim, Da Sol

2010-06-05

We investigated whether hypothalamic leptin alters beta-cell function and mass directly via the sympathetic nervous system (SNS) or indirectly as the result of altered insulin resistant states. The 90% pancreatectomized male Sprague Dawley rats had sympathectomy into the pancreas by applying phenol into the descending aorta (SNSX) or its sham operation (Sham). Each group was divided into two sections, receiving either leptin at 300ng/kgbw/h or artificial cerebrospinal fluid (aCSF) via intracerebroventricular (ICV) infusion for 3h as a short-term study. After finishing the infusion study, ICV leptin (3mug/kg bw/day) or ICV aCSF (control) was infused in rats fed 30 energy % fat diets by osmotic pump for 4weeks. At the end of the long-term study, glucose-stimulated insulin secretion and islet morphometry were analyzed. Acute ICV leptin administration in Sham rats, but not in SNSX rats, suppressed the first- and second-phase insulin secretion at hyperglycemic clamp by about 48% compared to the control. Regardless of SNSX, the 4-week administration of ICV leptin improved glucose tolerance during oral glucose tolerance tests and insulin sensitivity at hyperglycemic clamp, compared to the control, while it suppressed second-phase insulin secretion in Sham rats but not in SNSX rats. However, the pancreatic beta-cell area and mass were not affected by leptin and SNSX, though ICV leptin decreased individual beta-cell size and concomitantly increased beta-cell apoptosis in Sham rats. Leptin directly decreases insulin secretion capacity mainly through the activation of SNS without modulating pancreatic beta-cell mass.

Inhibition of beta-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules.

PubMed

Cheng, Guangmao; Qiao, Fei; Gallien, Thomas N; Kuppuswamy, Dhandapani; Cooper, George

2005-03-01

Decreased beta-adrenergic receptor (beta-AR) number occurs both in animal models of cardiac hypertrophy and failure and in patients. beta-AR recycling is an important mechanism for the beta-AR resensitization that maintains a normal complement of cell surface beta-ARs. We have shown that 1) in severe pressure overload cardiac hypertrophy, there is extensive microtubule-associated protein 4 (MAP4) decoration of a dense microtubule network; and 2) MAP4 microtubule decoration inhibits muscarinic acetylcholine receptor recycling in neuroblastoma cells. We asked here whether MAP4 microtubule decoration inhibits beta-AR recycling in adult cardiocytes. [(3)H]CGP-12177 was used as a beta-AR ligand, and feline cardiocytes were isolated and infected with adenovirus containing MAP4 (AdMAP4) or beta-galactosidase (Adbeta-gal) cDNA. MAP4 decorated the microtubules extensively only in AdMAP4 cardiocytes. beta-AR agonist exposure reduced cell surface beta-AR number comparably in AdMAP4 and Adbeta-gal cardiocytes; however, after agonist withdrawal, the cell surface beta-AR number recovered to 78.4 +/- 2.9% of the pretreatment value in Adbeta-gal cardiocytes but only to 56.8 +/- 1.4% in AdMAP4 cardiocytes (P < 0.01). This result was confirmed in cardiocytes isolated from transgenic mice having cardiac-restricted MAP4 overexpression. In functional terms of cAMP generation, beta-AR agonist responsiveness of AdMAP4 cells was 47% less than that of Adbeta-gal cells. We conclude that MAP4 microtubule decoration interferes with beta-AR recycling and that this may be one mechanism for beta-AR downregulation in heart failure.

Application of microalgal fucoxanthin for the reduction of colon cancer risk: inhibitory activity of fucoxanthin against beta-glucuronidase and DLD-1 cancer cells.

PubMed

Kawee-Ai, Arthitaya; Kim, Sang Moo

2014-07-01

Intestinal bacterial beta-glucuronidases are capable of retoxifying compounds that have been detoxified by liver glucuronidation and are also known to accelerate colon cancer invasion and metastasis. In this study, fucoxanthin extracted from the microalga Phaeodactylum tricornutum was investigated for its inhibitory activity against Escherichia coli beta-glucuronidase and DLD-1 cancer cells. Fucoxanthin inhibited beta-glucuronidase in a concentration-dependent manner with an IC50 value of 2.32 mM and a mixed inhibition type. Fucoxanthin had more potent inhibitory activity on beta-glucuronidase at 37 degrees C and in alkaline conditions. Fucoxanthin also inhibited the beta-glucuronidase activity of DLD-1 cancer cells at a concentration of 20-50 microM. The presence of beta-glucuronidase and substrate in the medium decreased the inhibitory activity of fucoxanthin against DLD-1 cancer cells. Therefore, microalgal fucoxanthin might prevent colon cancer because of its strong beta-glucuronidase inhibitory activity and could be utilized as a novel functional ingredient of food and pharmaceutical supplements.

Integrin-mediated transforming growth factor-beta activation regulates homeostasis of the pulmonary epithelial-mesenchymal trophic unit.

PubMed

Araya, Jun; Cambier, Stephanie; Morris, Alanna; Finkbeiner, Walter; Nishimura, Stephen L

2006-08-01

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL.

PubMed

Plaisance, Valérie; Brajkovic, Saška; Tenenbaum, Mathie; Favre, Dimitri; Ezanno, Hélène; Bonnefond, Amélie; Bonner, Caroline; Gmyr, Valéry; Kerr-Conte, Julie; Gauthier, Benoit R; Widmann, Christian; Waeber, Gérard; Pattou, François; Froguel, Philippe; Abderrahmani, Amar

2016-01-01

Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment.

Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis.

PubMed

Fantini, M C; Becker, C; Tubbe, I; Nikolaev, A; Lehr, H A; Galle, P; Neurath, M F

2006-05-01

The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25- T cells by treatment with transforming growth factor beta (TGF-beta). Here, we analysed the in vivo function of TGF-beta induced regulatory T (Ti-Treg) cells in experimental colitis. Ti-Treg cells were generated in cell culture in the presence or absence of TGF-beta and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. Ti-Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti-Treg cells showed that exogenous interleukin 2 (IL-2) was crucial for survival and expansion of these cells. Our data suggest that regulatory Ti-Treg cells expand by TGF-beta and exogenous IL-2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti-Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation.

CLIC4 Moves Into Nucleus to Stabilize Anti-Growth Signal | Center for Cancer Research

Cancer.gov

In cancer, the delicate balance of signaling pathways that control cell growth and function is disrupted. One signaling pathway commonly altered in cancer is the TGF-beta pathway. TGF-beta significantly inhibits growth of normal cells, particularly epithelial cells. Many cancer cells have developed ways to bypass one or more steps of this pathway in order to achieve uncontrolled growth.

CLIC4 Moves Into Nucleus to Stabilize Anti-Growth Signal | Center for Cancer Research

Cancer.gov

In cancer, the delicate balance of signaling pathways that control cell growth and function is disrupted. One signaling pathway commonly altered in cancer is the TGF-beta pathway. TGF-beta significantly inhibits growth of normal cells, particularly epithelial cells. Many cancer cells have developed ways to bypass one or more steps of this pathway in order to achieve

The influence of major dietary fatty acids on insulin secretion and action.

PubMed

López, Sergio; Bermúdez, Beatriz; Abia, Rocío; Muriana, Francisco J G

2010-02-01

To briefly summarize recent advances towards understanding the influence of major dietary fatty acids on beta-cell function and evaluate their implications for insulin resistance. Studies in humans have shown that beta-cell function and insulin sensitivity improve progressively in the postprandial period as the proportion of monounsaturated fatty acids (MUFAs) with respect to saturated fatty acids (SFAs) in dietary fats increases. However, cell-culture experiments have revealed a dichotomy in the ability of fatty acids to moderate hyperactivity of, and induce lipotoxicity in, beta-cells. There are also some novel findings regarding the ability of HDL to protect beta-cells against oxidized LDL-induced apoptosis in vitro and of reconstituted HDL to attenuate insulin resistance in vivo. These findings raise new questions regarding the contribution of dietary fatty acids to insulin secretion and action. These new findings point to a critical role for major dietary fatty acids in the etiology and pathogenesis of diabetes, which appears to be of particular relevance during postprandial periods and mainly depends on the fatty acid type. This underscores the importance of dietary fatty acids in standard diabetes management.

Grape seed procyanidin extract modulates proliferation and apoptosis of pancreatic beta-cells.

PubMed

Cedó, Lídia; Castell-Auví, Anna; Pallarès, Victor; Blay, Mayte; Ardévol, Anna; Arola, Lluís; Pinent, Montserrat

2013-05-01

Grape seed procyanidin extract (GSPE) modulates glucose homeostasis and insulinemia in several animal models. Under pathological conditions, insulin levels are dependent on pancreatic beta-cell functionality, as well as on the beta-cell mass expansion or apoptosis in the pancreas. In this study, we analysed the effects of GSPE on modulating apoptosis and proliferation in beta-cells. We tested the effects of GSPE in the INS-1E pancreatic beta-cell line, either under basal or altered conditions with high glucose, insulin or palmitate levels. GSPE enhanced the pro-apoptotic effect of high glucose and showed clear antiproliferative effects under high glucose, insulin and palmitate conditions. These antiproliferative effects are likely due to high molecular weight compounds contained in the extract. GSPE also modulated pro- and anti-apoptotic markers in the pancreas of rats fed a cafeteria diet, with the effect depending on the dose of GSPE and duration of treatment. Thus, GSPE is able to modulate apoptosis and proliferation of beta-cells under altered, but not basal, conditions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Targeted drug delivery and enhanced intracellular release using functionalized liposomes

NASA Astrophysics Data System (ADS)

Garg, Ashish

The ability to target cancer cells using an appropriate drug delivery system can significantly reduce the associated side effects from cancer therapies and can help in improving the overall quality of life, post cancer survival. Integrin alpha5beta1 is expressed on several types of cancer cells, including colon cancer and plays an important role in tumor growth and metastasis. Thus, the ability to target the integrin alpha 5beta1 using an appropriate drug delivery nano-vector can significantly help in inhibiting tumor growth and reducing tumor metastasis. The work in this thesis focuses on designing and optimizing, functionalized stealth liposomes (liposomes covered with polyethylene glycol (PEG)) that specifically target the integrin alpha5beta1. The PEG provides a steric barrier allowing the liposomes to circulate in the blood for longer duration and the functionalizing moiety, PR_b peptide specifically recognizes and binds to integrin alpha5beta1 expressing cells. The work demonstrates that by optimizing the amount of PEG and PR_b on the liposomal interface, nano-vectors can be engineered that bind to CT26.WT colon cancer cells in a specific manner and internalize through alpha 5beta1-mediated endocytosis. To further improve the efficacy of the system, PR_b functionalized pH-sensitive stealth liposomes that exhibit triggered release under mild acidic conditions present in endocytotic vesicles were designed. The study showed that PR_b functionalized pH-sensitive stealth liposomes, undergo destabilization under mildly acidic conditions and incorporation of the PR_b peptide does not significantly affect the pH-sensitivity of the liposomes. PR_b functionalized pH-sensitive stealth liposomes bind to CT26.WT colon carcinoma cells that express integrin alpha5beta 1, undergo cellular internalization, and release their load intracellularly in a short period of time as compared to other formulations. PR_b-targeted pH-sensitive stealth liposomes encapsulating 5-fluorouracil (5-FU) show significantly higher cytotoxicity than the PR_b-targeted inert stealth liposomes and the non-targeted stealth liposomes (both pH-sensitive and inert). The studies demonstrated that optimized PR_b functionalized pH sensitive liposomes have the potential to deliver a payload, such as chemotherapeutic agents, directly to colon cancer cells in an efficient and specific manner.

An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation

NASA Technical Reports Server (NTRS)

Wikramanayake, Athula H.; Hong, Melanie; Lee, Patricia N.; Pang, Kevin; Byrum, Christine A.; Bince, Joanna M.; Xu, Ronghui; Martindale, Mark Q.

2003-01-01

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

[Survey of recent clinical trials of the prevention and immunointervention of type 1 diabetes mellitus].

PubMed

Boerschmann, H; Walter, M; Achenbach, P; Ziegler, A-G

2010-02-01

Immunomodulatory strategies in the management of type 1 diabetes mellitus (T1DM) have as their primary target the prevention of initiating islet autoimmunity (primary-), the secondary one is the progression to diabetes (secondary-) in non-diabetic persons at risk, and the decline of beta-cell function in new-onset patients (tertiary-prevention). This article reviews four recent immunointervention trials in patients with T1DM. (1) The Pre-POINT study is a primary prevention trial that will test whether vaccination with oral or nasal insulin can prevent the progression of islet autoimmunity and of T1DM in autoantibody-negative children who are genetically at high diabetes risk. (2) The Cord Blood study is a tertiary immunointervention trial that will test whether administration of autologous umbilical cord blood to children with T1DM can lead to regeneration of pancreatic islet insulin-producing beta-cells and improved blood glucose control. (3) The GAD Vaccination study will test whether vaccination with alum-formulated rhGAD65 (recombinant human glutamic acid decarboxylate) can preserve beta-cell function in 320 children with newly diagnosed T1DM, as has been suggested in a recent phase II study. (4) The AIDA study will test the beta-cell protective effect of interleukin-1-receptor antagonist Anakinra in 80 patients with T1DM, which has recently been shown to improve beta-cell function in patients with type 2 diabetes. Copyright Georg Thieme Verlag KG Stuttgart . New York.

The resident macrophages in murine pancreatic islets are constantly probing their local environment, capturing beta cell granules and blood particles.

PubMed

Zinselmeyer, Bernd H; Vomund, Anthony N; Saunders, Brian T; Johnson, Michael W; Carrero, Javier A; Unanue, Emil R

2018-06-01

We studied here the interactions between the resident macrophages of pancreatic islets with beta cells and the blood vasculature. We also examined the immunological consequences of such interactions. Islets were isolated from C57BL/6 mice expressing CX3C motif chemokine receptor 1-green fluorescent protein (CX3CR-GFP) and examined live by two-photon microscopy. Islets were also examined by electron microscopy to study the relationship of the intra-islet macrophages with the beta cells. In NOD.Rag1 -/- mice and young (non-diabetic) male mice, the acquisition of beta cell granules was tested functionally by probing with CD4 + T cells directed against insulin epitopes. Two-photon microscopy showed that the islet resident macrophages were in close contact with blood vessels and had extensive filopodial activity. Some filopodia had direct access to the vessel lumen and captured microparticles. Addition of glucose at high concentration reduced the degree of filopodia sampling of islets. This finding applied to in vivo injection of glucose or to in vitro cultures. Ultrastructural examination showed the close contacts of macrophages with beta cells. Such macrophages contained intact dense core granules. Functional studies in NOD mice indicated that the macrophages presented insulin peptides to insulin-reactive T cells. Presentation was increased after glucose challenge either ex vivo or after an in vivo pulse. In agreement with the morphological findings, presentation was not affected by insulin receptor blockade. Islet resident macrophages are highly active, sampling large areas of the islets and blood contents and capturing beta cell granules. After such interactions, macrophages present immunogenic insulin to specific autoreactive T cells.

A critical role for transcription factor Smad4 in T cell function that is independent of transforming growth factor β receptor signaling.

PubMed

Gu, Ai-Di; Zhang, Song; Wang, Yunqi; Xiong, Hui; Curtis, Thomas A; Wan, Yisong Y

2015-01-20

Transforming growth factor-beta (TGF-β) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-β signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-βR) deletion and compromised T-cell-mediated tumor rejection. Although Smad4 was dispensable for T cell generation, homeostasis, and effector function, it was essential for T cell proliferation after activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-βR-independent role for Smad4 in promoting T cell function, autoimmunity, and anti-tumor immunity. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulated binding of PTP1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of beta-catenin

PubMed Central

1996-01-01

Cadherins are a family of cell-cell adhesion molecules which play a central role in controlling morphogenetic movements during development. Cadherin function is regulated by its association with the actin containing cytoskeleton, an association mediated by a complex of cytoplasmic proteins, the catenins: alpha, beta, and gamma. Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Consistent with this, we find that only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. Moreover, we demonstrate that a PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. Moreover, both direct binding experiments performed with phosphorylated and dephosphorylated molecules, and treatment of cells with tyrosine kinase inhibitors indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin, the association of N- cadherin with the actin containing cytoskeleton is lost and N-cadherin- mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, loss of the association of N-cadherin with the actin- containing cytoskeleton, and prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. We previously showed that the binding of two ligands to the cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase), the monoclonal antibody 1B11 and a proteoglycan with a 250-kD core protein, results in the accumulation of phosphorylated tyrosine residues on beta-catenin, uncoupling of N-cadherin from its association with the actin containing cytoskeleton, and loss of N- cadherin function. We now report that binding of these ligands to the GalNAcPTase results in the absence of the PTP1B-like phosphatase from its association with N-cadherin as well as the loss of the tyrosine kinase and tyrosine phosphatase activities that otherwise co- precipitate with N-cadherin. Control antibodies and proteoglycans have no such effect. This effect is similar to that observed with tyrosine kinase inhibitors, suggesting that the GalNAcPTase/proteoglycan interaction inhibits a tyrosine kinase, thereby preventing the phosphorylation of the PTP1B-like phosphatase, and its association with N-cadherin. Taken together these data indicate that a PTP1B-like tyrosine phosphatase can regulate N-cadherin function through its ability to dephosphorylate beta-catenin and that the association of the phosphatase with N-cadherin is regulated via the interaction of the GalNAcPTase with its proteoglycan ligand. In this manner the GalNAcPTase-proteoglycan interaction may play a major role in morphogenetic cell and tissue interactions during development. PMID:8707857

Expression and function of glycogen synthase kinase-3 in human hair follicles.

PubMed

Yamauchi, Koichi; Kurosaka, Akira

2010-05-01

Beta-catenin is involved in the hair follicle morphogenesis and stem cell differentiation, and inhibition of glycogen synthase kinase-3 (GSK-3) increases beta-catenin concentration in the cytoplasm. To examine the effects of GSK-3 inhibition on the hair follicle epithelium, we first examined the expression of GSK-3 in plucked human hair follicles by RT-PCR and found GSK-3 expression in hair follicles. Western blotting with a GSK-3beta-specific antibody, Y174, also demonstrated GSK-3beta expression in the follicles. Moreover, GSK-3beta immunostaining with Y174 showed that GSK-3beta colocalized with hair follicle bulge markers. Contrary to GSK-3beta, GSK-3 alpha was widely expressed throughout the follicles when immunostained with a specific antibody, EP793Y. We then investigated the influence of GSK-3 inhibition. A GSK-3 inhibitor, BIO, promoted the growth of human outer root sheath cells, which could be cultured for up to four passages. The BIO-treated cells exhibited smaller and more undifferentiated morphology than control cells. Moreover, in organ culture of plucked human hair, outer root sheath cells in the middle of a hair follicle proliferated when cultured with BIO. These results indicate that GSK-3beta is expressed in hair bulge stem cells and BIO promotes the growth of ORS cells, possibly by regulating the GSK-3 signaling pathway.

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin

PubMed Central

Wakae-Takada, N.; Xuan, S.; Watanabe, K.; Meda, P.; Leibel, R. L.

2014-01-01

Aims/hypothesis In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear β-catenin and D-cyclins. Methods We examined E-cadherin, nuclear β-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using β-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in β-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] βKO). Results In vitro, pseudo-islets of β-TC6 cells displayed increased E-cadherin but decreased nuclear β-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad βKO mice showed increased levels of D-cyclins and nuclear β-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. Conclusions/interpretation The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function. PMID:23354125

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin.

PubMed

Wakae-Takada, N; Xuan, S; Watanabe, K; Meda, P; Leibel, R L

2013-04-01

In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear β-catenin and D-cyclins. We examined E-cadherin, nuclear β-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using β-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in β-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] βKO). In vitro, pseudo-islets of β-TC6 cells displayed increased E-cadherin but decreased nuclear β-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad βKO mice showed increased levels of D-cyclins and nuclear β-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function.

Protective effects of St. John's wort extract and its component hyperforin against cytokine-induced cytotoxicity in a pancreatic beta-cell line.

PubMed

Menegazzi, Marta; Novelli, Michela; Beffy, Pascale; D'Aleo, Valentina; Tedeschi, Elisa; Lupi, Roberto; Zoratti, Elisa; Marchetti, Piero; Suzuki, Hisanori; Masiello, Pellegrino

2008-01-01

In both type 1 and type 2 diabetes, increased production of cytokines on autoimmune or metabolic basis is supposed to trigger an inflammatory process leading to dysfunction and death of pancreatic beta-cells. Therefore, anti-inflammatory pharmacological approaches aimed at blocking cytokine signalling pathways and consequent cytotoxicity in beta-cells are highly advisable. Based on previous evidence of cytokine antagonistic effects in other cell types, we explored the protective action of Hypericum perforatum (St-John's-wort) extract and its component hyperforin against cytokine-induced functional impairment and apoptosis in the INS-1E beta-cell line, searching for the underlying mechanisms. The results showed that either St-John's-wort extract or hyperforin (at 1-3 microM) prevented cytokine-induced impairment in glucose-stimulated insulin secretion and protected cells against apoptosis in a dose-dependent fashion. Inducible-NO-synthase expression was also potently hindered by the vegetal compounds. Interestingly, cytokine-induced activations of the signal-transducer-and-activator-of-transcription-1 (STAT-1) and the nuclear-factor-kappaB (NF-kappaB) were both down-regulated by SJW extract or HPF (range 0.5-5 microM) when evaluated by electrophoretic-mobility-shift-assay. Other transcription factors (CBF-1, SP-1) were unaffected. Components of SJW extract other than HPF were much less effective in down-regulating cytokine signalling. Significantly, inhibition of cytokine-elicited STAT-1 and NF-kappaB activation was confirmed in isolated rat and human islets incubated in the presence of these vegetal compounds. In conclusion, St-John's-wort extract and hyperforin are non-peptidyl compounds which, at low concentrations, target key mechanisms of cytokine-induced beta-cell injury, thereby improving beta-cell function and survival. Thus, they are potentially valuable for the prevention or limitation of beta-cell loss in diabetes.

Effects of steroid hormones on nuclear membrane and membrane-bound heterochromatin from breast cancer cells evaluated by fractal morphometry.

PubMed

Losa, G A; Graber, R; Baumann, G; Nonnenmacher, T F

1999-10-01

To evaluate the effect of steroid hormones on the ultrastructure of nuclear heterochromatin and perinuclear membranes in human MCF-7 breast cancer cells. MCF-7 cells were cultured briefly (five minutes) in the presence of 10(-9) M estrogen 17 beta-estradiol, a stimulator of cell proliferation and/or 10(-9) M glucocorticoid dexamethasone. Changes in the morphologic complexity of nuclear membrane-bound heterochromatin (NMBHC) and nuclear membranes (ENM) were assessed by means of the fractal capacity dimension, D, a noneuclidean geometric descriptor of complex, irregular bodies. 17 beta-estradiol (10(-9) M) enhanced the ultrastructural irregularity of NMBHC, as documented by the increased value of D, whereas dexamethasone (10(-9) M) reduced it when compared to NMBHC from untreated MCF-7 control cells. In contrast, neither steroid modified ENM ultrastructure. Changes in the nuclear heterochromatin complexity induced by estrogen 17 beta-estradiol occurred concomitantly with functional changes at the cell periphery, such as activation of the phospholipase C, a cell membrane-associated enzyme involved in signal transduction. Dexamethasone reduced the ultrastructural complexity of NMBHC without affecting functional processes. Fractal morphometry proved its usefulness in quantifying early ultrastructural changes in nuclear components induced in MCF-7 cells by steroid hormones, 17 beta-estradiol and dexamethasone.

E-cadherin interactions regulate beta-cell proliferation in islet-like structures.

PubMed

Carvell, Melanie J; Marsh, Phil J; Persaud, Shanta J; Jones, Peter M

2007-01-01

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca(2+)-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 beta-cell line that stably over- (S) and under-express (alphaS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not alphaS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of beta-cell proliferation. Copyright (c) 2007 S. Karger AG, Basel.

BH3-Only protein bmf is required for the maintenance of glucose homeostasis in an in vivo model of HNF1α-MODY diabetes.

PubMed

Pfeiffer, S; Halang, L; Düssmann, H; Byrne, M M; Prehn, Jhm

2015-01-01

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1α (HNF-1α) gene can lead to diminished amounts of functional HNF-1α, resulting in the onset of a particularly severe form of maturity-onset diabetes of the young (MODY). We have previously shown that induction of a dominant-negative mutant of HNF-1α (DNHNF-1α) results in the activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), preceding the onset of apoptosis and the induction of pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor) as a mediator of DNHNF-1α-induced apoptosis. Through the knockout of bmf in a transgenic mouse model with DNHNF-1α suppression of HNF-1α function in pancreatic beta-cells, this study aimed to examine the effect of loss-of-function of this BH3-only protein on the disease pathology and progression, and further elucidate the role of Bmf in mediating DNHNF-1α-induced beta-cell loss. Morphological analysis revealed an attenuation in beta-cell loss in bmf-deficient diabetic male mice and preserved insulin content. Surprisingly, bmf deficiency was found to exacerbate hyperglycemia in both diabetic male and hyperglycemic female mice, and ultimately resulted in a decreased glucose-stimulated insulin response, implicating a role for Bmf in glucose homeostasis regulation independent of an effect on beta-cell loss. Collectively, our data demonstrate that Bmf contributes to the decline in beta-cells in a mouse model of HNF1A-MODY but is also required for the maintenance of glucose homeostasis in vivo.

BH3-Only protein bmf is required for the maintenance of glucose homeostasis in an in vivo model of HNF1α-MODY diabetes

PubMed Central

Pfeiffer, S; Halang, L; Düssmann, H; Byrne, MM; Prehn, JHM

2015-01-01

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1α (HNF-1α) gene can lead to diminished amounts of functional HNF-1α, resulting in the onset of a particularly severe form of maturity-onset diabetes of the young (MODY). We have previously shown that induction of a dominant-negative mutant of HNF-1α (DNHNF-1α) results in the activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), preceding the onset of apoptosis and the induction of pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor) as a mediator of DNHNF-1α-induced apoptosis. Through the knockout of bmf in a transgenic mouse model with DNHNF-1α suppression of HNF-1α function in pancreatic beta-cells, this study aimed to examine the effect of loss-of-function of this BH3-only protein on the disease pathology and progression, and further elucidate the role of Bmf in mediating DNHNF-1α-induced beta-cell loss. Morphological analysis revealed an attenuation in beta-cell loss in bmf-deficient diabetic male mice and preserved insulin content. Surprisingly, bmf deficiency was found to exacerbate hyperglycemia in both diabetic male and hyperglycemic female mice, and ultimately resulted in a decreased glucose-stimulated insulin response, implicating a role for Bmf in glucose homeostasis regulation independent of an effect on beta-cell loss. Collectively, our data demonstrate that Bmf contributes to the decline in beta-cells in a mouse model of HNF1A-MODY but is also required for the maintenance of glucose homeostasis in vivo. PMID:27551471

Smad ubiquitination regulatory factor-2 in the fibrotic kidney: regulation, target specificity, and functional implication.

PubMed

Tan, Ruoyun; He, Weichun; Lin, Xia; Kiss, Lawrence P; Liu, Youhua

2008-05-01

Smad ubiquitination regulatory factor-2 (Smurf2) is an E3 ubiqutin ligase that plays a pivotal role in regulating TGF-beta signaling via selectively targeting key components of the Smad pathway for degradation. In this study, we have investigated the regulation of Smurf2 expression, its target specificity, and the functional implication of its induction in the fibrotic kidney. Immunohistochemical staining revealed that Smurf2 was upregulated specifically in renal tubules of kidney biopsies from patients with various nephropathies. In vitro, Smurf2 mRNA and protein were induced in human proximal tubular epithelial cells (HKC-8) upon TGF-beta1 stimulation. Ectopic expression of Smurf2 was sufficient to reduce the steady-state levels of Smad2, but not Smad1, Smad3, Smad4, and Smad7, in HKC-8 cells. Interestingly, Smurf2 was also able to downregulate the Smad transcriptional corepressors Ski, SnoN, and TG-interacting factor. Inhibition of the proteasomal pathway prevented Smurf2-mediated downregulation of Smad2 and Smad corepressors. Functionally, overexpression of Smurf2 enhanced the transcription of the TGF-beta-responsive promoter and augmented TGF-beta1-mediated E-cadherin suppression, as well as fibronectin and type I collagen induction in HKC-8 cells. These results indicate that Smurf2 specifically targets both positive and negative Smad regulators for destruction in tubular epithelial cells, thereby providing a complex fine-tuning of TGF-beta signaling. It appears that dysregulation of Smurf2 could contribute to an aberrant TGF-beta/Smad signaling in the pathogenesis of kidney fibrosis.

Effect of gel re-organization and tensional forces on alpha2beta1 integrin levels in dermal fibroblasts.

PubMed

Jenkins, G; Redwood, K L; Meadows, L; Green, M R

1999-07-01

Mechanical forces are known to play an important role in regulating cell function in a wide range of biological systems. This is of particular relevance to dermal fibroblast function, given that the skin is known to be held under an intrinsic natural tension. To understand more about the generation of force by dermal fibroblasts and their ability to respond to changes in it, we have studied the role of the beta1 integrin receptors expressed by dermal fibroblasts in their ability to generate tensional forces within a collagen type I matrix and the effect of altered tensional force on integrin expression by dermal fibroblasts. Using a purpose-built culture force monitor, function-blocking antibodies directed towards the beta1 receptors dramatically reduced the tensional forces generated by dermal fibroblasts in a 3D collagen I matrix. However, the specific involvement of alpha1 or alpha2 subunits could not be demonstrated. Analysis of cellular response demonstrated that cells isolated from contracting collagen gels expressed fourfold higher levels of alpha2 mRNA than cells isolated from fully restrained gels. The levels of beta1 messenger RNA were relatively unaffected by reductions in force. Cells exposed to single reductions in force, however, did not exhibit alterations in either alpha1 or beta1 mRNA levels. We propose, therefore that alpha2beta1 integrin receptor levels in dermal fibroblasts are not altered in response to single reductions of gel tension, but do change following a continual change in force and associated matrix re-organization

C/EBPβ Promotes STAT3 Expression and Affects Cell Apoptosis and Proliferation in Porcine Ovarian Granulosa Cells.

PubMed

Yuan, Xiaolong; Zhou, Xiaofeng; He, Yingting; Zhong, Yuyi; Zhang, Ailing; Zhang, Zhe; Zhang, Hao; Li, Jiaqi

2018-06-13

Previous studies suggest that signal transducer and activator of transcription 3 (STAT3) and CCAAT/enhancer binding protein beta (C/EBPβ) play an essential role in ovarian granulosa cells (GCs) for mammalian follicular development. Several C/EBPβ putative binding sites were previously predicted on the STAT3 promoter in mammals. However, the molecular regulation of C/EBPβ on STAT3 and their effects on cell proliferation and apoptosis remain virtually unexplored in GCs. Using porcine GCs as a model, the 5′-deletion, luciferase report assay, mutation, chromatin immunoprecipitation, Annexin-V/PI staining and EdU assays were applied to investigate the molecular mechanism for C/EBPβ regulating the expression of STAT3 and their effects on the cell proliferation and apoptosis ability. We found that over and interfering with the expression of C/EBPβ significantly increased and decreased the messenger RNA (mRNA) and protein levels of STAT3 , respectively. The dual luciferase reporter assay showed that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the mRNA and protein expressions of STAT3 . Both C/EBPβ and STAT3 were observed to inhibit cell apoptosis and promote cell proliferation. Furthermore, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3 . These results would be of great insight in further exploring the molecular mechanism of C/EBPβ and STAT3 on the function of GCs and the development of ovarian follicles in mammals.

Tumor suppression function of the Big-h3 gene in radiation carcinogenesis

NASA Astrophysics Data System (ADS)

Zhao, Y.; Piao, C.; Hei, T.

Interaction between cell and extracellular matrix (ECM) plays a crucial role in tumor invasiveness and metastasis. Using an immortalized human bronchial epithelial (BEP2D) cell model, we show here that expression of Big-h3 gene, a secreted adhesion molecule induced by transforming growth factor- beta (TGF-beta ), is markedly decreased in independently generated, high LET radiation-induced tumor cell lines (TL1-TL5) relative to parental BEP2D cells. Expression of this gene was restored to control level in fusion cell lines between the tumorigenic and parental BEP2D cells that were no longer tumorigenic in nude mice. Transfection of Big-h3 gene into tumor cells resulted in a significant reduction of tumor growth. While integrin receptor alpha 5/beta 1 was overexpressed in tumor cells, its expression was corrected to the level of control BEP2D cells after Big-h3 transfection. These data suggest that Big-h3 is involved in tumor progression by regulating integrin receptor alpha 5/beta 1. . WWee We further show that down regulation of Big-h3 results from loss of expression of TGFbeta1 in tumor cells. The findings provide strong evidence that the Big-h3 gene has tumor suppressor function in radiation induced tumorigenic human bronchial epithelial cells and suggest a potential target for interventional therapy.

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.

PubMed

De, Pradip; Carlson, Jennifer H; Wu, Hui; Marcus, Adam; Leyland-Jones, Brian; Dey, Nandini

2016-07-12

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronectin-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers

PubMed Central

De, Pradip; Carlson, Jennifer H.; Wu, Hui; Marcus, Adam; Leyland-Jones, Brian; Dey, Nandini

2016-01-01

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronection-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP. PMID:27281609

High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways

NASA Technical Reports Server (NTRS)

Wang, Minli; Hada, Megumi; Huff, Janice; Pluth, Janice M.; Anderson, Janniffer; ONeill, Peter; Cucinotta, Francis A.

2010-01-01

The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.

Alterations induced by E-cadherin and beta-catenin antibodies during the development of Bufo arenarum (Anura-Bufonidae).

PubMed

Izaguirre, M F; Adur, J F; Soler, A P; Casco, V H

2001-10-01

E(epithelial)-cadherin is a member of a calcium-dependent family of cell surface glycoproteins involved in cell-cell adhesion and morphogenesis. Catenins are a large family of proteins that connect the cadherins to the cytoskeleton. They are important for cadherin function and for transducing signals involved in specification of cell fate during embryogenesis. The best characterized catenins include alpha-, beta-, gamma-, and p120-catenin. Using specific antibodies, we studied the expression and distribution of E-cadherin, and alpha- and beta-catenin in developmental stages of Bufo arenarum toad. The three proteins were found co-localized in stages 19 to 41 of development. Surprisingly, E-cadherin was the only of these three proteins found earlier than stage 19. To test whether E-cadherin and beta-catenin have a functional role in Bufo arenarum embryogenesis, stage 17 whole embryos were incubated with anti-E-cadherin and beta-catenin antibodies. Both anti-E-cadherin and anti-beta-catenin antibodies induced severe morphological alterations. However, while alterations produced by the anti-beta-catenin antibody, showed some variability from the most severe (neural tube and notochord duplication) to a simple delay in development, the alterations with anti-E-cadherin were homogeneous. These observations suggest a critical role for E-cadherin and beta-catenin in the early embryonic development of the Bufo arenarum toad. Our results are consistent with the developmental role of these proteins in other species. One of the most surprising findings was the blockage with the anti-beta-catenin antibodies on later embryo stages, and we hypothesize that the partial axes duplication could be mediated by the notochord induction.

Biological effects of radiation, metabolic and replication kinetics alterations

NASA Technical Reports Server (NTRS)

Post, J.

1972-01-01

The biological effects of radiation upon normal and cancerous tissues were studied. A macromolecular precursor of DNA, 3ETdR, was incorporated into the cell nucleus during synthesis and provided intranuclear beta radiation. Tritium labeled cells were studied with autoradiographic methods; cell cycle kinetics were determined and cell functions modified by radiation dosage or by drugs were also evaluated. The long term program has included; (1) effects of radiation on cell replication and the correlation with incorporated dose levels, (2) radiation induced changes in cell function, viz., the response of beta irradiated spleen lymphocytes to antigenic stimulation by sheep red blood cells (SRBC), (3) kinetics of tumor and normal cell replication; and (4) megakaryocyte formation and modification by radiomimetic drugs.

Cross-talk between Smad and p38 MAPK signalling in transforming growth factor {beta} signal transduction in human glioblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dziembowska, Magdalena; Danilkiewicz, Malgorzata; Wesolowska, Aleksandra

2007-03-23

Transforming growth factor-beta (TGF-{beta}) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, and survival. Malignant tumour cells often do not respond to TGF-{beta} by growth inhibition, but retain responsiveness to cytokine in regulating extracellular matrix deposition, cell adhesion, and migration. We demonstrated that TGF-{beta}1 does not affect viability or proliferation of human glioblastoma T98G, but increases transcriptional responses exemplified by induction of MMP-9 expression. TGF-{beta} receptors were functional in T98G glioblastoma cells leading to SMAD3/SMAD4 nuclear translocation and activation of SMAD-dependent promoter. In parallel, a selective activation of p38 MAPK, and phosphorylation of its substrates: ATF2more » and c-Jun proteins were followed by a transient activation of AP-1 transcription factor. Surprisingly, an inhibition of p38 MAPK with a specific inhibitor, SB202190, abolished TGF-inducible activation of Smad-dependent promoter and decreased Smad2 phosphorylation. It suggests an unexpected interaction between Smad and p38 MAPK pathways in TGF-{beta}1-induced signalling.« less

Role of Jumonji c-domain containing protein 6 (JMJD6) in infectivity of foot-and-mouth disease virus

USDA-ARS?s Scientific Manuscript database

Foot-and-mouth disease virus (FMDV) can utilize as many as three distinct groups of receptor molecules to attach and enter a susceptible host cell. Four integrin heterodimers (alphavBeta1, alphavBeta3, alphavBeta6, and alphavBeta8) can function as the primary receptor for FMDV field strains. FMDV ...

Genome-wide analysis of DNA methylation variations caused by chronic glucolipotoxicity in beta-cells.

PubMed

Hu, Y; Xu, X-H; He, K; Zhang, L-L; Wang, S-K; Pan, Y-Q; He, B-S; Feng, T-T; Mao, X-M

2014-02-01

There is a growing body of literature suggesting the role of interactions between genes and the environment in development of type 2 diabetes mellitus (T2DM). However, the interplay between environment and genetic in developing and progressing T2MD is not fully understood. To determine the effects of high-glucose-lipid on the status of DNA methylation in beta cells, and clarify the mechanism of glucolipotoxicity on beta-cell deterioration, the DNA methylation profile was detected in beta-cells cultured with high-glucose-lipid medium.We utilized a high throughput NimbleGen RN34 CpG Island & Promoter Microarray to investigate the DNA methylation profile in beta-cells cultured with high-glucose-lipid medium. To validate the results of microarray, the immunoprecipitation (MeDIP) PCR was used to test the methylation status of some selected genes. The mRNA and protein expression of insulin and Tcf7l2 in these cells were quantified by RT-PCR and western blot, respectively.We have identified a lot of loci which experienced aberrant DNA methylation in beta-cells cultured with high-glucose-lipid medium. The results of MeDIP PCR were consistency to the microarray. An opposite regulation in transcription and translation of Tcf7l2 gene was found. Furthermore, the insulin mRNA and protein expression in beta-cells also decreased after cultured with high-glucose-lipid medium compared with the control cells.We conclude that chronic glucolipotoxicity could induce aberrant DNA methylation of some genes and may affect these genes expression in beta-cells, which might contribute to beta-cell function failure in T2DM and be helpful to explain, at least partially, the mechanism of glucolipotoxicity on beta-cells deterioration. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.

Agglutination of like-charged red blood cells induced by binding of beta2-glycoprotein I to outer cell surface.

PubMed

Lokar, Marusa; Urbanija, Jasna; Frank, Mojca; Hägerstrand, Henry; Rozman, Blaz; Bobrowska-Hägerstrand, Malgorzata; Iglic, Ales; Kralj-Iglic, Veronika

2008-08-01

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that beta(2)-glycoprotein I (beta(2)-GPI) may induce RBC discocyte-echinocyte-spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed beta(2)-GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of beta(2)-GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound beta(2)-GPI molecules. The suggested beta(2)-GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound beta(2)-GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.

Functional analysis of alpha5beta1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivalis using fluorescent beads coated with bacterial membrane vesicles.

PubMed

Tsuda, Kayoko; Furuta, Nobumichi; Inaba, Hiroaki; Kawai, Shinji; Hanada, Kentaro; Yoshimori, Tamotsu; Amano, Atsuo

2008-01-01

Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha5beta1 integrin and lipid rafts to invade host cells. However, it is unknown if the functional roles of these host components are distinct from one another during bacterial invasion. In the present study, we analyzed the mechanisms underlying P. gingivalis invasion, using fluorescent beads coated with bacterial membrane vesicles (MV beads). Cholesterol depletion reagents including methyl-beta-cyclodextrin (MbetaCD) drastically inhibited the entry of MV beads into epithelial cells, while they were less effective on bead adhesion to the cells. Bead entry was also abolished in CHO cells deficient in sphingolipids, components of lipid rafts, whereas adhesion was negligibly influenced. Following MbetaCD treatment, downstream events leading to actin polymerization were abolished; however, alpha5beta1 integrin was recruited to beads attached to the cell surface. Dominant-negative Rho GTPase Rac1 abolished cellular engulfment of the beads, whereas dominant-negative Cdc42 did not. Following cellular interaction with the beads, Rac1 was found to be translocated to the lipid rafts fraction, which was inhibited by MbetaCD. These results suggest that alpha5beta1 integrin, independent of lipid rafts, promotes P. gingivalis adhesion to epithelial cells, while the subsequent uptake process requires lipid raft components for actin organization, with Rho GTPase Rac1.

beta. -adrenergic relaxation of smooth muscle: differences between cells and tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scheid, C.R.

1987-09-01

The present studies were carried out in an attempt to resolve the controversy about the Na/sup +/ dependence of ..beta..-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of ..beta..-adrenergic agents, including a stimulatory effect on /sup 45/Ca efflux, were dependent on the presence of a normal transmembrane Na/sup +/ gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of ..beta..-adrenergic agents was Na/sup +/ independent. Uncertainty remained as to whether these discrepancies reflected differences betweenmore » cells and tissues or differences between species. Thus, in the present studies, the authors utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na/sup +/ dependence of ..beta..-adrenergic relaxation. They found that elimination of a normal Na/sup +/ gradient abolished ..beta..-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Thus the controversy concerning the mechanisms of ..beta..-adrenergic relaxation may reflect inherent differences between tissues and cells.« less

Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

PubMed

Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

2007-09-14

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

Beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing.

PubMed

Chen, Yan; Whetstone, Heather C; Lin, Alvin C; Nadesan, Puviindran; Wei, Qingxia; Poon, Raymond; Alman, Benjamin A

2007-07-31

Delayed fracture healing causes substantial disability and usually requires additional surgical treatments. Pharmacologic management to improve fracture repair would substantially improve patient outcome. The signaling pathways regulating bone healing are beginning to be unraveled, and they provide clues into pharmacologic management. The beta-catenin signaling pathway, which activates T cell factor (TCF)-dependent transcription, has emerged as a key regulator in embryonic skeletogenesis, positively regulating osteoblasts. However, its role in bone repair is unknown. The goal of this study was to explore the role of beta-catenin signaling in bone repair. Western blot analysis showed significant up-regulation of beta-catenin during the bone healing process. Using a beta-Gal activity assay to observe activation during healing of tibia fractures in a transgenic mouse model expressing a TCF reporter, we found that beta-catenin-mediated, TCF-dependent transcription was activated in both bone and cartilage formation during fracture repair. Using reverse transcription-PCR, we observed that several WNT ligands were expressed during fracture repair. Treatment with DKK1 (an antagonist of WNT/beta-catenin pathway) inhibited beta-catenin signaling and the healing process, suggesting that WNT ligands regulate beta-catenin. Healing was significantly repressed in mice conditionally expressing either null or stabilized beta-catenin alleles induced by an adenovirus expressing Cre recombinase. Fracture repair was also inhibited in mice expressing osteoblast-specific beta-catenin null alleles. In stark contrast, there was dramatically enhanced bone healing in mice expressing an activated form of beta-catenin, whose expression was restricted to osteoblasts. Treating mice with lithium activated beta-catenin in the healing fracture, but healing was enhanced only when treatment was started subsequent to the fracture. These results demonstrate that beta-catenin functions differently at different stages of fracture repair. In early stages, precise regulation of beta-catenin is required for pluripotent mesenchymal cells to differentiate to either osteoblasts or chondrocytes. Once these undifferentiated cells have become committed to the osteoblast lineage, beta-catenin positively regulates osteoblasts. This is a different function for beta-catenin than has previously been reported during development. Activation of beta-catenin by lithium treatment has potential to improve fracture healing, but only when utilized in later phases of repair, after mesenchymal cells have become committed to the osteoblast lineage.

Expression profile of senescence-associated beta-galactosidase and activation of telomerase in human ovarian surface epithelial cells undergoing immortalization.

PubMed

Litaker, J R; Pan, J; Cheung, Y; Zhang, D K; Liu, Y; Wong, S C; Wan, T S; Tsao, S W

1998-11-01

Senescence is a specific physiological stage of cells characterized by long population doubling time. It accounts for the inability of normal somatic cells to undergo indefinite cell division. As the number of population doublings increase, cell cycle regulatory mechanisms come into play and signal cells to exit the cell cycle and become senescent. Senescence has been implicated in the aging process and may function as a tumor suppressor mechanism in human cells. The ability to measure the degree of cellular senescence is important in understanding the biological processes regulating cell aging and immortalization. Senescent cells exhibit an enzyme termed senescence-associated histochemical staining. Cells immortalized by viral oncogenes often enter a stage of crisis at the early phase of immortalization. The cells at crisis have a long population doubling time. Cells at the crisis stage resemble senescent cells and the expression of SA- beta-Gal may be used to monitor the process of immortalization. In this study the expression profile of SA-beta-Gal was examined in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalization by the human papilloma viral oncogene E6 and E7 (HPV E6 and E7). Our results showed a low percentage (12.0%) of HOSE 6-3 cells expressing SA-beta-Gal activity at the pre-crisis stage. The percentage of HOSE 6-3 cells expressing SA-beta-Gal activity was highest (39.2%) at the crisis stage. When HOSE 6-3 cells achieved immortalized status there was a sharp decrease in cells (1. 3%) expressing SA-beta-Gal activity. In addition, an inverse relationship between the expression of SA-beta-Gal activity and telomerase activity was noted in cells undergoing immortalization. The results confirm that the SA-beta-Gal enzyme is a good marker for monitoring the population of cells undergoing senescence at different stages of immortalization and that telomerase activation is a characteristic feature of post-crisis cells.

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar

2012-08-17

Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protectsmore » against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.« less

Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.

PubMed Central

Gauldie, J; Richards, C; Harnish, D; Lansdorp, P; Baumann, H

1987-01-01

One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Images PMID:2444978

Human dendritic cells produce TGF-beta 1 under the influence of lung carcinoma cells and prime the differentiation of CD4+CD25+Foxp3+ regulatory T cells.

PubMed

Dumitriu, Ingrid E; Dunbar, Donald R; Howie, Sarah E; Sethi, Tariq; Gregory, Christopher D

2009-03-01

Dendritic cells (DCs) have a central role in the development of adaptive immune responses, including antitumor immunity. Factors present in the tumor milieu can alter the maturation of DCs and inhibit their capacity to activate T cells. Using gene expression analysis, we found that human DCs increased the expression of TGF-beta1 transcripts following culture with human lung carcinoma cells (LCCs). These DCs produced increased amounts of TGF-beta1 protein compared with DCs not exposed to tumor cells. LCCs also decreased the expression of CD86 and HLA-DR by immature DCs. Furthermore, LCCs decreased CD86 expression and the production of TNF-alpha and IL-12 p70 by mature DCs. Moreover, LCCs also converted mature DCs into cells producing TGF-beta1. These TGF-beta1-producing DCs were poor at eliciting the activation of naive CD4(+) T cells and sustaining their proliferation and differentiation into Th1 (IFN-gamma(+)) effectors. Instead, TGF-beta1-producing DCs demonstrated an increased ability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress the proliferation of T lymphocytes. These results identify a novel mechanism by which the function of human DCs is altered by tumor cells and contributes to the evasion of the immune response.

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions.

PubMed

Graser, R T; DiLorenzo, T P; Wang, F; Christianson, G J; Chapman, H D; Roopenian, D C; Nathenson, S G; Serreze, D V

2000-04-01

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Wenqing, E-mail: liangwenqing_1234@126.com; Yang, Chengwei; Qian, Yu

Highlights: Black-Right-Pointing-Pointer {beta}-Catenin expression were markedly down-regulated by CTNNB1 shRNA. Black-Right-Pointing-Pointer CTNNB1 shRNA could inhibit the proliferation of RPMI8226 cells. Black-Right-Pointing-Pointer Significantly profound apoptotic cell death in CTNNB1 shRNA cells. Black-Right-Pointing-Pointer In vivo, CTNNB1 silence led to a growth inhibition of myeloma growth. Black-Right-Pointing-Pointer c-myc and {beta}-catenin in the expression cells of cleaved caspase-3 were increased. -- Abstract: Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of {beta}-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between {beta}-catenin expression and MM remains unclear. In the present study, RNA interferencemore » was used to inhibit {beta}-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that {beta}-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of {beta}-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and {beta}-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that {beta}-catenin could be a new therapeutic agent that targets the biology of MM cells.« less

The TGF-beta-induced gene product, betaig-h3: its biological implications in peritoneal dialysis.

PubMed

Park, Sun-Hee; Choi, Soon-Youn; Kim, Mi-Hyung; Oh, Eun-Joo; Ryu, Hye Myung; Kim, Chan-Duck; Kim, In-San; Kim, Yong-Lim

2008-01-01

TGF-beta is involved in peritoneal changes during long-term peritoneal dialysis (PD). TGF-beta induces betaig-h3 in several cell lines, and betaig-h3 may be a marker for biologically active TGF-beta. However, no study has reported induction of betaig-h3 in human peritoneal mesothelial cells (HPMCs) or its involvement in PD-related peritoneal membrane changes. We used cultured HPMCs to investigate the biological roles of betaig-h3 during mesothelial cell injury and repair, employing the adhesion, spreading, scratching and cell migration assays. Changes in betaig-h3 expression after high glucose exposure in vivo were also evaluated using an animal chronic PD model. In vitro, TGF-beta1 induced betaig-h3 in cultured HPMCs, and betaig-h3-mediated mesothelial cell adhesion occurred via alphavbeta3 integrin. betaig-h3 enhanced mesothelial cell adhesion and migration and, in part, wound healing during mesothelial cell injury. The animal study demonstrated that compared to the control group, betaig-h3 concentrations in the dialysate effluent increased in the dialysis group with alterations in peritoneal structure and function during PD, and betaig-h3 positively correlated with peritoneal solute transport. Immunohistochemical and immunoblotting results showed that betaig-h3 localizes in the mesothelium and submesothelial matrix of the parietal peritoneum, and in the vascular endothelium of omentum. betaig-h3 protein expression was higher in the dialysis group. In vitro, betaig-h3 induced by TGF-beta1 in HPMCs improved adhesion and migration of HPMCs during wound healing. In the chronic infusion model of PD, betaig-h3 played a role in the functional deterioration of the peritoneal membrane, which is associated with fibrosis.

Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

2010-01-01

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cellmore » surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.« less

Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

PubMed Central

Neve, Bernadette; Fernandez-Zapico, Martin E.; Ashkenazi-Katalan, Vered; Dina, Christian; Hamid, Yasmin H.; Joly, Erik; Vaillant, Emmanuel; Benmezroua, Yamina; Durand, Emmanuelle; Bakaher, Nicolas; Delannoy, Valerie; Vaxillaire, Martine; Cook, Tiffany; Dallinga-Thie, Geesje M.; Jansen, Hans; Charles, Marie-Aline; Clément, Karine; Galan, Pilar; Hercberg, Serge; Helbecque, Nicole; Charpentier, Guillaume; Prentki, Marc; Hansen, Torben; Pedersen, Oluf; Urrutia, Raul; Melloul, Danielle; Froguel, Philippe

2005-01-01

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes. PMID:15774581

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit

PubMed Central

1992-01-01

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin. PMID:1500432

Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon-beta promoter.

PubMed

Jääskeläinen, Kirsi M; Kaukinen, Pasi; Minskaya, Ekaterina S; Plyusnina, Angelina; Vapalahti, Olli; Elliott, Richard M; Weber, Friedemann; Vaheri, Antti; Plyusnin, Alexander

2007-10-01

The S RNA genome segment of hantaviruses carried by Arvicolinae and Sigmodontinae rodents encodes the nucleocapsid (N) protein and has an overlapping (+1) open reading frame (ORF) for a putative nonstructural protein (NSs). The aim of this study was to determine whether the ORF is functional. A protein corresponding to the predicted size of Tula virus (TULV) NSs was detected using coupled in vitro transcription and translation from a cloned S segment cDNA, and a protein corresponding to the predicted size of Puumala virus (PUUV) NSs was detected in infected cells by Western blotting with an anti-peptide serum. The activities of the interferon beta (IFN-beta) promoter, and nuclear factor kappa B (NF-kappaB)- and interferon regulatory factor-3 (IRF-3) responsive promoters, were inhibited in COS-7 cells transiently expressing TULV or PUUV NSs. Also IFN-beta mRNA levels in IFN-competent MRC5 cells either infected with TULV or transiently expressing NSs were decreased. These data demonstrate that Tula and Puumala hantaviruses have a functional NSs ORF. The findings may explain why the NSs ORF has been preserved in the genome of most hantaviruses during their long evolution and why hantavirus-infected cells secrete relatively low levels of IFNs. (c) 2007 Wiley-Liss, Inc.

Mono-(2-ethylhexyl) phthalate (MEHP) regulates glucocorticoid metabolism through 11{beta}-hydroxysteroid dehydrogenase 2 in murine gonadotrope cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Dun; Department of Orthopedics, Taizhou Hospital, Wenzhou Medical College, Lin Hai, ZJ 317000; Li, Xing-Wang

2009-11-13

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite mono-(2-ethylhexyl) phthalate (MEHP) have been classified as toxicants to the reproductive system at the testis level and DEHP may also impair reproductive axis function at the pituitary levels. However, MEHP is 10-fold more potent than DEHP in toxicity and little is known about the toxicological effect of MEHP on pituitary. In this study, we demonstrated that 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD2), not 11{beta}-HSD1, is strongly expressed in murine gonadotrope L{beta}T2 cells. Interestingly, MEHP inhibited Hsd11b2 mRNA level and 11{beta}-HSD2 enzyme activity in L{beta}T2 cells at as low as 10{sup -7} M. Corticosterone (CORT) atmore » a concentration of 10{sup -6} M significantly inhibited L{beta}T2 cell proliferation after 2-day culture, and 10{sup -6} M RU486, an antagonist of glucocorticoid receptor (GR), reversed this inhibition. However, in the presence of 10{sup -5} or 10{sup -4} M MEHP, the minimal concentration of CORT to inhibit the proliferation of L{beta}T2 cells was lowered to 10{sup -7} M, and 10{sup -6} M RU486 was not able to completely reverse the CORT effect. In conclusion, along with the regulation of GR, 11{beta}-HSD2 may have a key role in glucocorticoid metabolism in L{beta}T2 cells. MEHP may participate in the glucocorticoid metabolism in L{beta}T2 cells through inhibition of 11{beta}-HSD2 enzyme activity. Such perturbation may be of pathological significance as MEHP may interfere with the reproductive system at pituitary level through regulation of glucocorticoid metabolism, especially in neonates with higher risk of phthalates exposure.« less

Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.

2006-02-17

Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when themore » entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.« less

EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi-Hwi; Kim, Eung-Hwi

Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H{sub 2}O{sub 2}. Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factormore » erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress. - Highlights: • EX4 protects against oxidative stress-induced pancreatic beta cell dysfunction. • EX4 increases antioxidant gene expression. • Antioxidative effect of EX4 is mediated by Nrf2. • EX4 increases Nrf2 level by stabilizing Nrf2 protein. • EX4 stabilizes Nrf2 by activation of PKCδ.« less

Human T-cell leukemia virus type I oncoprotein Tax represses Smad-dependent transforming growth factor beta signaling through interaction with CREB-binding protein/p300.

PubMed

Mori, N; Morishita, M; Tsukazaki, T; Giam, C Z; Kumatori, A; Tanaka, Y; Yamamoto, N

2001-04-01

Human T-cell leukemia virus type I (HTLV-I) Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-I- infected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-beta. Here it is shown that Tax can perturb Smad-dependent TGF-beta signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax-mediated inhibition of Smad transactivation. Furthermore, Smad could repress Tax transcriptional activation, indicating reciprocal repression between Tax and Smad. These results suggest that Tax interferes with the recruitment of CBP/p300 into transcription initiation complexes on TGF-beta-responsive elements through its binding to CBP/p300. The novel function of Tax as a repressor of TGF-beta signaling may contribute to HTLV-I leukemogenesis. (Blood. 2001;97:2137-2144)

The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

PubMed

van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

2002-06-21

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

Adiposity and family history of type 2 diabetes in an admixed population of adolescents: Associations with insulin sensitivity, beta-cell function, and hepatic insulin extraction in BRAMS study.

PubMed

Camilo, Daniella F; Vasques, Ana Carolina J; Hayashi, Keila; Tura, Andrea; da Silva, Cleliani de Cassia; Zambon, Mariana P; Antônio, Maria Ângela R de G Monteiro; Geloneze, Bruno

2018-03-01

Insulin resistance and beta-cell dysfunction manifest differently across racial/ethnic groups, and there is a lack of knowledge regarding the pathophysiology of type 2 diabetes mellitus (T2DM) for ethnically admixed adolescents. This study aimed to investigate the influence of adiposity and family history (FH) of T2DM on aspects of insulin sensitivity, beta-cell function, and hepatic insulin extraction in Brazilian adolescents. A total of 82 normoglycemic adolescents were assessed. The positive FH of T2DM was defined as the presence of at least one known family member with T2DM. The hyperglycemic clamp test consisted of a 120-min protocol. Insulin secretion and beta-cell function were obtained from C-peptide deconvolution. Analysis of covariance considered pubertal stage as a covariate. Both lean and overweight/obese adolescents had similar glycemic profiles and disposition indexes. Overweight/obese adolescents had about 1/3 the insulin sensitivity of lean adolescents (1.1 ± 0.2 vs. 3.4 ± 0.3 mg·kg·min·pmol ∗ 1000), which was compensated by an increase around 2.5 times in basal (130 ± 7 vs. 52 ± 10 pmol·l·min) and total insulin secretion (130,091 ± 12,230 vs. 59,010 ± 17,522 pmol·l·min), and in the first and second phases of insulin secretion; respectively (p < 0.001). This increase was accompanied by a mean reduction in hepatic insulin extraction of 35%, and a 2.7-time increase in beta-cell glucose sensitivity (p < 0.05). The positive FH of T2DM was not associated with derangements in insulin sensitivity, beta-cell function, and hepatic insulin extraction. In an admixed sample of adolescents, the hyperglycemic clamp test demonstrated that adiposity had a strong influence, and FH of T2DM had no direct influence, in different aspects of glucose metabolism. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of atorvastatin on pancreatic Beta-cell function and insulin resistance in type 2 diabetes mellitus patients: a randomized pilot study.

PubMed

Goyal, Aman; Singh, Surender; Tandon, Nikhil; Gupta, Nandita; Gupta, Yogendra Kumar

2014-12-01

Statins are commonly used for the management of dyslipidemia in type 2 diabetes mellitus patients. We hypothesized that atorvastatin could modulate the beta-cell function by altering the levels of proapoptotic and antiapoptotic lipoproteins and could also have an effect on insulin resistance. The aim of the present pilot study was to assess the effect of atorvastatin 10 mg on pancreatic beta-cell function and insulin resistance in patients with hyperlipidemia and type 2 diabetes by using the homeostasis model assessment-2 (HOMA2) index. Fifty-one type 2 diabetes patients receiving oral antidiabetes drugs, not taking statins, with baseline low-density lipoprotein cholesterol between 2.6 mmol/L and 4.1 mmol/L were included. Forty-three patients (21 in placebo group and 22 in atorvastatin group) completed the study and were taken up for final analysis. Fasting blood samples were obtained at baseline and at 12 weeks to determine levels of blood glucose, lipid profile, insulin, C-peptide and glycosylated hemoglobin (A1C). Atorvastatin nonsignificantly increased fasting serum insulin (+14.29%, p=0.18), accompanied by marginal nonsignificant increases in fasting plasma glucose and A1C. There was a decrease in HOMA2 percent beta-cell function (-2.9%, p=0.72) and increase in HOMA2 insulin resistance (+14%, p=0.16) in the atorvastatin group as compared with baseline, but the difference was not statistically significant. Atorvastatin in the dose used failed to produce significant change in pancreatic beta-cell function and insulin resistance in type 2 diabetes patients as assessed by the HOMA2 index. The possible explanations include absence of lipotoxicity at prevailing levels of dyslipidemia at baseline or inadequacy of statin dose used in the study. (Clinical Trials Registry-India: CTRI/2008/091/000099). Copyright © 2014 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

Intermittent Hypoxia Impairs Glucose Homeostasis in C57BL6/J Mice: Partial Improvement with Cessation of the Exposure

PubMed Central

Polak, Jan; Shimoda, Larissa A.; Drager, Luciano F.; Undem, Clark; McHugh, Holly; Polotsky, Vsevolod Y.; Punjabi, Naresh M.

2013-01-01

Objectives: Obstructive sleep apnea is associated with insulin resistance, glucose intolerance, and type 2 diabetes mellitus. Although several studies have suggested that intermittent hypoxia in obstructive sleep apnea may induce abnormalities in glucose homeostasis, it remains to be determined whether these abnormalities improve after discontinuation of the exposure. The objective of this study was to delineate the effects of intermittent hypoxia on glucose homeostasis, beta cell function, and liver glucose metabolism and to investigate whether the impairments improve after the hypoxic exposure is discontinued. Interventions: C57BL6/J mice were exposed to 14 days of intermittent hypoxia, 14 days of intermittent air, or 7 days of intermittent hypoxia followed by 7 days of intermittent air (recovery paradigm). Glucose and insulin tolerance tests were performed to estimate whole-body insulin sensitivity and calculate measures of beta cell function. Oxidative stress in pancreatic tissue and glucose output from isolated hepatocytes were also assessed. Results: Intermittent hypoxia increased fasting glucose levels and worsened glucose tolerance by 67% and 27%, respectively. Furthermore, intermittent hypoxia exposure was associated with impairments in insulin sensitivity and beta cell function, an increase in liver glycogen, higher hepatocyte glucose output, and an increase in oxidative stress in the pancreas. While fasting glucose levels and hepatic glucose output normalized after discontinuation of the hypoxic exposure, glucose intolerance, insulin resistance, and impairments in beta cell function persisted. Conclusions: Intermittent hypoxia induces insulin resistance, impairs beta cell function, enhances hepatocyte glucose output, and increases oxidative stress in the pancreas. Cessation of the hypoxic exposure does not fully reverse the observed changes in glucose metabolism. Citation: Polak J; Shimoda LA; Drager LF; Undem C; McHugh H; Polotsky VY; Punjabi NM. Intermittent hypoxia impairs glucose homeostasis in C57BL6/J mice: partial improvement with cessation of the exposure. SLEEP 2013;36(10):1483-1490. PMID:24082307

Calcium Channels in Postnatal Development of Rat Pancreatic Beta Cells and Their Role in Insulin Secretion

PubMed Central

García-Delgado, Neivys; Velasco, Myrian; Sánchez-Soto, Carmen; Díaz-García, Carlos Manlio; Hiriart, Marcia

2018-01-01

Pancreatic beta cells during the first month of development acquire functional maturity, allowing them to respond to variations in extracellular glucose concentration by secreting insulin. Changes in ionic channel activity are important for this maturation. Within the voltage-gated calcium channels (VGCC), the most studied channels are high-voltage-activated (HVA), principally L-type; while low-voltage-activated (LVA) channels have been poorly studied in native beta cells. We analyzed the changes in the expression and activity of VGCC during the postnatal development in rat beta cells. We observed that the percentage of detection of T-type current increased with the stage of development. T-type calcium current density in adult cells was higher than in neonatal and P20 beta cells. Mean HVA current density also increased with age. Calcium current behavior in P20 beta cells was heterogeneous; almost half of the cells had HVA current densities higher than the adult cells, and this was independent of the presence of T-type current. We detected the presence of α1G, α1H, and α1I subunits of LVA channels at all ages. The Cav 3.1 subunit (α1G) was the most expressed. T-type channel blockers mibefradil and TTA-A2 significantly inhibited insulin secretion at 5.6 mM glucose, which suggests a physiological role for T-type channels at basal glucose conditions. Both, nifedipine and TTA-A2, drastically decreased the beta-cell subpopulation that secretes more insulin, in both basal and stimulating glucose conditions. We conclude that changes in expression and activity of VGCC during the development play an important role in physiological maturation of beta cells. PMID:29556214

Regulation of calcium-permeable TRPV2 channel by insulin in pancreatic beta-cells.

PubMed

Hisanaga, Etsuko; Nagasawa, Masahiro; Ueki, Kohjiro; Kulkarni, Rohit N; Mori, Masatomo; Kojima, Itaru

2009-01-01

Calcium-permeable cation channel TRPV2 is expressed in pancreatic beta-cells. We investigated regulation and function of TRPV2 in beta-cells. Translocation of TRPV2 was assessed in MIN6 cells and cultured mouse beta-cells by transfecting TRPV2 fused to green fluorescent protein or TRPV2 containing c-Myc tag in the extracellular domain. Calcium entry was assessed by monitoring fura-2 fluorescence. In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition. Addition of exogenous insulin induced translocation and insertion of TRPV2 to the plasma membrane. Consistent with these observations, insulin increased calcium entry, which was inhibited by tranilast, an inhibitor of TRPV2, or by knockdown of TRPV2 using shRNA. A high concentration of glucose also induced translocation of TRPV2, which was blocked by nefedipine, diazoxide, and somatostatin, agents blocking glucose-induced insulin secretion. Knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2. Similarly, the effect of insulin on TRPV2 translocation was not observed in a beta-cell line derived from islets obtained from a beta-cell-specific insulin receptor knockout mouse. Knockdown of TRPV2 or addition of tranilast significantly inhibited insulin secretion induced by a high concentration of glucose. Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2. Finally, insulin-induced translocation of TRPV2 was observed in cultured mouse beta-cells, and knockdown of TRPV2 reduced insulin secretion induced by glucose. TRPV2 is regulated by insulin and is involved in the autocrine action of this hormone on beta-cells.

Sall2 knockdown exacerbates palmitic acid induced dysfunction and apoptosis of pancreatic NIT-1 beta cells.

PubMed

Wang, Ye; Liu, Jie; Liu, Zheng; Chen, Jing; Hu, Xuemei; Hu, Yimeng; Yuan, Yin; Wu, Guijun; Dai, Zhe; Xu, Yancheng

2018-05-18

Spalt-like (Sall) proteins are a class of transcription factors. The role of Sall2 in beta cells remain poorly understood. Here, we aimed to explore whether Sall2 involved in lipotoxicity-mediated dysfunction and apoptosis in pancreatic NIT-1 beta cells. Our results showed that high concentrations of palmitic acid (PA) led to impaired cell viability and decreased Sall2 expression in NIT-1 cells. Knocking down of Sall2 in NIT-1 cells resulted in increased sensitivity to lipotoxicity and caused higher rates of cell apoptosis following PA treatment. Additionally, Sall2 Knockdown impaired insulin synthesis and secretion in response to glucose. Further research indicated Sall2 knockdown attenuate antioxidant capacity and decreased expression level of Peroxiredoxin 2 in NIT-1 cells. These finding implicate that Sall2 may play a significant role in NIT-1 cell function and cell apoptosis under lipotoxic conditions. Therefore, the study of Sall2 in NIT-1 cells provided a new perspective for molecular mechanism of lipotoxicity mediating dysfunction and apoptosis of beta cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

On the coherent behavior of pancreatic beta cell clusters

NASA Astrophysics Data System (ADS)

Loppini, Alessandro; Capolupo, Antonio; Cherubini, Christian; Gizzi, Alessio; Bertolaso, Marta; Filippi, Simonetta; Vitiello, Giuseppe

2014-09-01

Beta cells in pancreas represent an example of coupled biological oscillators which via communication pathways, are able to synchronize their electrical activity, giving rise to pulsatile insulin release. In this work we numerically analyze scale free self-similarity features of membrane voltage signal power density spectrum, through a stochastic dynamical model for beta cells in the islets of Langerhans fine tuned on mouse experimental data. Adopting the algebraic approach of coherent state formalism, we show how coherent molecular domains can arise from proper functional conditions leading to a parallelism with “phase transition” phenomena of field theory.

Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model.

PubMed

Lee, Hong J; Kim, Kwang S; Kim, Eun J; Choi, Hyun B; Lee, Kwang H; Park, In H; Ko, Yong; Jeong, Sang W; Kim, Seung U

2007-05-01

We have generated stable, immortalized cell lines of human NSCs from primary human fetal telencephalon cultures via a retroviral vector encoding v-myc. HB1.F3, one of the human NSC lines, expresses a normal human karyotype of 46, XX, and nestin, a cell type-specific marker for NSCs. F3 has the ability to proliferate continuously and differentiate into cells of neuronal and glial lineage. The HB1.F3 human NSC line was used for cell therapy in a mouse model of intracerebral hemorrhage (ICH) stroke. Experimental ICH was induced in adult mice by intrastriatal administration of bacterial collagenase; 1 week after surgery, the rats were randomly divided into two groups so as to receive intracerebrally either human NSCs labeled with beta-galactosidase (n = 31) or phosphate-buffered saline (PBS) (n = 30). Transplanted NSCs were detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactoside histochemistry or double labeling with beta-galactosidase (beta-gal) and mitogen-activated protein (MAP)2, neurofilaments (both for neurons), or glial fibrillary acidic protein (GFAP) (for astrocytes). Behavior of the animals was evaluated for period up to 8 weeks using modified Rotarod tests and a limb placing test. Transplanted human NSCs were identified in the perihematomal areas and differentiated into neurons (beta-gal/MAP2(+) and beta-gal/NF(+)) or astrocytes (beta-gal/GFAP(+)). The NSC-transplanted group showed markedly improved functional performance on the Rotarod test and limb placing after 2-8 weeks compared with the control PBS group (p < .001). These results indicate that the stable immortalized human NSCs are a valuable source of cells for cell replacement and gene transfer for the treatment of ICH and other human neurological disorders. Disclosure of potential conflicts of interest is found at the end of this article.

Mouse Insulin Cells Expressing an Inducible RIPCre Transgene Are Functionally Impaired

PubMed Central

Teitelman, Gladys; Kedees, Mamdouh

2015-01-01

We used cre-lox technology to test whether the inducible expression of Cre minimize the deleterious effect of the enzyme on beta cell function. We studied mice in which Cre is linked to a modified estrogen receptor (ER), and its expression is controlled by the rat insulin promoter (RIP). Following the injection of tamoxifen (TM), CreER- migrates to the nucleus and promotes the appearance of a reporter protein, enhanced yellow fluorescent protein (EYFP), in cells. Immunocytochemical analysis indicated that 46.6 ± 2.1% insulin cells of adult RIPCreER- EYFP expressed EYFP. RIPCreER-EYFP (+TM) mice were normoglycemic throughout the study, and their glucose tolerance test results were similar to control CD-1 mice. However, an extended exposure to reagents that stimulate insulin synthesis was detrimental to the survival of IN+EYFP+cells. The administration of an inhibitor of the enzyme dipeptidyl-peptidase (DPP4i), which prevents the cleavage of glucagon-like peptide (GLP-1), to adult RIPCreER-EYFP mice lead to a decrease in the percentage of IN+EYFP+ to 17.5 ± 1.73 and a significant increase in apoptotic cells in islets. Similarly, a 2-week administration of the GLP-1 analog exendin 4 (ex-4) induced an almost complete ablation of IN+ expressing a different reporter protein and a significant decrease in the beta cell mass and rate of beta cell proliferation. Since normal beta cells do not die when induced to increase insulin synthesis, our observations indicate that insulin cells expressing an inducible RIPCre transgene are functionally deficient. Studies employing these mice should carefully consider the pitfalls of the Cre-Lox technique. PMID:25533471

Exercise training decreases pancreatic fat content and improves beta cell function regardless of baseline glucose tolerance: a randomised controlled trial.

PubMed

Heiskanen, Marja A; Motiani, Kumail K; Mari, Andrea; Saunavaara, Virva; Eskelinen, Jari-Joonas; Virtanen, Kirsi A; Koivumäki, Mikko; Löyttyniemi, Eliisa; Nuutila, Pirjo; Kalliokoski, Kari K; Hannukainen, Jarna C

2018-05-02

Pancreatic fat accumulation may contribute to the development of beta cell dysfunction. Exercise training improves whole-body insulin sensitivity, but its effects on pancreatic fat content and beta cell dysfunction are unclear. The aim of this parallel-group randomised controlled trial was to evaluate the effects of exercise training on pancreatic fat and beta cell function in healthy and prediabetic or type 2 diabetic participants and to test whether the responses were similar regardless of baseline glucose tolerance. Using newspaper announcements, a total of 97 sedentary 40-55-year-old individuals were assessed for eligibility. Prediabetes (impaired fasting glucose and/or impaired glucose tolerance) and type 2 diabetes were defined by ADA criteria. Of the screened candidates, 28 healthy men and 26 prediabetic or type 2 diabetic men and women met the inclusion criteria and were randomised into 2-week-long sprint interval or moderate-intensity continuous training programmes in a 1:1 allocation ratio using random permuted blocks. The primary outcome was pancreatic fat, which was measured by magnetic resonance spectroscopy. As secondary outcomes, beta cell function was studied using variables derived from OGTT, and whole-body insulin sensitivity and pancreatic fatty acid and glucose uptake were measured using positron emission tomography. The measurements were carried out at the Turku PET Centre, Finland. The analyses were based on an intention-to-treat principle. Given the nature of the intervention, blinding was not applicable. At baseline, the group of prediabetic or type 2 diabetic men had a higher pancreatic fat content and impaired beta cell function compared with the healthy men, while glucose and fatty acid uptake into the pancreas was similar. Exercise training decreased pancreatic fat similarly in healthy (from 4.4% [3.0%, 6.1%] to 3.6% [2.4%, 5.2%] [mean, 95% CI]) and prediabetic or type 2 diabetic men (from 8.7% [6.0%, 11.9%] to 6.7% [4.4%, 9.6%]; p = 0.036 for time effect) without any changes in pancreatic substrate uptake (p ≥ 0.31 for time effect in both insulin-stimulated glucose and fasting state fatty acid uptake). In prediabetic or type 2 diabetic men and women, both exercise modes similarly improved variables describing beta cell function. Two weeks of exercise training improves beta cell function in prediabetic or type 2 diabetic individuals and decreases pancreatic fat regardless of baseline glucose tolerance. This study shows that short-term training efficiently reduces ectopic fat within the pancreas, and exercise training may therefore reduce the risk of type 2 diabetes. ClinicalTrials.gov NCT01344928 FUNDING: This study was funded by the Emil Aaltonen Foundation, the European Foundation for the Study of Diabetes, the Finnish Diabetes Foundation, the Orion Research Foundation, the Academy of Finland (grants 251399, 256470, 281440, and 283319), the Ministry of Education of the State of Finland, the Paavo Nurmi Foundation, the Novo Nordisk Foundation, the Finnish Cultural Foundation, the Hospital District of Southwest Finland, the Turku University Foundation, and the Finnish Medical Foundation.

Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue

NASA Technical Reports Server (NTRS)

Barcellos-Hoff, M. H.; Ehrhart, E. J.; Kalia, M.; Jirtle, R.; Flanders, K.; Tsang, M. L.; Chatterjee, A. (Principal Investigator)

1995-01-01

The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.

The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus.

PubMed

Gerber, Philipp A; Rutter, Guy A

2017-04-01

Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene-environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn 2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501-518.

Smad7 induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis.

PubMed

Halder, Sunil K; Beauchamp, R Daniel; Datta, Pran K

2005-07-01

Smad proteins play a key role in the intracellular signaling of the transforming growth factor beta (TGF-beta) superfamily of extracellular polypeptides that initiate signaling to regulate a wide variety of biological processes. The inhibitory Smad, Smad7, has been shown to function as intracellular antagonists of TGF-beta family signaling and is upregulated in several cancers. To determine the effect of Smad7-mediated blockade of TGF-beta signaling, we have stably expressed Smad7 in a TGF-beta-sensitive, well-differentiated, and non-tumorigenic cell line, FET, that was derived from human colon adenocarcinoma. Smad7 inhibits TGF-beta-induced transcriptional responses by blocking complex formation between Smad 2/3 and Smad4. While Smad7 has no effect on TGF-beta-induced activation of p38 MAPK and ERK, it blocks the phosphorylation of Akt by TGF-beta and enhances TGF-beta-induced phosphorylation of c-Jun. FET cells expressing Smad7 show anchorage-independent growth and enhance tumorigenicity in athymic nude mice. Smad7 blocks TGF-beta-induced growth inhibition by preventing TGF-beta-induced G1 arrest. Smad7 inhibits TGF-beta-mediated downregulation of c-Myc, CDK4, and Cyclin D1, and suppresses the expression of p21(Cip1). As a result, Smad7 inhibits TGF-beta-mediated downregulation of Rb phosphorylation. Furthermore, Smad7 inhibits the apoptosis of these cells. Together, Smad7 may increase the tumorigenicity of FET cells by blocking TGF-beta-induced growth inhibition and by inhibiting apoptosis. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become refractory to tumor-suppressive actions of TGF-beta that might result in increased tumorigenicity.

PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3.

PubMed

Remy, Ingrid; Montmarquette, Annie; Michnick, Stephen W

2004-04-01

Transforming growth factor beta (TGF-beta) has a major role in cell proliferation, differentiation and apoptosis in many cell types. Integration of the TGF-beta pathway with other signalling cascades that control the same cellular processes may modulate TGF-beta responses. Here we report the discovery of a new functional link between TGF-beta and growth factor signalling pathways, mediated by a physical interaction between the serine-threonine kinase PKB (protein kinase B)/Akt and the transcriptional activator Smad3. Formation of the complex is induced by insulin, but inhibited by TGF-beta stimulation, placing PKB-Smad3 at a point of convergence between these two pathways. PKB inhibits Smad3 by preventing its phosphorylation, binding to Smad4 and nuclear translocation. In contrast, Smad3 does not inhibit PKB. Inhibition of Smad3 by PKB occurs through a kinase-activity-independent mechanism, resulting in a decrease in Smad3-mediated transcription and protection of cells against TGF-beta-induced apoptosis. Consistently, knockdown of the endogenous PKB gene with small-interfering RNA (siRNA) has the opposite effect. Our results suggest a very simple mechanism for the integration of signals arising from growth-factor- and TGF-beta-mediated pathways.

Functionally graded scaffolds for the engineering of interface tissues using hybrid twin screw extrusion/electrospinning technology

NASA Astrophysics Data System (ADS)

Erisken, Cevat

Tissue engineering is the application of the principles of engineering and life sciences for the development of biological alternatives for improvement or regeneration of native tissues. Native tissues are complex structures with functions and properties changing spatially and temporally, and engineering of such structures requires functionally graded scaffolds with composition and properties changing systematically along various directions. Utilization of a new hybrid technology integrating the controlled feeding, compounding, dispersion, deaeration, and pressurization capabilities of extrusion process with electrospinning allows incorporation of liquids and solid particles/nanoparticles into polymeric fibers/nanofibers for fabrication of functionally graded non-woven meshes to be used as scaffolds in engineering of tissues. The capabilities of the hybrid technology were demonstrated with a series of scaffold fabrication and cell culturing studies along with characterization of biomechanical properties. In the first study, the hybrid technology was employed to generate concentration gradations of beta-tricalcium phosphate (beta-TCP) nanoparticles in a polycaprolactone (PCL) binder, between two surfaces of nanofibrous scaffolds. These scaffolds were seeded with pre-osteoblastic cell line (MC3T3-E1) to attempt to engineer cartilage-bone interface, and after four weeks, the tissue constructs revealed formation of continuous gradations in extracellular matrix akin to cartilage-bone interface in terms of distributions of mineral concentrations and biomechanical properties. In a second demonstration of the hybrid technology, graded differentiation of stem cells was attempted by using insulin, a known stimulator of chondrogenic differentiation, and beta-glycerol phosphate (beta-GP), for mineralization. Concentrations of insulin and beta-GP in PCL were controlled to monotonically increase and decrease, respectively, along the length of scaffolds, which were then seeded with adipose derived stromal cells (h-ADSCs). Analysis of resulting tissue constructs revealed chondrocytic differentiation of h-ADSCs, with both the chondrocytic cell concentration and mineralization varying as a function of distributions of concentrations of insulin and beta-GP, respectively. The investigation also covered characterization of biomechanical properties of native bovine osteochondral tissue samples, which were then compared with biomechanical properties of tissue constructs at different stages of development. The hybrid technology developed in this thesis should provide another enabling platform for the fabrication of functionally graded scaffolds that aim to mimic the elegant gradations found in myriad native tissues.

Rab-coupling protein coordinates recycling of alpha5beta1 integrin and EGFR1 to promote cell migration in 3D microenvironments.

PubMed

Caswell, Patrick T; Chan, May; Lindsay, Andrew J; McCaffrey, Mary W; Boettiger, David; Norman, Jim C

2008-10-06

Here we show that blocking the adhesive function of alphavbeta3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with alpha5beta1 integrin and drove RCP-dependent recycling of alpha5beta1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in alpha5beta1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of alpha5beta1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, alpha5beta1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of alpha5beta1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices.

Putative signaling action of amelogenin utilizes the Wnt/beta-catenin pathway.

PubMed

Matsuzawa, M; Sheu, T-J; Lee, Y-J; Chen, M; Li, T-F; Huang, C T; Holz, J D; Puzas, J E

2009-06-01

While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells. To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a beta-galactosidase transgene under the control of a LEF/TCF and beta-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the beta-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and beta-catenin signaling using a TOPFLASH construct and the LacZ reporter gene. In these in vitro models, we showed that amelogenin can activate beta-catenin signaling. Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.

Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells.

PubMed

Jaiswal, Aruna S; Marlow, Benjamin P; Gupta, Nirupama; Narayan, Satya

2002-12-05

The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells.

Inhibition of phosphatidylcholine-specific phospholipase C prevents bone marrow stromal cell senescence in vitro.

PubMed

Sun, Chunhui; Wang, Nan; Huang, Jie; Xin, Jie; Peng, Fen; Ren, Yinshi; Zhang, Shangli; Miao, Junying

2009-10-01

Bone marrow stromal cells (BMSCs) can proliferate in vitro and can be transplanted for treating many kinds of diseases. However, BMSCs become senescent with long-term culture, which inhibits their application. To understand the mechanism underlying the senescence, we investigated the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and levels of integrin beta4, caveolin-1 and ROS with BMSC senescence. The activity of PC-PLC and levels of integrin beta4, caveolin-1 and ROS increased greatly during cell senescence. Selective inhibition of increased PC-PLC activity with D609 significantly decreased the number of senescence-associated beta galactosidase positive cells in BMSCs. Furthermore, D609 restored proliferation of BMSCs and their differentiation into adipocytes. Moreover, D609 suppressed the elevated levels of integrin beta4, caveolin-1 and ROS. The data suggest that PC-PLC is involved in senescence of BMSCs, and its function is associated with integrin beta4, caveolin-1 and ROS. (c) 2009 Wiley-Liss, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwasaki, Yuko; Iwasaki, Hitoshi; Yatoh, Shigeru

Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic {beta}-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, {beta}{epsilon}{tau}{alpha}2, MafA, andmore » IRS-2 were suppressed, partially explaining the loss and dysfunction of {beta}-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous {beta}-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts {beta}-cell mass and function.« less

Role of transforming growth factor-beta (TGF) beta in the physiopathology of rheumatoid arthritis.

PubMed

Gonzalo-Gil, Elena; Galindo-Izquierdo, María

2014-01-01

Transforming growth factor-beta (TGF-β) is a cytokine with pleiotropic functions in hematopoiesis, angiogenesis, cell proliferation, differentiation, migration and apoptosis. Although its role in rheumatoid arthritis is not well defined, TGF-β activation leads to functional immunomodulatory effects according to environmental conditions. The function of TGF-β in the development of arthritis in murine models has been extensively studied with controversial results. Recent findings point to a non-relevant role for TGF-β in a mice model of collagen-induced arthritis. The study of TGF-β on T-cell responses has shown controversial results as an inhibitor or promoter of the inflammatory response. This paper presents a review of the role of TGF-β in animal models of arthritis. Copyright © 2013 Elsevier España, S.L. All rights reserved.

Generation of stable cell line by using chitosan as gene delivery system.

PubMed

Şalva, Emine; Turan, Suna Özbaş; Ekentok, Ceyda; Akbuğa, Jülide

2016-08-01

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

A Plant-Based Dietary Intervention Improves Beta-Cell Function and Insulin Resistance in Overweight Adults: A 16-Week Randomized Clinical Trial.

PubMed

Kahleova, Hana; Tura, Andrea; Hill, Martin; Holubkov, Richard; Barnard, Neal D

2018-02-09

The aim of this study was to test the effect of a plant-based dietary intervention on beta-cell function in overweight adults with no history of diabetes. Participants ( n = 75) were randomized to follow a low-fat plant-based diet ( n = 38) or to make no diet changes ( n = 37) for 16 weeks. At baseline and 16 weeks, beta-cell function was quantified with a mathematical model. Using a standard meal test, insulin secretory rate was calculated by C-peptide deconvolution. The Homeostasis Model Assessment (HOMA-IR) index was used to assess insulin resistance while fasting. A marked increase in meal-stimulated insulin secretion was observed in the intervention group compared with controls (interaction between group and time, Gxt, p < 0.001). HOMA-IR index fell significantly ( p < 0.001) in the intervention group (treatment effect -1.0 (95% CI, -1.2 to -0.8); Gxt, p = 0.004). Changes in HOMA-IR correlated positively with changes in body mass index (BMI) and visceral fat volume ( r = 0.34; p = 0.009 and r = 0.42; p = 0.001, respectively). The latter remained significant after adjustment for changes in BMI ( r = 0.41; p = 0.002). Changes in glucose-induced insulin secretion correlated negatively with BMI changes ( r = -0.25; p = 0.04), but not with changes in visceral fat. Beta-cell function and insulin sensitivity were significantly improved through a low-fat plant-based diet in overweight adults.

TGF-beta1 stimulates expression of the aromatase (CYP19) gene in human osteoblast-like cells and THP-1 cells.

PubMed

Shozu, M; Zhao, Y; Simpson, E R

2000-02-25

Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-beta1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta1 increased IL-1beta+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-beta1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-beta1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta1. Increase in P450arom expression by TGF-beta1 was attributable to expression driven by promoter I.4. TGF-beta1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (-410/+14 and-340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-beta1. Deletion and mutation of the GRE in P450-I.4/Luc (-340/+14) abolished the TGF-beta1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta1. These results indicate that TGF-beta1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.

Re-engineering Islet Cell Transplantation

PubMed Central

Fotino, Nicoletta; Fotino, Carmen; Pileggi, Antonello

2015-01-01

We are living exciting times in the field of beta cell replacement therapies for the treatment of diabetes. While steady progress has been recorded thus far in clinical islet transplantation, novel approaches are needed to make cell-based therapies more reproducible and leading to long-lasting success. The multiple facets of diabetes impose the need for a transdisciplinary approach to attain this goal, by targeting immunity, promoting engraftment and sustained functional potency. We discuss herein the emerging technologies applied to beta cell replacement therapies. PMID:25814189

Neutrophil chemotaxis in sickle cell anaemia, sickle cell beta zero thalassaemia, and after splenectomy.

PubMed Central

Donadi, E A; Falcão, R P

1987-01-01

Neutrophil chemotaxis was evaluated in 28 patients with sickle cell anaemia, 10 patient with sickle cell beta zero thalassaemia, 25 patients who had undergone splenectomy, and 38 controls. The mean distance migrated by patients' neutrophils was not significantly different from that of neutrophils from controls. Although several immunological variables have been reported to be changed after loss of splenic function, we were unable to show a defect in neutrophil chemotaxis that could account for the increased susceptibility to infection. PMID:3611395

Role of the M3 muscarinic acetylcholine receptor in beta-cell function and glucose homeostasis.

PubMed

Gautam, D; Han, S-J; Duttaroy, A; Mears, D; Hamdan, F F; Li, J H; Cui, Y; Jeon, J; Wess, J

2007-11-01

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

NASA Astrophysics Data System (ADS)

Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A.; Ghosh, Partha Pratim; Mitra, Prasenjit

2016-06-01

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

PubMed Central

Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A; Ghosh, Partha Pratim; Mitra, Prasenjit

2016-01-01

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation. PMID:27282931

Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells.

PubMed

Nagasawa, Kunihiko; Chiba, Hideki; Fujita, Hiroki; Kojima, Takashi; Saito, Tsuyoshi; Endo, Toshiaki; Sawada, Norimasa

2006-07-01

Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed. Copyright 2006 Wiley-Liss, Inc.

Subcellular distribution of delta 5-3 beta-hydroxy steroid dehydrogenase in the granulosa cells of the domestic fowl (Gallus domesticus).

PubMed Central

Armstrong, D G

1979-01-01

1. The distribution of 3 beta-hydroxy steroid dehydrogenase was examined in the subcellular fractions of granulosa cells collected from the ovary of the domestic fowl. 2. 3 beta-hydroxy steroid dehydrogenase activity was observed in the mitochondrial (4000g for 20min) and microsomal (105 000g for 120min) fractions. 3. Approximately three times more 3 beta-hydroxy steroid dehydrogenase activity was associated with the cytochrome oxidase activity (a mitochondrial marker enzyme) in anteovulatory-follicle granulosa cells than with that of the postovulatory follicle. 4. Comparison of the latent properties of mitochondrial 3 beta-hydroxy steroid dehydrogenase with those of cytochrome oxidase and isocitrate dehydrogenase indicated that 3 beta-hydroxy steroid dehydrogenase is located extramitochondrially. 5. This apparent distribution of 3 beta-hydroxy steroid dehydrogenase is explained on the basis that the mitochondrial activity is either an artefact caused by a redistribution in the subcellular location of the enzyme, occurring during homogenization, or by the existence of a functionally heterogeneous endoplasmic reticulum that yields particles of widely differing sedimentation properties. PMID:518548

Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

PubMed Central

Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

2008-01-01

The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792

Cloning and characterization of a DNA polymerase beta gene from Trypanosoma cruzi.

PubMed

Venegas, Juan A; Aslund, Lena; Solari, Aldo

2009-06-01

A gene coding for a DNA polymerase beta from the Trypanosoma cruzi Miranda clone, belonging to the TcI lineage, was cloned (Miranda Tcpol beta), using the information from eight peptides of the T. cruzi beta-like DNA polymerase purified previously. The gene encodes for a protein of 403 amino acids which is very similar to the two T. cruzi CL Brener (TcIIe lineage) sequences published, but has three different residues in highly conserved segments. At the amino acid level, the identity of TcI-pol beta with mitochondrial pol beta and pol beta-PAK from other trypanosomatids was between 68-80% and 22-30%, respectively. Miranda Tc-pol beta protein has an N-terminal sequence similar to that described in the mitochondrial Crithidia fasciculata pol beta, which suggests that the TcI-pol beta plays a role in the organelle. Northern and Western analyses showed that this T. cruzi gene is highly expressed both in proliferative and non-proliferative developmental forms. These results suggest that, in addition to replication of kDNA in proliferative cells, this enzyme may have another function in non-proliferative cells, such as DNA repair role similar to that which has extensively been described in a vast spectrum of eukaryotic cells.

Immune Interventions to Preserve Beta Cell Function in Type 1 Diabetes

PubMed Central

Ehlers, Mario R.

2015-01-01

Type 1 diabetes (T1D) is a chronic autoimmune disease that leads to destruction of pancreatic beta cells, lifelong dependence on insulin, and increased morbidity and mortality from diabetes-related complications. Preservation of residual beta cells at diagnosis is a major goal because higher levels of endogenous insulin secretion are associated with better short- and long-term outcomes. Over the past 3 decades, a variety of immune interventions have been evaluated in the setting of new-onset T1D, including nonspecific immunosuppression, pathway-specific immune modulation, antigen-specific therapies, and cellular therapies. To date, no single intervention has produced durable remission off-therapy in the majority of treated patients, but the field has gained valuable insights into disease mechanisms and potential immunologic correlates of success. In particular, T cell-directed therapies, including therapies that lead to partial depletion or modulation of effector T (Teff) cells and preservation or augmentation of regulatory T (Treg) cells, have shown the most success and will likely form the backbone of future approaches. The next phase will see evaluation of rational combinations, comprising one or more of the following: a Teff-depleting or modulating drug, a cytokine-based tolerogenic (Treg-promoting) agent, and an antigen-specific component. The long-term goal is to reestablish immunologic tolerance to beta cells, thereby preserving residual beta cells early after diagnosis or enabling restoration of beta cell mass from autologous stem cells or induced neogenesis in patients with established T1D. PMID:26225763

Beta-catenin regulates vitamin C biosynthesis and cell survival in murine liver.

PubMed

Nejak-Bowen, Kari N; Zeng, Gang; Tan, Xinping; Cieply, Benjamin; Monga, Satdarshan P

2009-10-09

Because the Wnt/beta-catenin pathway plays multiple roles in liver pathobiology, it is critical to identify gene targets that mediate such diverse effects. Here we report a novel role of beta-catenin in controlling ascorbic acid biosynthesis in murine liver through regulation of expression of regucalcin or senescence marker protein 30 and L-gulonolactone oxidase. Reverse transcription-PCR, Western blotting, and immunohistochemistry demonstrate decreased regucalcin expression in beta-catenin-null livers and greater expression in beta-catenin overexpressing transgenic livers, HepG2 hepatoma cells (contain constitutively active beta-catenin), regenerating livers, and in hepatocellular cancer tissues that exhibit beta-catenin activation. Interestingly, coprecipitation and immunofluorescence studies also demonstrate an association of beta-catenin and regucalcin. Luciferase reporter and chromatin immunoprecipitation assays verified a functional TCF-4-binding site located between -163 and -157 (CTTTGCA) on the regucalcin promoter to be critical for regulation by beta-catenin. Significantly lower serum ascorbate levels were observed in beta-catenin knock-out mice secondary to decreased expression of regucalcin and also of L-gulonolactone oxidase, the penultimate and last (also rate-limiting) steps in the synthesis of ascorbic acid, respectively. These mice also show enhanced basal hepatocyte apoptosis. To test if ascorbate deficiency secondary to beta-catenin loss and regucalcin decrease was contributing to apoptosis, beta-catenin-null hepatocytes or regucalcin small interfering RNA-transfected HepG2 cells were cultured, which exhibited significant apoptosis that was alleviated by the addition of ascorbic acid. Thus, through regucalcin and L-gulonolactone oxidase expression, beta-catenin regulates vitamin C biosynthesis in murine liver, which in turn may be one of the mechanisms contributing to the role of beta-catenin in cell survival.

Overexpression of the VSSC-associated CAM, β-2, enhances LNCaP cell metastasis associated behavior.

PubMed

Jansson, Keith H; Lynch, Jill E; Lepori-Bui, Nadia; Czymmek, Kirk J; Duncan, Randall L; Sikes, Robert A

2012-07-01

Prostate cancer (PCa) is the second-leading cause of cancer death in American men. This is due largely to the "silent" nature of the disease until it has progressed to a highly metastatic and castrate resistant state. Voltage sensitive sodium channels (VSSCs) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two β subunits. The β-subunits modulate surface expression and gating kinetics of the channels but also have inherent cell adhesion molecule (CAM) functions. We hypothesize that PCa cells use VSSC β-subunits as CAMs during PCa progression and metastasis. We overexpressed the beta-2 isoform as a C-terminal fusion protein with enhanced cyan fluorescence protein (ECFP) in the weakly metastatic LNCaP cells. The effect of beta-2 overexpression on cell morphology was examined using confocal microscopy while metastasis-associated behavior was tested by performing several in vitro metastatic functional assays and in vivo subcutaneous tumor studies. We found that cells overexpressing beta-2 (2BECFP) converted to a bipolar fibroblastic morphology. 2BECFP cells were more adhesive than control (ECFP) to vitronectin (twofold) and Matrigel® (1.3-fold), more invasive through Matrigel® (3.6-fold in 72 hr), and had enhanced migration (2.1-fold in 96 hr) independent of proliferation in wound-healing assays. In contrast, 2BECFP cells have a reduced tumor-take and tumor volume in vivo even though the overexpression of beta-2 was maintained. Functional overexpression of VSSC β-subunits in PCa may be one mechanism leading to increased metastatic behavior while decreasing the ability to form localized tumor masses. Copyright © 2011 Wiley Periodicals, Inc.

Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors.

PubMed

Sergeeva, Olga A; Kletke, Olaf; Kragler, Andrea; Poppek, Anja; Fleischer, Wiebke; Schubring, Stephan R; Görg, Boris; Haas, Helmut L; Zhu, Xin-Ran; Lübbert, Hermann; Gisselmann, Günter; Hatt, Hanns

2010-07-30

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

Mediation of glucolipotoxicity in INS-1 rat insulinoma cells by small heterodimer partner interacting leucine zipper protein (SMILE).

PubMed

Lee, Kyeong-Min; Seo, Ye Jin; Kim, Mi-Kyung; Seo, Hyun-Ae; Jeong, Ji-Yun; Choi, Hueng-Sik; Lee, In-Kyu; Park, Keun-Gyu

2012-03-23

Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

Reactive hypoglycemia in lean young women with PCOS and correlations with insulin sensitivity and with beta cell function.

PubMed

Altuntas, Yuksel; Bilir, Muammer; Ucak, Sema; Gundogdu, Sadi

2005-04-01

Reactive hypoglycemia (RH), which is a postprandial hypoglycemic state, occurs within 2-5 h after food intake. It is classified as idiopathic, alimentary, or diabetic reactive hypoglycemia. We studied the incidence of reactive hypoglycemia and looked for any correlations between it and the presence of insulin sensitivity and/or beta cell function in young lean polycystic ovary syndrome (PCOS) patients. This study was designed as a cross-sectional study in 64 lean young women with PCOS (BMI < or = 25 kg/m2). Various indices of insulin sensitivity and beta cell function derived from the oral glucose tolerance test (OGTT) results were used. We found the rate of RH to be 50% in lean young women with PCOS. DHEA-S and PRL levels were found to be lower in subjects with RH (P < 0.05 and P > 0.05, respectively). Beta cell function indices such as the insulinogenic index (at 120 min), CIR (at 120 min) and HOMA beta cell index were found to be insignificantly higher in the RH group than the nonreactive hypoglycemia (NRH) group. The 4 h glucose level, but not the 3 h glucose level, was significantly correlated with insulin resistance indices, such as fasting insulin level, HOMA-IR, Quicky index, and FIRI in the RH group. Significantly decreased DHEA-S levels were an interesting finding. In conclusion, there is an urgent need to investigate RH in lean young women with PCOS. Our results indicate that more definite insulin resistance occurs in subjects with RH in the fourth hour of the OGTT than those with RH in the third hour. In addition, RH in the fourth hour together with a low DHEA-S level may be predictive of future diabetes in young women with PCOS even when they are not obese.

Integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansing, Hope A.; Sarkeshik, Ali; Yates, John R.

2011-03-11

Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomicsmore » screen of proteins recruited to clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.« less

Beta-Cell Replacement: Pancreas and Islet Cell Transplantation.

PubMed

Niclauss, Nadja; Meier, Raphael; Bédat, Benoît; Berishvili, Ekaterine; Berney, Thierry

2016-01-01

Pancreas and islet transplantation are 2 types of beta-cell replacement therapies for type 1 diabetes mellitus. Since 1966, when pancreas transplantation was first performed, it has evolved to become a highly efficient procedure with high success rates, thanks to advances in surgical technique and immunosuppression. Pancreas transplantation is mostly performed as simultaneous pancreas-kidney transplantation in patients with end-stage nephropathy secondary to diabetes. In spite of its efficiency, pancreas transplantation is still a major surgical procedure burdened by high morbidity, which called for the development of less invasive and hazardous ways of replacing beta-cell function in the past. Islet transplantation was developed in the 1970s as a minimally invasive procedure with initially poor outcomes. However, since the report of the 'Edmonton protocol' in 2000, the functional results of islet transplantation have substantially and constantly improved and are about to match those of whole pancreas transplantation. Islet transplantation is primarily performed alone in nonuremic patients with severe hypoglycemia. Both pancreas transplantation and islet transplantation are able to abolish hypoglycemia and to prevent or slow down the development of secondary complications of diabetes. Pancreas transplantation and islet transplantation should be seen as two complementary, rather than competing, therapeutic approaches for beta-cell replacement that are able to optimize organ donor use and patient care. © 2016 S. Karger AG, Basel.

The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus

PubMed Central

Gerber, Philipp A.

2017-01-01

Abstract Significance: Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Critical Issues: Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene–environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Future Directions: Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501–518. PMID:27225690

β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype.

PubMed

Azizi, Zahra; Lange, Claudia; Paroni, Federico; Ardestani, Amin; Meyer, Anke; Wu, Yonghua; Zander, Axel R; Westenfelder, Christof; Maedler, Kathrin

2016-08-02

Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow-derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes.Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment.With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP.After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes.

Pancreas Volume and Fat Deposition in Diabetes and Normal Physiology: Consideration of the Interplay Between Endocrine and Exocrine Pancreas.

PubMed

Saisho, Yoshifumi

2016-01-01

The pancreas is comprised of exocrine and endocrine components. Despite the fact that they are derived from a common origin in utero, these two compartments are often studied individually because of the different roles and functions of the exocrine and endocrine pancreas. Recent studies have shown that not only type 1 diabetes (T1D), but also type 2 diabetes (T2D), is characterized by a deficit in beta-cell mass, suggesting that pathological changes in the pancreas are critical events in the natural history of diabetes. In both patients with T1D and those with T2D, pancreas mass and exocrine function have been reported to be reduced. On the other hand, pancreas volume and pancreatic fat increase with obesity. Increased beta-cell mass with increasing obesity has also been observed in humans, and ectopic fat deposits in the pancreas have been reported to cause beta-cell dysfunction. Moreover, neogenesis and transdifferentiation from the exocrine to the endocrine compartment in the postnatal period are regarded as a source of newly formed beta-cells. These findings suggest that there is important interplay between the endocrine and exocrine pancreas throughout life. This review summarizes the current knowledge on physiological and pathological changes in the exocrine and endocrine pancreas (i.e., beta-cell mass), and discusses the potential mechanisms of the interplay between the two compartments in humans to understand the pathophysiology of diabetes better.

Pancreas Volume and Fat Deposition in Diabetes and Normal Physiology: Consideration of the Interplay Between Endocrine and Exocrine Pancreas

PubMed Central

Saisho, Yoshifumi

2016-01-01

The pancreas is comprised of exocrine and endocrine components. Despite the fact that they are derived from a common origin in utero, these two compartments are often studied individually because of the different roles and functions of the exocrine and endocrine pancreas. Recent studies have shown that not only type 1 diabetes (T1D), but also type 2 diabetes (T2D), is characterized by a deficit in beta-cell mass, suggesting that pathological changes in the pancreas are critical events in the natural history of diabetes. In both patients with T1D and those with T2D, pancreas mass and exocrine function have been reported to be reduced. On the other hand, pancreas volume and pancreatic fat increase with obesity. Increased beta-cell mass with increasing obesity has also been observed in humans, and ectopic fat deposits in the pancreas have been reported to cause beta-cell dysfunction. Moreover, neogenesis and transdifferentiation from the exocrine to the endocrine compartment in the postnatal period are regarded as a source of newly formed beta-cells. These findings suggest that there is important interplay between the endocrine and exocrine pancreas throughout life. This review summarizes the current knowledge on physiological and pathological changes in the exocrine and endocrine pancreas (i.e., beta-cell mass), and discusses the potential mechanisms of the interplay between the two compartments in humans to understand the pathophysiology of diabetes better. PMID:28012279

Regulation of dendritic cell function through Toll-like receptors.

PubMed

Kaisho, Tsuneyasu; Akira, Shizuo

2003-06-01

Higher animals establish host defense by orchestrating innate and adaptive immunity. This is mediated by professional antigen presenting cells, i.e. dendritic cells (DCs). DCs can incorporate pathogens, produce a variety of cytokines, maturate, and present pathogen-derived peptides to T cells, thereby inducing T cell activation and differentiation. These responses are triggered by microbial recognition through type I transmembrane proteins, Toll-like receptors (TLRs) on DCs. TLRs consist of ten members and each TLR is involved in recognizing a variety of microorganism-derived molecular structures. TLR ligands include cell wall components, proteins, nucleic acids, and synthetic chemical compounds, all of which can activate DCs as immune adjuvants. Each TLR can activate DCs in a similar, but distinct manner. For example, TLRs can be divided into subgroups according to their type I interferon (IFN) inducing ability. TLR2 cannot induce IFN-alpha or IFN-beta, but TLR4 can lead to IFN-beta production. Meanwhile, TLR3, TLR7, and TLR9 can induce both IFN-alpha and IFN-beta. Recent evidences suggest that cytoplamic adapters for TLRs are especially crucial for this functional heterogeneity. Clarifying how DC function is regulated by TLRs should provide us with critical information for manipulating the host defense against a variety of diseases.

Beta-Adrenergic Receptor Population is Up-Regulated in Chicken Skeletal Muscle Cells Treated with Forskolin

NASA Technical Reports Server (NTRS)

Bridge, K. Y.; Young, R. B.; Vaughn, J. R.

1998-01-01

Skeletal muscle hypertrophy is promoted by in vivo administration of beta-adrenergic receptor (betaAR) agonists. These compounds presumably exert their physiological action through the betaAR, and alterations in the population of betaAR could potentially change the ability of the cell to respond to the betaAR agonists. Since the intracellular chemical signal generated by the betaAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of functional betaAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 microM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the betaAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 microM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in betaAR population, with a maximum increase of approximately 50% at 10 microM. This increase in PAR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of betaAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 microM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

Dual role of Ski in pancreatic cancer cells: tumor-promoting versus metastasis-suppressive function.

PubMed

Wang, Peng; Chen, Zhen; Meng, Zhi-Qiang; Fan, Jie; Luo, Jian-Min; Liang, Wang; Lin, Jun-Hua; Zhou, Zhen-Hua; Chen, Hao; Wang, Kun; Shen, Ye-Hua; Xu, Zu-De; Liu, Lu-Ming

2009-09-01

Ski used to be defined as an oncogene that contributes to the resistance of tumor cells to transforming growth factor-beta (TGF-beta)-induced growth arrest. As TGF-beta has a dual effect on tumor growth with both tumor-suppressing and -promoting activity depending on the stage of carcinogenesis and the cell type, the precise role of Ski in carcinogenesis remains unclear. In this study, we show that downregulation of Ski through lentivirus-mediated RNA interference decreases tumor growth both in vitro and in vivo, yet promotes cell invasiveness in vitro, and lung metastasis in vivo in the pancreatic cancer cell line SW1990, which contain wild-type Smad4 expression, and the BxPC3 cell line, which is Smad4 deficient. We also show that the downregulation of Ski increases TGF-beta-induced transcriptional activity, which is associated with increased TGF-beta-dependent Smad2/3 phosphorylation, and results in an altered expression profile of TGF-beta-inducible genes involved in metastasis, angiogenesis and cell proliferation and epithelial-mesenchymal transition. Immunohistochemical analysis of specimens from 71 patients with pancreatic adenocarcinoma showed a significant association between overexpression of Ski and decreased patient survival time (P = 0.0024). Our results suggest that Ski may act as a tumor proliferation-promoting factor or as a metastatic suppressor in human pancreatic cancer.

CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells.

PubMed

Baughn, Linda B; Di Liberto, Maurizio; Niesvizky, Ruben; Cho, Hearn J; Jayabalan, David; Lane, Joseph; Liu, Fang; Chen-Kiang, Selina

2009-02-15

Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.

Neurotensin receptor 1 gene activation by the Tcf/beta-catenin pathway is an early event in human colonic adenomas.

PubMed

Souazé, Frédérique; Viardot-Foucault, Véronique; Roullet, Nicolas; Toy-Miou-Leong, Mireille; Gompel, Anne; Bruyneel, Erik; Comperat, Eva; Faux, Maree C; Mareel, Marc; Rostène, William; Fléjou, Jean-François; Gespach, Christian; Forgez, Patricia

2006-04-01

Alterations in the Wnt/APC (adenomatous polyposis coli) signalling pathway, resulting in beta-catenin/T cell factor (Tcf)-dependent transcriptional gene activation, are frequently detected in familial and sporadic colon cancers. The neuropeptide neurotensin (NT) is widely distributed in the gastrointestinal tract. Its proliferative and survival effects are mediated by a G-protein coupled receptor, the NT1 receptor. NT1 receptor is not expressed in normal colon epithelial cells, but is over expressed in a number of cancer cells and tissues suggesting a link to the outgrowth of human colon cancer. Our results demonstrate that the upregulation of NT1 receptor occurring in colon cancer is the result of Wnt/APC signalling pathway activation. We first established the functionality of the Tcf response element within the NT1 receptor promoter. Consequently, we observed the activation of NT1 receptor gene by agents causing beta-catenin cytosolic accumulation, as well as a strong decline of endogenous receptor when wt-APC was restored. At the cellular level, the re-establishment of wt-APC phenotype resulted in the impaired functionality of NT1 receptor, like the breakdown in NT-induced intracellular calcium mobilization and the loss of NT pro-invasive effect. We corroborated the Wnt/APC signalling pathway on the NT1 receptor promoter activation with human colon carcinogenesis, and showed that NT1 receptor gene activation was perfectly correlated with nuclear or cytoplasmic beta-catenin localization while NT1 receptor was absent when beta-catenin was localized at the cell-cell junction in early adenomas of patients with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer and loss of heterozygosity tumours. In this report we establish a novel link in vitro between the Tcf/beta-catenin pathway and NT1 receptor promoter activation.

Functional cloning of the proto-oncogene brain factor-1 (BF-1) as a Smad-binding antagonist of transforming growth factor-beta signaling.

PubMed

Rodriguez, C; Huang, L J; Son, J K; McKee, A; Xiao, Z; Lodish, H F

2001-08-10

Using the plasminogen activator inhibitor (PAI) promoter to drive the expression of a reporter gene (mouse CD2), we devised a system to clone negative regulators of the transforming growth factor-beta (TGF-beta) signaling pathway. We infected a TGF-beta-responsive cell line (MvLu1) with a retroviral cDNA library, selecting by fluorescence-activated cell sorter single cells displaying low PAI promoter activity in response to TGF-beta. Using this strategy we cloned the proto-oncogene brain factor-1 (BF-1). BF-1 represses the PAI promoter in part by associating with both unphosphorylated Smad3 (in the cytoplasm) and phosphorylated Smad3 (in the nucleus), thus preventing its binding to DNA. BF-1 also associates with Smad1, -2, and -4; the Smad MH2 domain binds to BF-1, and the C-terminal segment of BF-1 is uniquely and solely required for binding to Smads. Further, BF-1 represses another TGF-beta-induced promoter (p15), it up-regulates a TGF-beta-repressed promoter (Cyclin A), and it reverses the growth arrest caused by TGF-beta. Our results suggest that BF-1 is a general inhibitor of TGF-beta signaling and as such may play a key role during brain development.

CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

NASA Technical Reports Server (NTRS)

Sharina, Iraida G.; Martin, Emil; Thomas, Anthony; Uray, Karen L.; Murad, Ferid

2003-01-01

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

Incretin Receptor Null Mice Reveal Key Role of GLP-1 but Not GIP in Pancreatic Beta Cell Adaptation to Pregnancy

PubMed Central

Moffett, R. Charlotte; Vasu, Srividya; Thorens, Bernard; Drucker, Daniel J.; Flatt, Peter R.

2014-01-01

Islet adaptations to pregnancy were explored in C57BL6/J mice lacking functional receptors for glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). Pregnant wild type mice and GIPRKO mice exhibited marked increases in islet and beta cell area, numbers of medium/large sized islets, with positive effects on Ki67/Tunel ratio favouring beta cell growth and enhanced pancreatic insulin content. Alpha cell area and glucagon content were unchanged but prohormone convertases PC2 and PC1/3 together with significant amounts of GLP-1 and GIP were detected in alpha cells. Knockout of GLP-1R abolished these islet adaptations and paradoxically decreased pancreatic insulin, GLP-1 and GIP. This was associated with abolition of normal pregnancy-induced increases in plasma GIP, L-cell numbers, and intestinal GIP and GLP-1 stores. These data indicate that GLP-1 but not GIP is a key mediator of beta cell mass expansion and related adaptations in pregnancy, triggered in part by generation of intra-islet GLP-1. PMID:24927416

Complementary deoxyribonucleic acid cloning of spermatogonial stem cell renewal factor.

PubMed

Miura, Takeshi; Ohta, Takashi; Miura, Chiemi I; Yamauchi, Kohei

2003-12-01

Spermatogonial mitosis can be subdivided into two processes: spermatogonial stem cell renewal and spermatogonial proliferation toward meiosis. Recently it has been indicated that estrogen, estradiol-17beta, is involved in regulating the renewal of spermatogonial stem cells in eel. To determine the genes that directly regulate this process, we used expression screening to identify genes whose expression is regulated by estradiol-17beta in testes. We detected a previously unidentified cDNA clone that is up-regulated by estradiol-17beta stimulation and named it eel spermatogenesis-related substances 34 (eSRS34) cDNA. Homology searching showed that eSRS34 shares amino acid sequence similarity with human platelet-derived endothelial cell growth factor. We examined the function of eSRS34 using several in vitro systems. Recombinant eSRS34 produced by a baculovirus system induced spermatogonial mitosis in testicular organ culture. Furthermore, the addition of an antibody specific for eSRS34 prevented spermatogonial mitosis induced by estradiol-17beta stimulation in a germ cell/somatic cell coculture system. We therefore conclude that eSRS34 is a "spermatogonial stem cell renewal factor."

Opposing actions of Arx and Pax4 in endocrine pancreas development.

PubMed

Collombat, Patrick; Mansouri, Ahmed; Hecksher-Sorensen, Jacob; Serup, Palle; Krull, Jens; Gradwohl, Gerard; Gruss, Peter

2003-10-15

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in alpha, beta-precursor, and delta cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine alpha cells with a concomitant increase in beta-and delta-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for alpha-cell fate acquisition and a repressive action on beta-and delta-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

The low density lipoprotein receptor-related protein 1B retains beta-amyloid precursor protein at the cell surface and reduces amyloid-beta peptide production.

PubMed

Cam, Judy A; Zerbinatti, Celina V; Knisely, Jane M; Hecimovic, Silva; Li, Yonghe; Bu, Guojun

2004-07-09

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family that shares high homology with the LDL receptor-related protein (LRP). LRP1B was originally described as a putative tumor suppressor in lung cancer cells; however, its expression profile in several regions of adult human brain suggests it may have additional functions in the central nervous system. Since LRP1B has overlapping ligand binding properties with LRP, we investigated whether LRP1B, like LRP, could interact with the beta-amyloid precursor protein (APP) and modulate its processing to amyloid-beta peptides (Abetas). Using an LRP1B minireceptor (mLRP1B4) generated to study the trafficking of LRP1B, we found that mLRP1B4 and APP form an immunoprecipitable complex. Furthermore mLRP1B4 bound and facilitated the degradation of a soluble isoform of APP containing a Kunitz proteinase inhibitor domain but not soluble APP lacking a Kunitz proteinase inhibitor domain. A functional consequence of mLRP1B4 expression was a significant accumulation of APP at the cell surface, which is likely related to the slow endocytosis rate of LRP1B. More importantly, mLRP1B4-expressing cells that accumulated cell surface APP produced less Abeta and secreted more soluble APP. These findings reveal that LRP1B is a novel binding partner of APP that functions to decrease APP processing to Abeta. Consequently LRP1B expression could function to protect against the pathogenesis of Alzheimer's disease.

The role of oestrogens in the adaptation of islets to insulin resistance.

PubMed

Nadal, Angel; Alonso-Magdalena, Paloma; Soriano, Sergi; Ropero, Ana B; Quesada, Ivan

2009-11-01

Pregnancy is characterized by peripheral insulin resistance, which is developed in parallel with a plasma increase of maternal hormones; these include prolactin, placental lactogens, progesterone and oestradiol among others. Maternal insulin resistance is counteracted by the adaptation of the islets of Langerhans to the higher insulin demand. If this adjustment is not produced, gestational diabetes may be developed. The adaptation process of islets is characterized by an increase of insulin biosynthesis, an enhanced glucose-stimulated insulin secretion (GSIS) and an increase of beta-cell mass. It is not completely understood why, in some individuals, beta-cell mass and function fail to adapt to the metabolic demands of pregnancy, yet a disruption of the beta-cell response to maternal hormones may play a key part. The role of the maternal hormone 17beta-oestradiol (E2) in this adaptation process has been largely unknown. However, in recent years, it has been demonstrated that E2 acts directly on beta-cells to increase insulin biosynthesis and to enhance GSIS through different molecular mechanisms. E2 does not increase beta-cell proliferation but it is involved in beta-cell survival. Classical oestrogen receptors ERalpha and ERbeta, as well as the G protein-coupled oestrogen receptor (GPER) seem to be involved in these adaptation changes. In addition, as the main production of E2 in post-menopausal women comes from the adipose tissue, E2 may act as a messenger between adipocytes and islets in obesity.

Opposite actions of transforming growth factor-beta 1 on the gene expression of atrial natriuretic peptide biological and clearance receptors in a murine thymic stromal cell line.

PubMed

Agui, T; Xin, X; Cai, Y; Shim, G; Muramatsu, Y; Yamada, T; Fujiwara, H; Matsumoto, K

1995-09-01

The regulation of the gene expression of the atrial natriuretic peptide receptor (ANPR) subtypes, ANPR-A, ANPR-B, and ANPR-C, was investigated in a murine thymic stromal cell line, MRL 104.8a. When MRL 104.8a cells were cultured with transforming growth factor (TGF)-beta1, [125I]ANP binding sites increased with increasing dose of TGF-beta1. These binding sites were identified as ANPR-C by a displacement experiment with ANPR-C-specific ligand, C-ANF, and by the affinity cross-linking of the [125I]ANP binding sites with a chemical cross-linker to determine the molecular weight of the ANPR. This augmentation of the ANPR-C expression was elucidated to occur at the transcriptional level by Northern blot experiment, comparison of the relative amounts of mRNA by reverse transcription (RT)-PCR, and in vitro nuclear transcription assay. Conversely, the expression of the ANP biological receptors, ANPR-A and ANPR-B, was shown to be down-regulated by TGF-beta1. These data suggest that TGF-beta1 regulates the gene expression of ANPRs in the thymic stromal cells and that ANP and TGF-beta1 might affect the thymic stromal cell functions.

Rhesus lymphocryptovirus latent membrane protein 2A activates {beta}-catenin signaling and inhibits differentiation in epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siler, Catherine A.; Raab-Traub, Nancy; Lineberger Comprehensive Cancer Center, CB7295, University of North Carolina-Chapel Hill, 450 West Drive, Chapel Hill, NC 27599-7295

2008-08-01

Rhesus lymphocryptovirus (LCV) is a {gamma}-herpesvirus closely related to Epstein-Barr virus (EBV). The rhesus latent membrane protein 2A (LMP2A) is highly homologous to EBV LMP2A. EBV LMP2A activates the phosphatidylinositol 3-kinase (PI3K) and {beta}-catenin signaling pathways in epithelial cells and affects differentiation. In the present study, the biochemical and biological properties of rhesus LMP2A in epithelial cells were investigated. The expression of rhesus LMP2A in epithelial cells induced Akt activation, GSK3{beta} inactivation and accumulation of {beta}-catenin in the cytoplasm and nucleus. The nuclear translocation, but not accumulation of {beta}-catenin was dependent on Akt activation. Rhesus LMP2A also impaired epithelial cellmore » differentiation; however, this process was not dependent upon Akt activation. A mutant rhesus LMP2A lacking six transmembrane domains functioned similarly to wild-type rhesus LMP2A indicating that the full number of transmembrane domains is not required for effects on {beta}-catenin or cell differentiation. These results underscore the similarity of LCV to EBV and the suitability of the macaque as an animal model for studying EBV pathogenesis.« less

Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matzuk, M.M.; Krieger, M.; Corless, C.L.

1987-09-01

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ..cap alpha.. subunit but differ in their hormone-specific ..beta..-subunits. The ..beta.. subunit of hCG (hCG..beta..) is unique among the ..beta.. subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCG..beta.. gene alone or together with the hCG..cap alpha.. gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results revealmore » that hCG..beta.. can be secreted normally in the absence of its O-linked oligosaccharides. hCG..beta.. devoid of O-linked carbohydrate can also combine efficiently with hCG..cap alpha.. and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCG..beta.. O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.« less

A synergistic role for IL-1beta and TNFalpha in monocyte-derived IFNgamma inducing activity.

PubMed

Raices, Raquel M; Kannan, Yashaswini; Sarkar, Anasuya; Bellamkonda-Athmaram, Vedavathi; Wewers, Mark D

2008-11-01

Although much is known about classic IFNgamma inducers, little is known about the IFNgamma inducing capability of inflammasome-activated monocytes. In this study, supernatants from LPS/ATP-stimulated human monocytes were analyzed for their ability to induce IFNgamma production by KG-1 cells. Unexpectedly, monocyte-derived IFN inducing activity was detected, but it was completely inhibited by IL-1beta, not IL-18 blockade. Moreover, size-fractionation of the monocyte conditioned media dramatically reduced the IFNgamma inducing activity of IL-1beta, suggesting that IL-1beta requires a cofactor to induce IFNgamma production in KG-1 cells. Because TNFalpha is known to synergize with IL-1beta for various gene products, it was studied as the putative IL-1beta synergizing factor. Although recombinant TNFalpha (rTNFalpha) alone had no IFNgamma inducing activity, neutralization of TNFalpha in the monocyte conditioned media inhibited the IFNgamma inducing activity. Furthermore, rTNFalpha restored the IFNgamma inducing activity of the size-fractionated IL-1beta. Finally, rTNFalpha synergized with rIL-1beta, as well as with rIL-1alpha and rIL-18, for KG-1 IFNgamma release. These studies demonstrate a synergistic role between TNFalpha and IL-1 family members in the induction of IFNgamma production and give caution to interpretations of KG-1 functional assays designed to detect functional IL-18.

Deletion of CXCR4 in cardiomyocytes exacerbates cardiac dysfunction following isoproterenol administration

PubMed Central

Wang, ER; Jarrah, AA; Benard, L; Chen, J; Schwarzkopf, M; Hadri, L; Tarzami, ST

2014-01-01

Altered alpha- and beta-adrenergic receptor signaling is associated with cardiac hypertrophy and failure. Stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXCR4 have been reported to mediate cardioprotection after injury through the mobilization of stem cells into injured tissue. However, little is known regarding whether SDF-1/CXCR4 induces acute protection following pathological hypertrophy and if so, by what molecular mechanism. We have previously reported that CXCR4 physically interacts with the beta-2 adrenergic receptor and modulates its down stream signaling. Here we have shown that CXCR4 expression prevents beta-adrenergic receptor induced hypertrophy. Cardiac beta-adrenergic receptors were stimulated with the implantation of a subcutaneous osmotic pump administrating isoproterenol and CXCR4 expression was selectively abrogated in cardiomyocytes using Cre-loxP-mediated gene recombination. CXCR4 knockout mice showed worsened fractional shortening and ejection fraction. CXCR4 ablation increased susceptibility to isoproterenol-induced heart failure, by upregulating apoptotic markers and reducing mitochondrial function; cardiac function decreases while fibrosis increases. Additionally, CXCR4 expression was rescued with the use of cardiotropic Adeno-associated viral-9 (AAV9) vectors. CXCR4 gene transfer reduced cardiac apoptotic signaling, improved mitochondrial function and resulted in a recovered cardiac function. Our results represent the first evidence that SDF-1/CXCR4 signaling mediates acute cardioprotection through modulating beta-adrenergic receptor signaling in vivo. PMID:24646609

Expression of the alpha and beta subunits of Ca2+/calmodulin kinase II in the cerebellum of jaundiced Gunn rats during development: a quantitative light microscopic analysis.

PubMed

Conlee, J W; Shapiro, S M; Churn, S B

2000-04-01

The homozygous (jj) jaundiced Gunn rat model for hyperbilirubinemia displays pronounced cerebellar hypoplasia. To examine the cellular mechanisms involved in bilirubin toxicity, this study focused on the effect of hyperbilirubinemia on calcium/calmodulin-dependent kinase II (CaM kinase II). CaM kinase II is a neuronally enriched enzyme which performs several important functions. Immunohistochemical analysis of alternating serial sections were performed using monoclonal antibodies for the alpha and beta subunits of CaM kinase II. Measurements were made of the total numbers of stained cells in each of the deep cerebellar nuclei and of Purkinje and granule cell densities in cerebellar lobules II, VI, and IX. The beta subunit was present in Purkinje cells and deep cerebellar nuclei of both groups at all ages, but only granule cells which had migrated through the Purkinje cell layer showed staining for beta subunit; external granule cells were completely negative. Many Purkinje cells had degenerated in the older animals, and the percent of granule cells stained for beta subunit was significantly reduced. The alpha subunit was found exclusively in Purkinje cells, although its appearance was delayed in the jaundiced animals. Sulfadimethoxine was administered to some jj rats 24 h or 15 days prior to sacrifice to increase brain bilirubin concentration. Results showed that bilirubin exposure modulated both alpha and beta CaM kinase II subunit expression in selective neuronal populations, but sulfadimethoxine had no acute effect on enzyme immunoreactivity. Thus, developmental expression of the alpha and beta subunits of CaM kinase II was affected by chronic bilirubin exposure during early postnatal development of jaundiced Gunn rats.

Transforming growth factor-beta in the chicken fundal layers: an immunohistochemical study.

PubMed

Mathis, Ute; Schaeffel, Frank

2010-06-01

In the chicken model of myopia, it has first been shown that imposing defocus to the retina results in active remodelling of the sclera which, in turn, results in axial length changes of the eye. Transforming growth factor-beta (TGF-beta) is one of the scleral growth modulators but its cellular localization in the fundal layers, colocalization and function are not well known. The aim of the current study was to investigate the cellular distribution of the three isoforms TGF-beta1, 2 and 3 by immunohistochemical labelling. Furthermore, the effects of visual experience that induces refractive errors on TGF-beta2 labelling were examined. Transversal cryostat sections of the fundal layers were analyzed by indirect immunofluorescent labelling and cell counts. Visual experience was changed by having the chicks wear either diffusers, or positive or negative lenses of 7D power in front of the right eyes for various periods of time. Left eyes served as uncovered controls. All TGF-beta isoforms were localized in both scleral layers. In choroid, diffuse labelling of all isoforms was found. In retina, TGF-beta1 and 3 were detected in bipolar, amacrine and ganglion cells and TGF-beta2 in amacrine and ganglion cells. To further characterize these cells, double-labelling with known amacrine and bipolar cell markers was performed (calbindin, cellular retinoic acid binding protein (CRABP), Islet1, Lim3 and protein kinase C (PKC)). TGF-beta1, 2 and 3 could be colocalized with calbindin and CRABP in single amacrine cells. TGF-beta1-positive bipolar cells were immunoreactive to Lim3. TGF-beta1 and 3 were never colocalized with PKC in bipolar cells. Also, colocalization with peptides known to be involved in myopia development in chicks, such as glucagon, or vasointestinal polypeptide and the key enzyme for dopamine synthesis, tyrosine hydroxylase, was not observed. Lenses or diffusers, worn by the chicks for various periods of time, had no effect on TGF-beta2 immunoreactivity in choroid or sclera, or on the number of TGF-beta2 (active and latent form) expressing amacrine cells. This result did not change when the two identified populations of TGF-beta2 expressing amacrine cells (one calbindin-positive and the other CRABP-positive) were separately considered. Also no modulation was seen in choroid, although an earlier study had found changes in TGF-beta2 mRNA after lens treatment. The lack of any visually-induced changes in retina or choroid suggests that TGF-beta may not represent a key molecule in the retino-choroidal signalling cascade although it has previously been shown to have a primary role in scleral remodelling. Copyright 2010 Elsevier Ltd. All rights reserved.

Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin.

PubMed

Parekh, T; Saxena, B; Reibman, J; Cronstein, B N; Gold, L I

1994-03-01

TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimately retarding cellular migration and modulating the chemotactic response.

Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and {beta}4 integrin function in MDA-MB-231 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, W.-J.; Chen, W.-K.; Wang, C.-J.

2008-01-15

Hepatocyte growth factor (HGF) and its receptor, Met, known to control invasive growth program have recently been shown to play crucial roles in the survival of breast cancer patients. The diet-derived flavonoids have been reported to possess anti-invasion properties; however, knowledge on the pharmacological and molecular mechanisms in suppressing HGF/Met-mediated tumor invasion and metastasis is poorly understood. In our preliminary study, we use HGF as an invasive inducer to investigate the effect of flavonoids including apigenin, naringenin, genistein and kaempferol on HGF-dependent invasive growth of MDA-MB-231 human breast cancer cells. Results show that apigenin presents the most potent anti-migration andmore » anti-invasion properties by Boyden chamber assay. Furthermore, apigenin represses the HGF-induced cell motility and scattering and inhibits the HGF-promoted cell migration and invasion in a dose-dependent manner. The effect of apigenin on HGF-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis, it shows that apigenin blocks the HGF-induced Akt phosphorylation but not Met, ERK, and JNK phosphorylation. In addition to MDA-MB-231 cells, apigenin exhibits inhibitory effect on HGF-induced Akt phosphorylation in hepatoma SK-Hep1 cells and lung carcinoma A549 cells. By indirect immunofluorescence microscopy assay, apigenin inhibits the HGF-induced clustering of {beta}4 integrin at actin-rich adhesive site and lamellipodia through PI3K-dependent manner. Treatment of apigenin inhibited HGF-stimulated integrin {beta}4 function including cell-matrix adhesion and cell-endothelial cells adhesion in MDA-MB-231 cells. By Akt-siRNA transfection analysis, it confirmed that apigenin inhibited HGF-promoted invasive growth involving blocking PI3K/Akt pathway. Finally, we evaluated the effect of apigenin on HGF-promoted metastasis by lung colonization of tumor cells in nude mice and organ metastasis of tumor cells in chick embryo. By histological and gross examination of mouse lung and real-time PCR analysis of human alu in host tissues, it showed that apigenin, wortmannin, as well as anti-{beta}4 antibody all inhibit HGF-promoted metastasis. These data support the inhibitory effect of apigenin on HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and integrin {beta}4 function.« less

IKKβ inhibition prevents fat-induced beta cell dysfunction in vitro and in vivo in rodents.

PubMed

Ivovic, Aleksandar; Oprescu, Andrei I; Koulajian, Khajag; Mori, Yusaku; Eversley, Judith A; Zhang, Liling; Nino-Fong, Rodolfo; Lewis, Gary F; Donath, Marc Y; Karin, Michael; Wheeler, Michael B; Ehses, Jan; Volchuk, Allen; Chan, Catherine B; Giacca, Adria

2017-10-01

We have previously shown that oxidative stress plays a causal role in beta cell dysfunction induced by fat. Here, we address whether the proinflammatory kinase inhibitor of (nuclear factor) κB kinase β (IKKβ), which is activated by oxidative stress, is also implicated. Fat (oleate or olive oil) was infused intravenously in Wistar rats for 48 h with or without the IKKβ inhibitor salicylate. Thereafter, beta cell function was evaluated in vivo using hyperglycaemic clamps or ex vivo in islets isolated from fat-treated rats. We also exposed rat islets to oleate in culture, with or without salicylate and 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS, another inhibitor of IKKβ) and evaluated beta cell function in vitro. Furthermore, oleate was infused in mice treated with BMS and in beta cell-specific Ikkb-null mice. 48 h infusion of fat impaired beta-cell function in vivo, assessed using the disposition index (DI), in rats (saline: 1.41 ± 0.13; oleate: 0.95 ± 0.11; olive oil [OLO]: 0.87 ± 0.15; p < 0.01 for both fats vs saline) and in mice (saline: 2.51 ± 0.39; oleate: 1.20 ± 0.19; p < 0.01 vs saline) and ex vivo (i.e., insulin secretion, units are pmol insulin islet -1  h -1 ) in rat islets (saline: 1.51 ± 0.13; oleate: 1.03 ± 0.10; OLO: 0.91 ± 0.13; p < 0.001 for both fats vs saline) and the dysfunction was prevented by co-infusion of salicylate in rats (oleate + salicylate: 1.30 ± 0.09; OLO + salicylate: 1.33 ± 0.23) or BMS in mice (oleate + BMS: 2.25 ± 0.42) in vivo and by salicylate in rat islets ex vivo (oleate + salicylate: 1.74 ± 0.31; OLO + salicylate: 1.54 ± 0.29). In cultured islets, 48 h exposure to oleate impaired beta-cell function ([in pmol insulin islet -1  h -1 ] control: 0.66 ± 0.12; oleate: 0.23 ± 0.03; p < 0.01 vs saline), an effect prevented by both inhibitors (oleate + salicylate: 0.98 ± 0.08; oleate + BMS: 0.50 ± 0.02). Genetic inhibition of IKKβ also prevented fat-induced beta-cell dysfunction ex vivo ([in pmol insulin islet -1  h -1 ] control saline: 0.16 ± 0.02; control oleate: 0.10 ± 0.02; knockout oleate: 0.17 ± 0.04; p < 0.05 control saline vs. control oleate) and in vivo (DI: control saline: 3.86 ± 0.40; control oleate: 1.95 ± 0.29; knockout oleate: 2.96 ± 0.24; p < 0.01 control saline vs control oleate). Our results demonstrate a causal role for IKKβ in fat-induced beta cell dysfunction in vitro, ex vivo and in vivo.

Peroxisome proliferator-activated receptor gamma and transforming growth factor-beta pathways inhibit intestinal epithelial cell growth by regulating levels of TSC-22.

PubMed

Gupta, Rajnish A; Sarraf, Pasha; Brockman, Jeffrey A; Shappell, Scott B; Raftery, Laurel A; Willson, Timothy M; DuBois, Raymond N

2003-02-28

Peroxisome proliferator-activated receptor gamma (PPARgamma) and transforming growth factor-beta (TGF-beta) are key regulators of epithelial cell biology. However, the molecular mechanisms by which either pathway induces growth inhibition and differentiation are incompletely understood. We have identified transforming growth factor-simulated clone-22 (TSC-22) as a target gene of both pathways in intestinal epithelial cells. TSC-22 is member of a family of leucine zipper containing transcription factors with repressor activity. Although little is known regarding its function in mammals, the Drosophila homolog of TSC-22, bunched, plays an essential role in fly development. The ability of PPARgamma to induce TSC-22 was not dependent on an intact TGF-beta1 signaling pathway and was specific for the gamma isoform. Localization studies revealed that TSC-22 mRNA is enriched in the postmitotic epithelial compartment of the normal human colon. Cells transfected with wild-type TSC-22 exhibited reduced growth rates and increased levels of p21 compared with vector-transfected cells. Furthermore, transfection with a dominant negative TSC-22 in which both repressor domains were deleted was able to reverse the p21 induction and growth inhibition caused by activation of either the PPARgamma or TGF-beta pathways. These results place TSC-22 as an important downstream component of PPARgamma and TGF-beta signaling during intestinal epithelial cell differentiation.

DNase I hypersensitivity and epsilon-globin transcriptional enhancement are separable in locus control region (LCR) HS1 mutant human beta-globin YAC transgenic mice.

PubMed

Shimotsuma, Motoshi; Okamura, Eiichi; Matsuzaki, Hitomi; Fukamizu, Akiyoshi; Tanimoto, Keiji

2010-05-07

Expression of the five beta-like globin genes (epsilon, Ggamma, Agamma, delta, beta) in the human beta-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5'HS1 through 5'HS5). We report here a novel enhancer activity in 5'HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5'HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human beta-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, epsilon-, but not gamma-globin, gene expression in primitive erythroid cells was severely attenuated, while adult beta-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5'HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5'HS1 is critical for efficient epsilon-globin gene expression, hypersensitive site formation per se is independent of 5'HS1 function, if any, in definitive erythroid cells.

High fat programming of beta cell compensation, exhaustion, death and dysfunction.

PubMed

Cerf, Marlon E

2015-03-01

Programming refers to events during critical developmental windows that shape progeny health outcomes. Fetal programming refers to the effects of intrauterine (in utero) events. Lactational programming refers to the effects of events during suckling (weaning). Developmental programming refers to the effects of events during both fetal and lactational life. Postnatal programming refers to the effects of events either from birth (lactational life) to adolescence or from weaning (end of lactation) to adolescence. Islets are most plastic during the early life course; hence programming during fetal and lactational life is most potent. High fat (HF) programming is the maintenance on a HF diet (HFD) during critical developmental life stages that alters progeny metabolism and physiology. HF programming induces variable diabetogenic phenotypes dependent on the timing and duration of the dietary insult. Maternal obesity reinforces HF programming effects in progeny. HF programming, through acute hyperglycemia, initiates beta cell compensation. However, HF programming eventually leads to chronic hyperglycemia that triggers beta cell exhaustion, death and dysfunction. In HF programming, beta cell dysfunction often co-presents with insulin resistance. Balanced, healthy nutrition during developmental windows is critical for preserving beta cell structure and function. Thus early positive nutritional interventions that coincide with the development of beta cells may reduce the overwhelming burden of diabetes and metabolic disease. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Expression of beta-dystroglycan is reduced or absent in many human carcinomas.

PubMed

Cross, S S; Lippitt, J; Mitchell, A; Hollingsbury, F; Balasubramanian, S P; Reed, M W R; Eaton, C; Catto, J W; Hamdy, F; Winder, S J

2008-11-01

Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

PubMed

Hindié, M; Vayssade, M; Dufresne, M; Quéant, S; Warocquier-Clérout, R; Legeay, G; Vigneron, P; Olivier, V; Duval, J-L; Nagel, M-D

2006-09-01

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Difference between beta1-adrenoceptor autoantibodies of human and animal origin-Limitations detecting beta1-adrenoceptor autoantibodies using peptide based ELISA technology.

PubMed

Wenzel, Katrin; Schulze-Rothe, Sarah; Müller, Johannes; Wallukat, Gerd; Haberland, Annekathrin

2018-01-01

Cell-based analytics for the detection of the beta1-adrenoceptor autoantibody (beta1-AAB) are functional, yet difficult to handle, and should be replaced by easily applicable, routine lab methods. Endeavors to develop solid-phase-based assays such as ELISA to exploit epitope moieties for trapping autoantibodies are ongoing. These solid-phase-based assays, however, are often unreliable when used with human patient material, in contrast to animal derived autoantibodies. We therefore tested an immunogen peptide-based ELISA for the detection of beta1-AAB, and compared commercially available goat antibodies against the 2nd extracellular loop of human beta1-adrenoceptor (ADRB1-AB) to autoantibodies enriched from patient material. The functionality of these autoantibodies was tested in a cell based assay for comparison and their structural appearance was investigated using 2D gel electrophoresis. The ELISA showed a limit of detection for ADRB1-AB of about 1.5 nmol antibody/L when spiked in human control serum and only about 25 nmol/L when spiked in species identical (goat) matrix material. When applied to samples of human origin, the ELISA failed to identify the specific beta1-AABs. A low concentration of beta1-AAB, together with structural inconsistency of the patient originated samples as seen from the 2D Gel appearance, might contribute to the failure of the peptide based ELISA technology to detect human beta1-AABs.

Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

PubMed Central

Geib, Sandrine; Sandoz, Guillaume; Mabrouk, Kamel; Matavel, Alessandra; Marchot, Pascale; Hoshi, Toshinori; Villaz, Michel; Ronjat, Michel; Miquelis, Raymond; Lévêque, Christian; de Waard, Michel

2002-01-01

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit. PMID:11988102

Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Eun Hee; Gorman, Amanda A.; Singh, Puja

2015-12-04

HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that ismore » impaired in the HNF1α mutation-driven diabetes. - Highlights: • We identified AES as a transcriptional repressor for HNF1α in pancreatic beta-cell. • AES's repressive activity was HNF1α-specific and was not observed with HNF1β. • AES interacts with the transactivation domain of HNF1α. • Small molecules can be designed or discovered to disrupt this interaction and improve insulin secretion and glucose homeostasis.« less

Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis.

PubMed

Neef, Markus; Ledermann, Monika; Saegesser, Hans; Schneider, Vreni; Reichen, Juerg

2006-12-01

Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis. Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5 mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-alpha1, transforming growth factor-beta1 (TGF-beta1) and beta2 was quantified by RT-PCR. Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-beta1 mRNA was downregulated in TAA, TGF-beta2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats. Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.

The Synthesis of N-Acetyllactosamine Functionalized Dendrimers, and the Functionalization of Silica Surfaces Using Tunable Dendrons and beta-Cyclodextrins

NASA Astrophysics Data System (ADS)

Ennist, Jessica Helen

Galectin-3 is beta-galactoside binding protein which is found in many healthy cells. In cancer, the galectin-3/tumor-associated Thomsen-Friedenreich antigen (TF antigen) interaction has been implicated in heterotypic and homotypic cellular adhesion and apoptotic signaling pathways. However, a stronger mechanistic understanding of the role of galectin-3 in these processes is needed. N-acetyllactosamine (LacNAc) is a non-native ligand for galectin-3 which binds with comparable affinity to the TF antigen and therefore an important ligand to study galectin-3 mediated processes. To study galectin-3 mediated homotypic cellular aggregation, four generations of polyamidoamine (PAMAM) dendrimers were functionalized with N-acetyllactosamine using a four-step chemoenzymatic route. The enzymatic step controlled the regiochemistry of the galactose addition to N-acetylglucosamine functionalized dendrimers using a recombinant beta-1,4-Galactosyltransferase-/UDP-4'-Gal Epimerase Fusion Protein (lgtB-galE). Homotypic cellular aggregation, which is promoted by the presence of galectin-3 as it binds to glycosides at the cell surface, was studied using HT-1080 fibrosarcoma, A549 lung, and DU-145 prostate cancer cell lines. In the presence of small LacNAc functionalized PAMAM dendrimers, galectin-3 induced cancer cellular aggregation was inhibited. However, the larger glycodendrimers induced homotypic cellular aggregation. Additionally, novel poly(aryl ether) dendronized silica surfaces designed for reversible adsorbtion of targeted analytes were synthesized, and characterization using X-ray Photoelectron Spectroscopy (XPS) was performed. Using a Cu(I) mediated cycloaddition "click" reaction, beta-cyclodextrin was appended to dendronized surfaces via triazole formation and also to a non-dendronized surface for comparison purposes. First generation G(1) dendrons have more than 6 times greater capacity to adsorb targeted analytes than slides functionalized with monomeric beta-cyclodextrin and are 2 times greater than slides functionalized with larger generation dendrons. This study reported beta-cyclodextrin functionalized surfaces can undergo a triggered release of the adsorbent, but otherwise retained the targeted analyte through multiple aqueous washes. Therefore, a new generation of G(1) dendronized surfaces capable of reversible adsorption were developed by heterogeneously appending sulfonic acid/pyridine end-groups. Auger Electron Spectroscopy (AES) was used to quantify the ratio of groups installed. Furthermore, G(1) dendronized surfaces were functionalized homogenously with sulfonic acid and pyridine for comparison and with chiral amino acids for chiral recognition studies.

Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane.

PubMed

Filardo, E; Quinn, J; Pang, Y; Graeber, C; Shaw, S; Dong, J; Thomas, P

2007-07-01

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17beta-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17beta-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

Inhibition of Interferon-beta Responses in Multiple Sclerosis Immune Cells Associated With High-Dose Statins

PubMed Central

Feng, Xuan; Han, Diana; Kilaru, Bharat K.; Franek, Beverly S.; Niewold, Timothy B.; Reder, Anthony T.

2014-01-01

Objective To determine whether statins affect type 1 interferon responses in relapsing-remitting multiple sclerosis (RRMS). Design Study effects of atorvastatin on type 1 interferon responses in Jurkat cells, mononuclear cells (MNCs) from therapy-naive patients with RRMS in vitro, and MNCs from interferon-treated RRMS patients in vivo in 4 conditions: no drug, statin only, interferon-beta only, and statin added on to interferon-beta therapy. Patients The study examined clinically stable patients with RRMS: 21 therapy-naive patients and 14 patients receiving interferon-beta with a statin. Interventions Statin effects on in vitro and in vivo interferon-beta–induced STAT1 transcription factor activation, expression of interferon-stimulated proteins in MNCs, and serum type 1 interferon activity. Results In vitro, atorvastatin dose dependently inhibited expression of interferon-stimulated P-Y-STAT1 by 44% (P< .001), interferon regulatory factor 1 protein by 30% (P= .006), and myxovirus resistance 1 protein by 32% (P=.004) compared with no-statin control in MNCs from therapy-naive RRMS patients. In vivo, 9 of 10 patients who received high-dose statins (80 mg) had a significant reduction in interferon-beta therapy–induced serum interferon-α/β activity, whereas only 2 of 4 patients who received medium-dose statins (40 mg) had reductions. High-dose add-on statin therapy significantly blocked interferon-beta function, with less P-Y-STAT1 transcription factor activation, and reduced myxovirus resistance 1 protein and viperin protein production. Medium doses of statins did not change STAT1 activation. Conclusions High-dose add-on statin therapy significantly reduces interferon-beta function and type 1 interferon responses in RRMS patients. These data provide a putative mechanism for how statins could counteract the beneficial effects of interferon-beta and worsen disease. PMID:22801747

Dynamics of beta-cell turnover: evidence for beta-cell turnover and regeneration from sources of beta-cells other than beta-cell replication in the HIP rat.

PubMed

Manesso, Erica; Toffolo, Gianna M; Saisho, Yoshifumi; Butler, Alexandra E; Matveyenko, Aleksey V; Cobelli, Claudio; Butler, Peter C

2009-08-01

Type 2 diabetes is characterized by hyperglycemia, a deficit in beta-cells, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). These characteristics are recapitulated in the human IAPP transgenic (HIP) rat. We developed a mathematical model to quantify beta-cell turnover and applied it to nondiabetic wild type (WT) vs. HIP rats from age 2 days to 10 mo to establish 1) whether beta-cell formation is derived exclusively from beta-cell replication, or whether other sources of beta-cells (OSB) are present, and 2) to what extent, if any, there is attempted beta-cell regeneration in the HIP rat and if this is through beta-cell replication or OSB. We conclude that formation and maintenance of adult beta-cells depends largely ( approximately 80%) on formation of beta-cells independent from beta-cell duplication. Moreover, this source adaptively increases in the HIP rat, implying attempted beta-cell regeneration that substantially slows loss of beta-cell mass.

Electron-microscopic study of the secretion of the ependymal cells in the domestic cat (ependymin-beta cells).

PubMed

Gonzalez-Santander, R

1979-01-01

We have studied, by electron microscopy, the ultrastructural aspects of secretion (neurosecretion) of the ependyma of the third ventricle of the domestic cat. We have found cytoplasmic protrusions and isolated masses of cytoplasm, some with homogeneous cytoplasm and others with very dense granulation (protein-beta?). Axons, synaptic terminals and free secretory granules in the ventricular lumen were also seen. The existence of ependymin-beta cells (ependymocyte-beta) and axohormonal buttons is suggested. The ependymal cells are classified into seven types: (1) covering ependymocytes, (2) tanycyt ependymocytes, (3) secretory ependymocytes, (4) ependymocytes-beta, (5) neurosecretory ependymocytes, (6) neurosensorial ependymocytes (crown-like) and (7) supraependymal microgial ependymocytes. A neurohormonal hypothesis and the possible existence of one or more cerebral hormones (neurohormones) are suggested. These hormones would flow into the CSF through some of the ependymal cells (by microapocrine secretion, liberation of neurosecretion granules, or by axohormonal buttons): this could be the most important link in the endocrine system, assuring the functional unity throughout the ventricular system of the cerebrospinal axis which it winds through, although its basic influence is exercised) on the hypophysis level as a vertex of the classical endocrine system.

Interferon β induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice.

PubMed

Chort, Alice; Alves, Sandro; Marinello, Martina; Dufresnois, Béatrice; Dornbierer, Jean-Gabriel; Tesson, Christelle; Latouche, Morwena; Baker, Darren P; Barkats, Martine; El Hachimi, Khalid H; Ruberg, Merle; Janer, Alexandre; Stevanin, Giovanni; Brice, Alexis; Sittler, Annie

2013-06-01

We showed previously, in a cell model of spinocerebellar ataxia 7, that interferon beta induces the expression of PML protein and the formation of PML protein nuclear bodies that degrade mutant ataxin 7, suggesting that the cytokine, used to treat multiple sclerosis, might have therapeutic value in spinocerebellar ataxia 7. We now show that interferon beta also induces PML-dependent clearance of ataxin 7 in a preclinical model, SCA7(266Q/5Q) knock-in mice, and improves motor function. Interestingly, the presence of mutant ataxin 7 in the mice induces itself the expression of endogenous interferon beta and its receptor. Immunohistological studies in brains from two patients with spinocerebellar ataxia 7 confirmed that these modifications are also caused by the disease in humans. Interferon beta, administered intraperitoneally three times a week in the knock-in mice, was internalized with its receptor in Purkinje and other cells and translocated to the nucleus. The treatment induced PML protein expression and the formation of PML protein nuclear bodies and decreased mutant ataxin 7 in neuronal intranuclear inclusions, the hallmark of the disease. No reactive gliosis or other signs of toxicity were observed in the brain or internal organs. The performance of the SCA7(266Q/5Q) knock-in mice was significantly improved on two behavioural tests sensitive to cerebellar function: the Locotronic® Test of locomotor function and the Beam Walking Test of balance, motor coordination and fine movements, which are affected in patients with spinocerebellar ataxia 7. In addition to motor dysfunction, SCA7(266Q/5Q) mice present abnormalities in the retina as in patients: ataxin 7-positive neuronal intranuclear inclusions that were reduced by interferon beta treatment. Finally, since neuronal death does not occur in the cerebellum of SCA7(266Q/5Q) mice, we showed in primary cell cultures expressing mutant ataxin 7 that interferon beta treatment improves Purkinje cell survival.

Activation of PPAR{delta} up-regulates fatty acid oxidation and energy uncoupling genes of mitochondria and reduces palmitate-induced apoptosis in pancreatic {beta}-cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, Jun; Jiang, Li; Lue, Qingguo

2010-01-15

Recent evidence indicates that decreased oxidative capacity, lipotoxicity, and mitochondrial aberrations contribute to the development of insulin resistance and type 2 diabetes. The goal of this study was to investigate the effects of peroxisome proliferator-activated receptor {delta} (PPAR{delta}) activation on lipid oxidation, mitochondrial function, and insulin secretion in pancreatic {beta}-cells. After HIT-T15 cells (a {beta}-cell line) were exposed to high concentrations of palmitate and GW501516 (GW; a selective agonist of PPAR{delta}), we found that administration of GW increased the expression of PPAR{delta} mRNA. GW-induced activation of PPAR{delta} up-regulated carnitine palmitoyltransferase 1 (CPT1), long-chain acyl-CoA dehydrogenase (LCAD), pyruvate dehydrogenase kinase 4more » (PDK4), and uncoupling protein 2 (UCP2); alleviated mitochondrial swelling; attenuated apoptosis; and reduced basal insulin secretion induced by increased palmitate in HIT cells. These results suggest that activation of PPAR{delta} plays an important role in protecting pancreatic {beta}-cells against aberrations caused by lipotoxicity in metabolic syndrome and diabetes.« less

Artesunate protects pancreatic beta cells against cytokine-induced damage via SIRT1 inhibiting NF-κB activation.

PubMed

Yu, L; Chen, J F; Shuai, X; Xu, Y; Ding, Y; Zhang, J; Yang, W; Liang, X; Su, D; Yan, C

2016-01-01

Artesunate (ART) has been known as the most effective and safe reagents to treat malaria for many years. In this study, we explored whether ART could protect pancreatic beta-cell against cytokine-induced damage. The production of nitrite (NO) was detected with the Griess Assay Kit. SIRT1 and inducible nitric oxide synthase (iNOS) expression were determined with Western blot. The transcriptional activity of NF-κB was evaluated by luciferase reporter assay. The expression of Sirt1 was silenced by RNA interference. Glucose-stimulated insulin secretion (GSIS) and potassium-stimulated insulin secretion (KSIS) assays were performed to measure the effect of ART on pancreatic beta-cells' function. The effect of ART on beta-cells apoptosis was evaluated by using Hochest/PI staining and TUNEL assay. ART enhanced GSIS (KSIS) and reduced apoptosis of pancreatic beta-cells induced by IL-1β. Further study showed that ART inhibited IL-1β-induced increase of NF-κB activity, iNOS expression, and NO production. Moreover, ART up-regulated SIRT1 expression in INS-1 cells and islets exposed to IL-1β. Inhibition of SIRT1 expression could partially abolished the inhibitory effect of ART on NF-κB activity in IL-1β-treated beta-cells. More importantly, the protective effect of ART on cytokine-induced damage was reversed by silencing SIRT1 expression. ART can elicit a protective effect on beta-cells exposed to IL-1β by stimulating SIRT1 expression, which resulted in the decrease of NF-κB activity, iNOS expression, and NO production. Hence, ART might be an effective drug for diabetes.

Mucosal tolerance to experimental autoimmune myasthenia gravis is associated with down-regulation of AChR-specific IFN-gamma-expressing Th1-like cells and up-regulation of TGF-beta mRNA in mononuclear cells.

PubMed

Ma, C G; Zhang, G X; Xiao, B G; Wang, Z Y; Link, J; Olsson, T; Link, H

1996-02-13

Oral and nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) resulted in prevention or marked decrease of the severity of experimental autoimmune myasthenia gravis (EAMG) and suppression of AChR-specific B-cell responses and of AChR-reactive T-cell function. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabeled cDNA oligonucleotide proves was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine interferon-gamma (IFN-gamma), the B cell-stimulating interleukin-4 (IL-4), and the immunosuppressive transforming growth factor-beta (TGF-beta). Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells, compared to control rats receiving PBS orally or nasally and injected with CFA only. Oral and nasal tolerance was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs when compared to nontolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG and that TGF-beta plays an important role in tolerance induction to EAMG.

Pharmacological characterization of P2X7 receptors in rat peritoneal cells.

PubMed

Chen, Y-W; Donnelly-Roberts, D L; Namovic, M T; Gintant, G A; Cox, B F; Jarvis, M F; Harris, R R

2005-03-01

P2X(7) receptor activation by ATP results in the release of IL-1beta and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X(7) receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X(7) receptor (1321rP2X(7)-11). RPC were isolated from rats by lavage. P2X(7) agonist induced pore formation in RPC was measured by EtBr uptake. P2X(7)-stimulated pore formation and Ca(++) influx in 1321rP2X(7)-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl -2'-4'-disulfonic acid (PPADS) on pore formation and Ca(++) influx were examined in both RPC and 1321rP2X(7)-11. P2X(7)-mediated IL-1beta release in RPC and the effect of PPADS were determined. RPC express functional P2X(7) receptors that were activated by ATP analogs with a rank order of potency of 2'- 3'-O-(4-Benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) > ATP > alpha,beta-methylene ATP. Activation of P2X(7) receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X(7)-11 cells. Activation of P2X(7) receptors on RPC resulted in IL-1 beta secretion, which was inhibited by PPADS. RPC express functional P2X(7) receptors that form pores and mediate the release of IL-1beta.

An endocrine pathway in the prostate, ERbeta, AR, 5alpha-androstane-3beta,17beta-diol, and CYP7B1, regulates prostate growth.

PubMed

Weihua, Zhang; Lathe, Richard; Warner, Margaret; Gustafsson, Jan-Ake

2002-10-15

Epithelial proliferation of the ventral prostate in rodents peaks between 2 and 4 weeks of age, and by week 8, proliferating cells are rare. We have used ERbeta(-/-) and CYP7B1(-/-) mice to investigate the role of ERbeta and one of its ligands, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), in growth of the ventral prostate. Before puberty, ERbeta was found in quiescent but not in proliferating cells, and proliferating cells occurred more frequently in ventral prostates of ERbeta(-/-) mice than in wild-type littermates. Treatment with 3betaAdiol decreased proliferation in wild-type but not in ERbeta(-/-) mice. In rats, treatment with 3betaAdiol from postnatal day 2 to 28 resulted in reduction in growth of ventral prostates. The prostates of CYP7B1(-/-) mice were hypoproliferative before puberty and smaller than those of their wild-type littermates after puberty. Because CYP7B1 represents the major pathway for inactivating 3betaAdiol in the prostate, we suggest that ERbeta, 3betaAdiol, and CYP7B1 are the components of a pathway that regulates growth of the rodent ventral prostate. In this pathway, ERbeta is an antiproliferative receptor, 3betaAdiol is an ERbeta ligand, and CYP7B1 is the enzyme that regulates ERbeta function by regulating the level of 3betaAdiol.

Nanomaterial Solutions for the Protection of Insulin Producing Beta Cells

NASA Astrophysics Data System (ADS)

Atchison, Nicole Ann

Islet transplantation is a promising treatment for type 1 diabetes. However, even with the many successes, islet transplantation has yet to reach its full potential. Limited islet sources, loss of cell viability during isolation and culture, and post-transplant graft loss are a few of the issues preventing extensive use of islet transplantation. The application of biomaterial systems to alleviate some of the stresses affecting islet viability has led to improvements in isolation and transplantation outcomes, but problems persist. In this work we approach two distinct issues affecting islet viability; ischemic conditions and immunological attack post-transplant. Ischemic conditions have been linked to a loss of islet graft function and occur during organ preservation, islet isolation and culture, and after islets are transplanted. We show that liposomal delivery of adenosine triphosphate (ATP) to beta cells can limit cell death and loss of function in ischemic conditions. We demonstrate that by functionalizing liposomes with the fibronectin-mimetic peptide PR_b, delivery of liposomes to porcine islets and rat beta cells is increased compared to nontargeted controls. Additionally, liposomes are shown to protect by providing both ATP and lipids to the ischemic cells. The delivery of ATP was investigated here but application of PR_b functionalized liposomes could be extended to other interesting cargos as well. The second area of investigation involves encapsulation of islets with silica nanoparticles to create a permselective barrier. Silica nanoparticles are an interesting material for encapsulation given their ability to be fine-tuned and further functionalized. We demonstrate that size-tunable, fluorescent silica nanoparticles can be assembled layer-by-layer on the surface of cells and that silica nanoparticle encapsulated islets are able to secrete insulin in response to a glucose challenge.

Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mTOR complex 2 (mTORC2).

PubMed

Barlow, A D; Xie, J; Moore, C E; Campbell, S C; Shaw, J A M; Nicholson, M L; Herbert, T P

2012-05-01

Rapamycin (sirolimus) is one of the primary immunosuppressants for islet transplantation. Yet there is evidence that the long-term treatment of islet-transplant patients with rapamycin may be responsible for subsequent loss of islet graft function and viability. Therefore, the primary objective of this study was to elucidate the molecular mechanism of rapamycin toxicity in beta cells. Experiments were performed on isolated rat and human islets of Langerhans and MIN6 cells. The effects of rapamycin and the roles of mammalian target of rapamycin complex 2 (mTORC2)/protein kinase B (PKB) on beta cell signalling, function and viability were investigated using cell viability assays, insulin ELISA assays, kinase assays, western blotting, pharmacological inhibitors, small interfering (si)RNA and through the overproduction of a constitutively active mutant of PKB. Rapamycin treatment of MIN6 cells and islets of Langerhans resulted in a loss of cell function and viability. Although rapamycin acutely inhibited mTOR complex 1 (mTORC1), the toxic effects of rapamycin were more closely correlated to the dissociation and inactivation of mTORC2 and the inhibition of PKB. Indeed, the overproduction of constitutively active PKB protected islets from rapamycin toxicity whereas the inhibition of PKB led to a loss of cell viability. Moreover, the selective inactivation of mTORC2 using siRNA directed towards rapamycin-insensitive companion of target of rapamycin (RICTOR), mimicked the toxic effects of chronic rapamycin treatment. This report provides evidence that rapamycin toxicity is mediated by the inactivation of mTORC2 and the inhibition of PKB and thus reveals the molecular basis of rapamycin toxicity and the essential role of mTORC2 in maintaining beta cell function and survival.

The species origin of the cellular microenvironment influences markers of beta cell fate and function in EndoC-βH1 cells.

PubMed

Jeffery, N; Richardson, S; Beall, C; Harries, L W

2017-12-15

Interaction between islet cell subtypes and the extracellular matrix influences beta-cell function in mammals. The tissue architecture of rodent islets is very different to that of human islets; cell-to-cell communication and interaction with the extracellular matrix may vary between species. In this work, we have compared the responses of the human EndoC-βH1 cell line to non-human and human-derived growth matrices in terms of growth morphology, gene expression and glucose-stimulated insulin secretion (GSIS). EndoC-βH1 cells demonstrated a greater tendency to form cell clusters when cultured in a human microenvironment and exhibited reduced alpha cell markers at the mRNA level; mean expression difference - 0.23 and - 0.51; p = 0.009 and 0.002 for the Aristaless-related homeobox (ARX) and Glucagon (GCG) genes respectively. No differences were noted in the protein expression of mature beta cell markers such as Pdx1 and NeuroD1 were noted in EndoC-βH1 cells grown in a human microenvironment but cells were however more sensitive to glucose (4.3-fold increase in insulin secretion following glucose challenge compared with a 1.9-fold increase in cells grown in a non-human microenvironment; p = 0.0003). Our data suggests that the tissue origin of the cellular microenvironment has effects on the function of EndoC-βH1 cells in vitro, and the use of a more human-like culture microenvironment may bring benefits in terms of increased physiological relevance. Copyright © 2017 Elsevier Inc. All rights reserved.

Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein

PubMed Central

1996-01-01

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton- insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells. PMID:8647901

[Role of Ski/SnoN protein in the regulation of TGF-beta signal pathway].

PubMed

Lu, Zhao-hui; Chen, Jie

2003-04-01

TGF-beta signal pathway plays an important role in the cell growth, differentiation, formation of extracellular matrix, embryo development and carcinogenesis, etc. However, the regulation of TGF-beta pathway is not totally understood. In 1999, three independent research groups found that Ski/SnoN protein could inhibit the TGF-beta mediated transcription by recruiting N-CoR, a transcription co-repressor. Later studies suggested that TGF-beta and SMADs degraded the Ski/SnoN protein by mediating ubiquitin linkage, showing negative feedback regulation. The important findings in Ski/SnoN laid the theoretical foundation for demonstrating the function of TGF-beta signal pathway.

Tumor suppression in basal keratinocytes via dual non-cell-autonomous functions of a Na,K-ATPase beta subunit

PubMed Central

Hatzold, Julia; Beleggia, Filippo; Herzig, Hannah; Altmüller, Janine; Nürnberg, Peter; Bloch, Wilhelm; Wollnik, Bernd; Hammerschmidt, Matthias

2016-01-01

The molecular pathways underlying tumor suppression are incompletely understood. Here, we identify cooperative non-cell-autonomous functions of a single gene that together provide a novel mechanism of tumor suppression in basal keratinocytes of zebrafish embryos. A loss-of-function mutation in atp1b1a, encoding the beta subunit of a Na,K-ATPase pump, causes edema and epidermal malignancy. Strikingly, basal cell carcinogenesis only occurs when Atp1b1a function is compromised in both the overlying periderm (resulting in compromised epithelial polarity and adhesiveness) and in kidney and heart (resulting in hypotonic stress). Blockade of the ensuing PI3K-AKT-mTORC1-NFκB-MMP9 pathway activation in basal cells, as well as systemic isotonicity, prevents malignant transformation. Our results identify hypotonic stress as a (previously unrecognized) contributor to tumor development and establish a novel paradigm of tumor suppression. DOI: http://dx.doi.org/10.7554/eLife.14277.001 PMID:27240166

Delta-catenin/NPRAP: A new member of the glycogen synthase kinase-3beta signaling complex that promotes beta-catenin turnover in neurons.

PubMed

Bareiss, Sonja; Kim, Kwonseop; Lu, Qun

2010-08-15

Through a multiprotein complex, glycogen synthase kinase-3beta (GSK-3beta) phosphorylates and destabilizes beta-catenin, an important signaling event for neuronal growth and proper synaptic function. delta-Catenin, or NPRAP (CTNND2), is a neural enriched member of the beta-catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that delta-catenin expression is also affected by GSK-3beta signaling and participates in the molecular complex regulating beta-catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of delta-catenin with members of the molecular destruction complex: GSK-3beta, beta-catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK-3beta formed a complex with delta-catenin, and its inhibition resulted in increased delta-catenin and beta-catenin expression levels. LY294002 and amyloid peptide, known activators of GSK-3beta signaling, reduced delta-catenin expression levels. Furthermore, delta-catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of delta-catenin, like that of beta-catenin, is regulated by proteasome-mediated degradation. Coimmunoprecipitation experiments showed that delta-catenin overexpression promoted GSK-3beta and beta-catenin interactions. Primary cortical neurons and PC12 cells expressing delta-catenin treated with proteasome inhibitors showed increased ubiquitinated beta-catenin forms. Consistent with the hypothesis that delta-catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing delta-catenin showed enhanced beta-catenin turnover. These studies identify delta-catenin as a new member of the GSK-3beta signaling pathway and further suggest that delta-catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of beta-catenin in neuronal cells. (c) 2010 Wiley-Liss, Inc.

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi

2008-10-20

In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissuesmore » in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.« less

Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats.

PubMed Central

Wiedermann, U; Hanson, L A; Kahu, H; Dahlgren, U I

1993-01-01

We have previously reported that vitamin A deficiency resulted in a reduced IgA antibody response to cholera toxin (CT) after per-oral immunization. In the present investigation we have studied the in vivo and in vitro immune response in vitamin A-deficient rats to two parenterally applied antigens, beta-lactoglobulin (beta-LG) and picrylsulphonic acid (TNP)-Ficoll. The serum IgG and IgM antibody responses to the T-cell dependent antigen beta-LG were significantly lower in the vitamin A-deficient rats than in the pair-fed control rats. No such differences were seen with the IgG and IgM responses to the T-cell independent antigen TNP-Ficoll. However, the biliary IgA and the serum IgE antibodies against both antigens were decreased in the vitamin A-deficient rats. In vitro lymphocyte stimulation with concanavalin A (Con A) or beta-LG gave higher T-cell proliferation rates in the vitamin A-deficient than in the control rats. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) levels in supernatants from Con A-stimulated mesenteric lymph node cells were also higher in the vitamin A-deficient rats, while IL-6 levels were decreased, which is consistent with an up-regulated Th1 activity. Proliferation studies on purified accessory cells and T cells from the deficient and the control rats, mixed in different combinations, showed that the T cells, but not the accessory cells, were disturbed in the vitamin A-deficient rats. Despite the increased T-cell activity in vitro the vitamin A-deficient rats had a lower delayed-type hypersensitivity (DTH) reaction than the pair-fed control rats. In conclusion, the increased IL-2 and IFN-gamma levels may reflect an up-regulation of Th1 cell function, while the decreased IgA, IgE and IL-6 levels indicate a suppression of Th2 cells. The disturbed T-lymphocyte function is manifested in vivo as a decreased DTH reaction and suppressed antibody production, the latter possibly due to a lack of B-cell switching and proliferation factors in vitamin A-deficient rats. PMID:8307607

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Loss of intra-islet heparan sulfate is a highly sensitive marker of type 1 diabetes progression in humans.

PubMed

Simeonovic, Charmaine J; Popp, Sarah K; Starrs, Lora M; Brown, Debra J; Ziolkowski, Andrew F; Ludwig, Barbara; Bornstein, Stefan R; Wilson, J Dennis; Pugliese, Alberto; Kay, Thomas W H; Thomas, Helen E; Loudovaris, Thomas; Choong, Fui Jiun; Freeman, Craig; Parish, Christopher R

2018-01-01

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

Stevioside improves pancreatic beta-cell function during glucotoxicity via regulation of acetyl-CoA carboxylase.

PubMed

Chen, Jianguo; Jeppesen, Per Bendix; Nordentoft, Iver; Hermansen, Kjeld

2007-06-01

Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity influences ACC and whether this action can be counteracted by SVS are not known. To investigate this, we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose, respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid (OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5-Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in beta-cells via a unique mechanism of action.

Adaptive changes in pancreas post Roux-en-Y gastric bypass induced weight loss.

PubMed

Lautenbach, A; Wernecke, M; Riedel, N; Veigel, J; Yamamura, J; Keller, S; Jung, R; Busch, P; Mann, O; Knop, F K; Holst, J J; Meier, J J; Aberle, J

2018-05-16

Obesity has been shown to trigger adaptive increases in pancreas parenchymal and fat volume. Consecutively, pancreatic steatosis may lead to beta-cell dysfunction. However, it is not known, whether the pancreatic tissue components decrease with weight loss and pancreatic steatosis is reversible following RYGB. Therefore, the objective of the study was to investigate the effects of RYGB-induced weight loss on pancreatic volume and glucose homeostasis. 11 patients were recruited in the Obesity Centre of the University Medical Centre Hamburg-Eppendorf. Before and 6 months after RYGB, total GLP-1 levels were measured during OGTT. To assess changes in visceral adipose tissue and pancreatic volume, MRI was performed. Measures of glucose homeostasis and insulin indices were assessed. Fractional beta-cell area was estimated by correlation with the C-peptide-to-glucose ratio, beta-cell mass was calculated by the product of beta-cell area and pancreas parenchymal weight. Pancreas volume decreased from 83.8 (75.7-92.0) to 70.5 (58.8-82.3) cm 3 [mean (95% CI), p=0.001]. The decrease in total volume was associated with a significant decrease in fat volume. Fasting insulin and C-peptide were lower post RYGB. HOMA-IR levels decreased, whereas insulin sensitivity increased (p=0.03). This was consistent with a reduction in the estimated beta-cell area and mass. Following RYGB, pancreatic volume and steatosis adaptively decreased to "normal" levels with accompanying improvement in glucose homeostasis. Moreover, obesity-driven beta-cell expansion seems to be reversible, however future studies must define a method to more accurately estimate functional beta-cell mass to increase our understanding of glucose homeostasis after RYGB. This article is protected by copyright. All rights reserved.

Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves.

PubMed Central

Mauch, F.; Staehelin, L. A.

1989-01-01

Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens. PMID:12359894

Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation.

PubMed

Ren, Jiaqiang; Jin, Ping; Wang, Ena; Liu, Eric; Harlan, David M; Li, Xin; Stroncek, David F

2007-01-03

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called beta cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding beta-cell function at the molecular level will likely facilitate the development of techniques to manufacture beta-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release.

Milk composition and lactation of beta-casein-deficient mice.

PubMed Central

Kumar, S; Clarke, A R; Hooper, M L; Horne, D S; Law, A J; Leaver, J; Springbett, A; Stevenson, E; Simons, J P

1994-01-01

beta-Casein is a major protein component of milk and, in conjunction with the other caseins, it is assembled into micelles. The casein micelles determine many of the physical characteristics of milk, which are important for stability during storage and for milk-processing properties. There is evidence that suggests that beta-casein may also possess other, nonnutritional functions. To address the function of beta-casein, the mouse beta-casein gene was disrupted by gene targeting in embryonic stem cells. Homozygous beta-casein mutant mice are viable and fertile; females can lactate and successfully rear young. beta-Casein was expressed at a reduced level in heterozygotes and was completely absent from the milk of homozygous mutant mice. Despite the deficiency of beta-casein, casein micelles were assembled in heterozygous and homozygous mutants, albeit with reduced diameters. The absence of beta-casein expression was reflected in a reduced total protein concentration in milk, although this was partially compensated for by an increased concentration of other proteins. The growth of pups feeding on the milk of homozygous mutants was reduced relative to those feeding on the milk of wild-type mice. Various genetic manipulations of caseins have been proposed for the qualitative improvement of cow's milk composition. The results presented here demonstrate that beta-casein has no essential function and that the casein micelle is remarkably tolerant of changes in composition. Images PMID:8016126

Proliferation of prostate cancer cells and activity of neutral endopeptidase is regulated by bombesin and IL-1beta with IL-1beta acting as a modulator of cellular differentiation.

PubMed

Albrecht, Martin; Doroszewicz, Jolanta; Gillen, Sonja; Gomes, Iara; Wilhelm, Beate; Stief, Thomas; Aumüller, Gerhard

2004-01-01

Neutral endopeptidase (NEP) is a cell-surface bound enzyme that cleaves and inactivates neuropeptides such as bombesin and substance P and is involved in the transition from hormonally regulated androgen-dependent prostate cancer (PC) to androgen-independent PC. Neuropeptides are implicated in growth regulation of different cell types and function as transmitters between the neuroendocrine and the immune system. NEP-expression, enzymatic activity of the membrane bound protein, cell proliferation, procalcitonin (PCT) production, and secretion as well as changes in cell morphology of prostatic cells were evaluated after treatment with the immunomodulatory cytokine interleukin-1beta (IL-1beta), neuropeptides (bombesin, substance P), and neuropeptide-conditioned media derived from a human neuroendocrine cell line. Incubation of LNCaP tumor cells with IL-1beta resulted in a diminished proliferative activity, induction of neurite-like outgrowth which was accompanied by the formation of tubular-type mitochondria typical for neuronal/neuroendocrine cells, and an increased production and secretion of PCT. Conversely, proliferation of prostatic stromal cells was enhanced by the cytokine coming along with an increased number of Golgi-apparatuses and ER-cisternae. Bombesin had an antimitotic effect on LNCaP, but not on stromal cells. Substance P did not influence the growth of any of the cell types investigated, whereas neuropeptide-conditioned media exerted a slightly mitogenic effect on both cell types. The activity of LNCaP cell-surface bound NEP was enhanced by bombesin, but was diminished by substance P and neuropeptide-conditioned media. Proliferation and activity of neuropeptide degrading NEP is regulated differently by immunomodulatory substances in PC cells and cells derived from the prostatic stroma with IL-1beta being a potent modulator of cellular differentiation and a potential target for anticancer drug design in PC cells. Copyright 2003 Wiley-Liss, Inc.

Distinctive postprandial modulation of beta cell function and insulin sensitivity by dietary fats: monounsaturated compared with saturated fatty acids.

PubMed

López, Sergio; Bermúdez, Beatriz; Pacheco, Yolanda M; Villar, José; Abia, Rocío; Muriana, Francisco J G

2008-09-01

Exaggerated and prolonged postprandial triglyceride concentrations are associated with numerous conditions related to insulin resistance, including obesity, type 2 diabetes, and the metabolic syndrome. Although dietary fats profoundly affect postprandial hypertriglyceridemia, limited data exist regarding their effects on postprandial glucose homeostasis. We sought to determine whether postprandial glucose homeostasis is modulated distinctly by high-fat meals enriched in saturated fatty acids (SFAs) or monounsaturated fatty acids (MUFAs). Normotriglyceridemic subjects with normal fasting glucose and normal glucose tolerance were studied. Blood samples were collected over the 8 h after ingestion of a glucose and triglyceride tolerance test meal (GTTTM) in which a panel of dietary fats with a gradual change in the ratio of MUFAs to SFAs was included. On 5 separate occasions, basal and postprandial concentrations of glucose, insulin, triglyceride, and free fatty acids (FFAs) were measured. High-fat meals increased the postprandial concentrations of insulin, triglycerides, and FFAs, and they enhanced postprandial beta cell function while decreasing insulin sensitivity (as assessed with different model-based and empirical indexes: insulinogenic index, insulinogenic index/homeostasis model assessment of insulin resistance, area under the curve for insulin/area under the curve for glucose, homeostasis model assessment for beta cell function, and GTTTM-determined insulin sensitivity, oral glucose insulin sensitivity, and the postprandial Belfiore indexes for glycemia and blood FFAs. These effects were significantly ameliorated, in a direct linear relation, when MUFAs were substituted for SFAs. The data presented here suggest that beta cell function and insulin sensitivity progressively improve in the postprandial state as the proportion of MUFAs with respect to SFAs in dietary fats increases.

The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

PubMed

Tsonkova, Violeta Georgieva; Sand, Fredrik Wolfhagen; Wolf, Xenia Asbæk; Grunnet, Lars Groth; Kirstine Ringgaard, Anna; Ingvorsen, Camilla; Winkel, Louise; Kalisz, Mark; Dalgaard, Kevin; Bruun, Christine; Fels, Johannes Josef; Helgstrand, Charlotte; Hastrup, Sven; Öberg, Fredrik Kryh; Vernet, Erik; Sandrini, Michael Paolo Bastner; Shaw, Allan Christian; Jessen, Carsten; Grønborg, Mads; Hald, Jacob; Willenbrock, Hanni; Madsen, Dennis; Wernersson, Rasmus; Hansson, Lena; Jensen, Jan Nygaard; Plesner, Annette; Alanentalo, Tomas; Petersen, Maja Borup Kjær; Grapin-Botton, Anne; Honoré, Christian; Ahnfelt-Rønne, Jonas; Hecksher-Sørensen, Jacob; Ravassard, Philippe; Madsen, Ole D; Rescan, Claude; Frogne, Thomas

2018-02-01

To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-βH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates. Copyright © 2017 Novo Nordisk A/S. Published by Elsevier GmbH.. All rights reserved.

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy

PubMed Central

Schraenen, A.; Lemaire, K.; de Faudeur, G.; Hendrickx, N.; Granvik, M.; Van Lommel, L.; Mallet, J.; Vodjdani, G.; Gilon, P.; Binart, N.; in’t Veld, P.

2010-01-01

Aims/hypothesis Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. Methods RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. Results mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). Conclusions/interpretation A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified. Electronic supplementary material The online version of this article (doi:10.1007/s00125-010-1913-7) contains supplementary material, which is available to authorised users. PMID:20938637

HGF/scatter factor selectively promotes cell invasion by increasing integrin avidity.

PubMed

Trusolino, L; Cavassa, S; Angelini, P; Andó, M; Bertotti, A; Comoglio, P M; Boccaccio, C

2000-08-01

Hepatocyte growth factor/scatter factor (HGF/SF) controls a genetic program known as 'invasive growth', which involves as critical steps cell adhesion, migration, and trespassing of basement membranes. We show here that in MDA-MB-231 carcinoma cells, these steps are elicited by HGF/SF but not by epidermal growth factor (EGF). Neither factor substantially alters the production or activity of extracellular matrix proteases. HGF/SF, but not EGF, selectively promotes cell adhesion on laminins 1 and 5, fibronectin, and vitronectin through a PI3-K-dependent mechanism. Increased adhesion is followed by enhanced invasiveness through isolated matrix proteins as well as through reconstituted basement membranes. Inhibition assays using function-blocking antibodies show that this phenomenon is mediated by multiple integrins including beta1, beta3, beta4, and beta5. HGF/SF triggers clustering of all these integrins at actin-rich adhesive sites and lamellipodia but does not quantitatively modify their membrane expression. These data suggest that HGF/SF promotes cell adhesion and invasiveness by increasing the avidity of integrins for their specific ligands.

Age-related changes in expression of transforming growth factor-beta and receptors in cells of intervertebral discs.

PubMed

Matsunaga, Shunji; Nagano, Satoshi; Onishi, Toshiyuki; Morimoto, Norio; Suzuki, Shusaku; Komiya, Setsuro

2003-01-01

The authors conducted a study to determine age-related changes in expression of transforming growth factor (TGF)-beta1, -beta2, -beta3, and Type I and Type II receptors in various cells in the nucleus pulposus and anulus fibrosus. Immunolocalization of TGFbetas and Type I and II receptors was examined during the aging process of cervical intervertebral discs in senescence-accelerated mice (SAM). The TGFbeta family has important roles for cellular function of various tissues. Its role in disc aging, however, is unknown. Detailed information on the temporal and spatial localization of TGFbetas and their receptors in discs is required before discussing introduction of them clinically into the intervertebral disc. Three groups of five SAM each were used. The groups of SAM were age 8, 24, and 50 weeks, respectively. Hematoxylin and eosin staining and immunohistochemical study involving specific antibodies for TGFbeta1, -beta2, -beta3, and Types I and II TGF receptors were performed. Intervertebral discs exhibited degenerative change with advancing age. The TGFbetas and their receptors were present in the fibrocartilaginous cells within the anulus fibrosus and notochord-like cells within the nucleus pulposus of young mice. Expression of TGFbetas and Type I and Type II receptors changed markedly in the cells within the anulus fibrosus during the aging process. The TGFbetas and their receptors were present in cells within the nucleus pulposus and the anulus fibrosus of young mice, and their expression decreased with age.

Beta-lactams and their potential use as novel anticancer chemotherapeutics drugs.

PubMed

Kuhn, Deborah; Coates, Cristina; Daniel, Kenyon; Chen, Di; Bhuiyan, Mohammad; Kazi, Aslamuzzaman; Turos, Edward; Dou, Q Ping

2004-09-01

The discovery of natural and synthetic antibiotics is one of the most important medical breakthroughs in human history. Many diseases, such as bacterial meningitis, pneumonia, and septicemia, are now curable with the use of antibiotics. Antibiotics are efficacious, generally well tolerated in patients, and have a low toxicity level. It is for these reasons antibiotics remain an attractive target for drug discovery. Traditional beta-lactam antibiotics (e.g. penicillins, penems, cephalosporins) have a bicyclic ring structure that is conformationally rigid and functions to inhibit bacterial cell wall synthesis. In addition to the bactericidal action of antibiotics, it has been discovered that many antibiotics are capable of inhibiting tumor cell growth. There are currently many antitumor antibiotics approved for cancer therapy, which work to inhibit tumor cell growth by DNA intercalation. The use of beta-lactams as prodrugs has also met with success by aiding delivery of the chemotherapeutic directly to tumor sites. Recently, a novel class of N-thiolated monobactams, so termed because they possess a monocyclic ring instead of the bicyclic ring, has been found to induce apoptosis potently and specifically in many tumor cell lines but not in normal, non-transformed cell lines. Other beta-lactams, such as the polyaromatics, have been found to slow or inhibit tumor cell growth, and the 4-alkylidene beta-lactams are capable of inhibiting matrix metalloproteinases and leukocyte elactase activity. These data indicate that synthesis and evaluation of beta-lactams are a promising area for further development in anticancer research.

Extracellular matrix and hormones transcriptionally regulate bovine. beta. -casein 5 prime sequences in stably transfected mouse mammary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidhauser, C. Bissell, M.J.; Myers, C.A.; Casperson, G.F.

1990-12-01

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-2D has been developed in which more than 35% of the cells express {beta}-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete {beta}-casein undirectionally into a lumen. These cells were stably transfected with amore » series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine {beta}-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency. This regulation occurered primarily at the transcriptional level and was dependent on the length of the 5{prime} flanking region of the {beta}-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous {beta}-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.« less

Improvement of insulin sensitivity and beta-cell function by nateglinide and repaglinide in type 2 diabetic patients - a randomized controlled double-blind and double-dummy multicentre clinical trial.

PubMed

Li, J; Tian, H; Li, Q; Wang, N; Wu, T; Liu, Y; Ni, Z; Yu, H; Liang, J; Luo, R; Li, Y; Huang, L

2007-07-01

To evaluate the efficacy of nateglinide vs. repaglinide in blood glucose (BG) control and the effect on insulin resistance and beta-Cell function in patients with type 2 diabetes. A randomized controlled double-blind and double-dummy multicentre clinical trial was conducted. A total of 230 Chinese patients with type 2 diabetes were enrolled in five clinical centres. The patients were divided randomly into group A [repaglinide 1.0 mg three times daily (t.i.d.), n = 115] or group B (nateglinide 90 mg t.i.d., n = 115). At baseline and end of the 12-week clinical trial, standard mixed meal tolerance tests were performed. A total of 223 patients (96.9%) completed the trial. There was no significant difference between repaglinide and nateglinide groups in the effects of reducing fasting blood glucose (FBG), 30-, 60- and 120-min BG during 12 weeks (p > 0.05). At week 12, no significant difference was shown between the two groups in BG or haemoglobin A(1c) (HbA(1c)) (p > 0.05). However, the effect on HbA(1c) in repaglinide group was stronger than that in nateglinide group (p < 0.05). After 12-week treatment, area under the curve (AUC) of BG decreased (p < 0.05), and AUC of insulin and C-peptide (CP) increased in both groups (p < 0.05). The effects of nateglinide on AUC of BG, insulin and CP were similar to that of repaglinide (p > 0.05). There was no significant difference between the two groups in AUC of BG, insulin or CP in week 12 (p > 0.05). Furthermore, homeostasis model assessment of insulin resistance (HOMA-IR) and beta-cell function indexes measured by HOMA-beta, DeltaI(30)/DeltaG(30) and (DeltaI(30)/DeltaG(30))/HOMA-IR were improved significantly in both groups during 12 weeks (p < 0.05). The effects of improving HOMA-IR and beta-cell function indexes in nateglinide group were comparable with that of repaglinide group (p > 0.05). The efficacy of repaglinide and nateglinide in FBG, postprandial glucose excursion and early-phase insulin secretion is similar. But the effect of repaglinide 1.0 mg t.i.d. on HbA(1c) is stronger than that of nateglinide 90 mg t.i.d.. This trial had shown that nateglinide and repaglinide could comparably improve insulin sensitivity and beta-cell function.

Bioinformatics Knowledge Map for Analysis of Beta-Catenin Function in Cancer

PubMed Central

Arighi, Cecilia N.; Wu, Cathy H.

2015-01-01

Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge “maps” of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease. PMID:26509276

The receptor protein tyrosine phosphatase (RPTP){beta}/{zeta} is expressed in different subtypes of human breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Pinera, Pablo; Garcia-Suarez, Olivia; Instituto Universitario de Oncologia del Principado de Asturias, Oviedo

2007-10-12

Increasing evidence suggests mutations in human breast cancer cells that induce inappropriate expression of the 18-kDa cytokine pleiotrophin (PTN, Ptn) initiate progression of breast cancers to a more malignant phenotype. Pleiotrophin signals through inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, leading to increased tyrosine phosphorylation of different substrate proteins of RPTP{beta}/{zeta}, including {beta}-catenin, {beta}-adducin, Fyn, GIT1/Cat-1, and P190RhoGAP. PTN signaling thus has wide impact on different important cellular systems. Recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway; this discovery potentially is very important, since constitutive ALK activity of nucleophosmin (NPM)-ALK fusionmore » protein is causative of anaplastic large cell lymphomas, and, activated ALK is found in other malignant cancers. Recently ALK was identified in each of 63 human breast cancers from 22 subjects. We now demonstrate that RPTP{beta}/{zeta} is expressed in each of these same 63 human breast cancers that previously were found to express ALK and in 10 additional samples of human breast cancer. RPTP{beta}/{zeta} furthermore was localized not only in its normal association with the cell membrane but also scattered in cytoplasm and in nuclei in different breast cancer cells and, in the case of infiltrating ductal carcinomas, the distribution of RPTP{beta}/{zeta} changes as the breast cancer become more malignant. The data suggest that the PTN/RPTP{beta}/{zeta} signaling pathway may be constitutively activated and potentially function to constitutively activate ALK in human breast cancer.« less

Vitronectin and fibronectin function as glucan binding proteins augmenting macrophage responses to Pneumocystis carinii.

PubMed

Vassallo, R; Kottom, T J; Standing, J E; Limper, A H

2001-08-01

beta-glucans represent major structural components of fungal cell walls. We recently reported that Pneumocystis carinii beta-glucans stimulate alveolar macrophages to release proinflammatory cytokines. Macrophage activation by beta-glucan is augmented by serum, implying the presence of circulating factors that interact with beta-glucans and enhance their ability to stimulate macrophages. Using beta-glucan-enriched cell wall fractions from P. carinii and Saccharomyces cerevisiae, two prominent proteins were precipitated from serum and demonstrated to be vitronectin (VN) and fibronectin (FN) by immune analysis. Preincubation of beta-glucan with VN or FN enhanced macrophage activation in response to this cell wall component. Because VN and FN accumulate in the lungs during P. carinii pneumonia, we further investigated hepatic and pulmonary expression of VN and FN messenger RNA during infection. P. carinii pneumonia in rodents is associated with increased hepatic expression of VN and FN as well as increased local expression of FN in the lung. Because interleukin (IL)-6 represents the major regulator of VN and FN expression during inflammatory conditions, we measured macrophage IL-6 release in response to stimulation with P. carinii beta-glucan. Stimulation of macrophages with P. carinii beta-glucan induced significant release of IL-6. Elevated concentrations of IL-6 were noted in the blood of infected animals compared with uninfected control animals. These studies indicate that VN and FN bind to beta-glucan components of P. carinii and augment macrophage inflammatory responses. P. carinii cell wall beta-glucan stimulates secretion of IL-6 by macrophages, thereby enhancing hepatic synthesis of both VN and FN, and lung synthesis of FN during pneumonia.

Maternal obesity accelerates fetal pancreatic beta-cell but not alpha-cell development in sheep: prenatal consequences.

PubMed

Ford, Stephen P; Zhang, Liren; Zhu, Meijun; Miller, Myrna M; Smith, Derek T; Hess, Bret W; Moss, Gary E; Nathanielsz, Peter W; Nijland, Mark J

2009-09-01

Maternal obesity affects offspring weight, body composition, and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high-energy diet on fetal pancreatic development. Sixty days prior to breeding, ewes were assigned to control [100% of National Research Council (NRC) recommendations] or obesogenic (OB; 150% NRC) diets. At 75 days gestation, OB ewes exhibited elevated insulin-to-glucose ratios at rest and during a glucose tolerance test, demonstrating insulin resistance compared with control ewes. In fetal studies, ewes ate their respective diets from 60 days before to 75 days after conception when animals were euthanized under general anesthesia. OB and control ewes increased in body weight by approximately 43% and approximately 6%, respectively, from diet initiation until necropsy. Although all organs were heavier in fetuses from OB ewes, only pancreatic weight increased as a percentage of fetal weight. Blood glucose, insulin, and cortisol were elevated in OB ewes and fetuses on day 75. Insulin-positive cells per unit pancreatic area were 50% greater in fetuses from OB ewes as a result of increased beta-cell mitoses rather than decreased programmed cell death. Lambs of OB ewes were born earlier but weighed the same as control lambs; however, their crown-to-rump length was reduced, and their fat mass was increased. We conclude that increased systemic insulin in fetuses from OB ewes results from increased glucose exposure and/or cortisol-induced accelerated fetal beta-cell maturation and may contribute to premature beta-cell function loss and predisposition to obesity and metabolic disease in offspring.

The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, H.; Grubb, J.H.; Sly, W.S.

1990-10-01

The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less

Purification and characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52.

PubMed

Masuda, N; Oda, H; Tanaka, H

1983-01-04

An NADP-dependent 7 beta-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7 beta-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (Km) value of 0.05 mM for 3 alpha, 7 beta-dihydroxy-5 beta-cholanoic acid whereas higher values were obtained with 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl glycine (0.20 mM), and 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and had a Km value of 0.30 mM. A molecular weight of 64000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.

The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

PubMed

Berthouze, Magali; Venkataramanan, Vidya; Li, Yi; Shenoy, Sudha K

2009-06-17

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

The 17 beta-oestradiol dehydrogenase of pig endometrial cells is localized in specialized vesicles.

PubMed Central

Adamski, J; Husen, B; Marks, F; Jungblut, P W

1993-01-01

Two monoclonal antibodies against the 17 beta-oestradiol dehydrogenase of pig endometrial cells have been used in localization studies with immunogold electron microscopy. The antibodies attach both to a fraction of dehydrogenase-rich cytoplasmic vesicles isolated from homogenates and to vesicles of similar appearance in cells. The vesicles are filled with electron-dense material. Their tagging intensity indicates a high degree of specialization. Endometrial cells from mature animals contain a host of dehydrogenase vesicles, and cells from prepubertal animals only a few. Functional aspects of the novel organelle are discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8457206

V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.

PubMed

Alvarez, J D; Anderson, S J; Loh, D Y

1995-08-01

Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.

The role of stress and beta-adrenergic system in melanoma: current knowledge and possible therapeutic options.

PubMed

Colucci, Roberta; Moretti, Silvia

2016-05-01

The aim of the present review was to discuss recent findings on the role of beta-adrenergic system in melanoma, in order to provide information on the biological responses elicited by its activation and its potential application for melanoma treatment. A literature search was performed, and evidences regarding the involvement of stress and beta-adrenergic system in cancer and melanoma were found and discussed. Our search pointed out that beta-adrenergic system is a key regulator of important biological processes involved in the onset and progression of some solid tumors. In the last decade, functional beta-adrenoceptors have been also identified on melanoma cells, as well as on their microenvironment cells. Similarly to other common cancers too, the activation of such adrenoceptors by catecholamines, usually released under stress conditions, has been found to trigger pro-tumorigenic pathways contributing to cell proliferation and motility, immune system regulation, apoptosis, epithelial-mesenchymal transition, invasion and neoangiogenesis. The biological evidences we found clarify and sustain the clinical evidences reporting the involvement of chronic stress in melanoma onset and progression. In such scenario, it is conceivable that a therapeutic approach targeting beta-adrenergic system could constitute a novel and promising strategy for melanoma treatment.

Regulation of. beta. -cell glucose transporter gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ling; Alam, Tausif; Johnson, J.H.

1990-06-01

It has been postulated that a glucose transporter of {beta} cells (GLUT-2) may be important in glucose-stimulated insulin secretion. To determine whether this transporter is constitutively expressed or regulated, the authors subjected conscious unrestrained Wistar rats to perturbations in glucose homeostasis and quantitated {beta}-cell GLUT-2 mRNA by in situ hybridization. After 3 hr of hypoglycemia, GLUT-2 and proinsulin mRNA signal densities were reduced by 25% of the level in control rats. After 4 days, GLUT-2 and proinsulin mRNA densities were reduced by 85% and 65%, respectively. After 12 days of hypoglycemia, the K{sub m} for 3-O-methyl-D-glucose transport in isolated ratmore » islets, normally 18-20 mM, was 2.5 mM. This provides functional evidence of a profound reduction of high K{sub m} glucose transporter in {beta} cells. In contrast, GLUT-2 was only slightly reduced by hypoglycemia in liver. To determine the effect of prolonged hyperglycemia, they also infused animals with 50% (wt/vol) glucose for 5 days. Hyperglycemic clamping increased GLUT-2 mRNA by 46% whereas proinsulin mRNA doubled. They conclude that GLUT-2 expression in {beta} cells, but not liver, is subject to regulation by certain perturbations in blood glucose homeostasis.« less

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform.

PubMed

Candiello, Joseph; Grandhi, Taraka Sai Pavan; Goh, Saik Kia; Vaidya, Vimal; Lemmon-Kishi, Maya; Eliato, Kiarash Rahmani; Ros, Robert; Kumta, Prashant N; Rege, Kaushal; Banerjee, Ipsita

2018-05-25

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering. Copyright © 2018. Published by Elsevier Ltd.

Nuclear import of glucokinase in pancreatic beta-cells is mediated by a nuclear localization signal and modulated by SUMOylation.

PubMed

Johansson, Bente Berg; Fjeld, Karianne; Solheim, Marie Holm; Shirakawa, Jun; Zhang, Enming; Keindl, Magdalena; Hu, Jiang; Lindqvist, Andreas; Døskeland, Anne; Mellgren, Gunnar; Flatmark, Torgeir; Njølstad, Pål Rasmus; Kulkarni, Rohit N; Wierup, Nils; Aukrust, Ingvild; Bjørkhaug, Lise

2017-10-15

The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal ( 30 LKKVMRR 36 ) in the human enzyme. Substituting the residues KK 31,32 and RR 35,36 with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell. Copyright © 2017 Elsevier B.V. All rights reserved.

Oncogenic mechanisms of Evi-1 protein.

PubMed

Hirai, H; Izutsu, K; Kurokawa, M; Mitani, K

2001-08-01

Although Evi-1 is thought to promote growth or block differentiation in some cell types, its biological functions have not been elucidated. To explore the mechanisms underlying Evi-1-induced oncogenesis, we investigated whether Evi-1 affects the signaling of transforming growth factor beta (TGF-beta), which inhibits proliferation of a wide range of cell types and is one of the most studied growth regulatory factors. We demonstrated that Evi-1 represses TGF-beta signaling and antagonizes its growth-inhibitory effects. Two separate regions of Evi-1 are responsible for this repression, one of which is the first zinc-finger domain. Through this domain, Evi-1 physically interacts with Smad3, an intracellular mediator of TGF-beta signaling, thereby suppressing the transcriptional activity of Smad3. These results define a novel function of Evi-1 as a repressor of signaling components of TGF-beta. We also demonstrated that Evi-1 represses Smad-induced transcriptional activation by recruiting CtBP as a corepressor. Evi-1 associates with CtBP1 through one of the CtBP-binding consensus motifs within the region from amino acid 544 to 607, and this association is required for the efficient inhibition of TGF-beta signaling. A specific histone deacetylase (HDAc) inhibitor, trichostatin A (TSA), alleviates Evi-1-mediated repression of TGF-beta signaling, suggesting that HDAc is involved in transcriptional repression by Evi-1. This identifies a novel function of Evi-1 as a member of corepressor complexes and suggests that aberrant recruitment of corepressors is one of the mechanisms involved in Evi-1-induced leukemogenesis. These results indicate that specific HDAc inhibitors may be useful in the treatment of Evi-1-induced neoplastic tumors, including myeloid leukemias.

Development of magnetic resonance imaging based detection methods for beta amyloids via sialic acid-functionalized magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Kouyoumdjian, Hovig

The development of a non-invasive method for the detection of Alzheimer's disease is of high current interest, which can be critical in early diagnosis and in guiding preventive treatment of the disease. The aggregates of beta amyloids are a pathological hallmark of Alzheimer's disease. Carbohydrates such as sialic acid terminated gangliosides have been shown to play significant roles in initiation of amyloid aggregation. Herein, we report a biomimetic approach using sialic acid coated iron oxide superparamagnetic nanoparticles for in vitro detection in addition to the assessment of the in vivo mouse-BBB (Blood brain barrier) crossing of the BSA (bovine serum albumin)-modified ones. The sialic acid functionalized dextran nanoparticles were shown to bind with beta amyloids through several techniques including ELISA (enzyme linked immunosorbent assay), MRI (magnetic resonance imaging), TEM (transmission electron microscopy), gel electrophoresis and tyrosine fluorescence assay. The superparamagnetic nature of the nanoparticles allowed easy detection of the beta amyloids in mouse brains in both in vitro and ex vivo model by magnetic resonance imaging. Furthermore, the sialic acid nanoparticles greatly reduced beta amyloid induced cytotoxicity to SH-SY5Y neuroblastoma cells, highlighting the potential of the glyconanoparticles for detection and imaging of beta amyloids. Sialic acid functionalized BSA (bovine serum albumin) nanoparticles also showed significant binding to beta amyloids, through ELISA and ex vivo mouse brain MRI experiments. Alternatively, the BBB crossing was demonstrated by several techniques such as confocal microscopy, endocytosis, exocytosis assays and were affirmed by nanoparticles transcytosis assays through bEnd.3 endothelial cells. Finally, the BBB crossing was confirmed by analyzing the MRI signal of nanoparticle-injected CD-1 mice.

Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deckert, Katelyn; Budiardjo, S. Jimmy; Brunner, Luke C.

2012-08-07

Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492Gmore » in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.« less

Calmodulin is a phospholipase C-beta interacting protein.

PubMed

McCullar, Jennifer S; Larsen, Shana A; Millimaki, Ryan A; Filtz, Theresa M

2003-09-05

Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways. Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3. Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates. CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity. There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.

The Tregs' world according to GARP.

PubMed

Battaglia, Manuela; Roncarolo, Maria Grazia

2009-12-01

Naturally occurring CD4+CD25(high) regulatory T cells (nTreg) are essential for maintaining tolerance. FOXP3 has been established as a molecular marker of nTreg; however, FOXP3 cannot be used as a reliable marker for bona fide human nTreg since effector T cells also up-regulate FOXP3 expression upon activation. Despite the important function of nTreg, the underlying molecular mechanisms of nTreg-mediated suppression are far from defined. Previous studies have demonstrated that the TGF-beta latency-associated peptide (LAP) is expressed on the surface of nTreg, and that immunosuppression can be mediated by membrane TGF-beta; however, it remains unknown how LAP is bound to nTreg and what is the functional significance of its selective expression on activated nTreg. The nTreg's world may now change according to GARP, an orphan toll-like receptor composed of leucine-rich repeats. In this issue of the European Journal of Immunology, a study provides further demonstration that GARP is selectively expressed only in activated human nTreg and nTreg cell clones but not in activated effector T cells, confirming GARP as a bona fide nTreg marker. In addition, GARP binds directly to LAP; yet, GARP over-expression is insufficient to induce modification of latent TGF-beta into active TGF-beta further clarifying its role in nTreg-mediated suppression.

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.

PubMed

de Graaf, M; Boven, E; Oosterhoff, D; van der Meulen-Muileman, I H; Huls, G A; Gerritsen, W R; Haisma, H J; Pinedo, H M

2002-03-04

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug. Copyright 2002 Cancer Research UK

Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah

Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cellsmore » by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.« less

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

PubMed

Takayama, S; White, M F; Kahn, C R

1988-03-05

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Booth, Brian W., E-mail: brbooth@clemson.edu; Institute for Biological Interfaces of Engineering, Clemson University, Clemson, SC 29634; Boulanger, Corinne A.

2010-02-01

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D {beta}-geo (CD{beta}geo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CD{beta}geo cells and that the mitogen activated protein kinase signalingmore » pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG{sup -/-} mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.« less

TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function

NASA Technical Reports Server (NTRS)

Lu, L.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

2001-01-01

BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle fabrication and after growth factor release. At an optimal TGF-beta1 dosage of 1.0 ng/ml after 3 days, the released TGF-beta1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells over 21 days of culture, with increased total cell number, alkaline phosphatase activity, and osteocalcin production. CONCLUSIONS: PLGA/PEG blend microparticles can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor enhances marrow stromal cell proliferation and osteoblastic differentiation in vitro. CLINICAL RELEVANCE: Controlled release of TGF-beta1 from PLGA/PEG microparticles is representative of emerging tissue engineering technologies that may modulate cellular responses to encourage bone regeneration at a skeletal defect site.

Monodora myristica (African nutmeg) modulates redox homeostasis and alters functional chemistry in sickled erythrocytes.

PubMed

Erukainure, O L; Ajiboye, J A; Abbah, U A; Asieba, G O; Mamuru, S; Zaruwa, M Z; Manhas, N; Singh, P; Islam, M S

2018-05-01

The antioxidative effect of Monodora myristica seed acetone extract and its effect on chemical functional groups were investigated in sickled erythrocytes as well as molecular modeling of the antisickling potentials of its secondary metabolites. The extract was subjected to gas chromatography-mass spectrometry to identify the compounds present, which were then docked into the allosteric-binding site of deoxy-hemoglobin. The extract was incubated with sickled erythrocytes at 37°C for 6, 12, and 24 h and were subjected to antioxidative analysis for reduced glutathione (GSH), superoxide dismutase (SOD), catalase, and lipid peroxidation (LPO). Chemical functional group of the treated cells was analyzed via Fourier transform infrared spectroscopy (FTIR). The predominant compounds identified were 17-octadecynoic acid; oleic acid, androstan-3-one, 17-hydroxy-2-methyl- (2.beta.,5.beta.,17.beta.)-; estran-3-one, 17-(acetyloxy)-2-methyl-, (2.alpha., 5.alpha., 17.beta.), and (+)-3-carene, 10-(acetylmethyl)-. They all fitted well within the active site of Hb with good binding affinity, as evidenced by the negative CDocker interaction energies of their complexes ranging between -54.4 and -26.7 kcal/mol. Treatment with the extract exacerbated SOD and catalase activities as well as GSH level, while LPO was suppressed. This antioxidative activity was time and/or dose dependent, with 6 and 12 h incubation showing the optimum activity. FTIR analysis of the treated cells showed the presence of hydrophobic functional groups. The synergetic molecular interaction of the major compounds of the extract with the α-dimer of Hb depicts an antisickling effect of M. myristica acetone extract. This is accompanied by exacerbation of endogenous antioxidant enzymes activity and modification of the functional chemistry of the cells.

Estrogen induced {beta}-1,4-galactosyltransferase 1 expression regulates proliferation of human breast cancer MCF-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Hee-Jung; Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan-city, Gyeongsangnam-do; Chung, Tae-Wook

2012-10-05

Highlights: Black-Right-Pointing-Pointer We examined the regulation and biological functions of B4GALT1 expression induced by estrogen. Black-Right-Pointing-Pointer Estrogen-induced B4GALT1 expression through the direct binding of ER-{alpha} to ERE in MCF-7 cells. Black-Right-Pointing-Pointer B4GALT1 expression activates the proliferation of MCF-7 cells via its receptor function. Black-Right-Pointing-Pointer Thus, we suggest B4GALT1 as a molecular target for inhibiting breast cancer proliferation. -- Abstract: Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose {beta}-1,4-N-acetylglucosamine (Gal{beta}1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressedmore » by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Gal{beta}1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-{alpha}-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells. Considering these results, we propose that estrogen regulates the expression of B4GALT1 through the direct binding of ER-{alpha} to ERE and that the expressed B4GALT1 plays a crucial role in the proliferation of MCF-7 cells through its activity as a membrane receptor.« less

Lycopene and Beta-Carotene Induce Growth Inhibition and Proapoptotic Effects on ACTH-Secreting Pituitary Adenoma Cells

PubMed Central

Leite de Oliveira, Felipe; Soares, Nathália; de Mattos, Rômulo Medina; Hecht, Fábio; Dezonne, Rômulo Sperduto; Vairo, Leandro; Goldenberg, Regina Coeli dos Santos; Gomes, Flávia Carvalho Alcântara; de Carvalho, Denise Pires; Gadelha, Mônica R.; Nasciutti, Luiz Eurico; Miranda-Alves, Leandro

2013-01-01

Pituitary adenomas comprise approximately 10–15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27kip1 in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2) and increased expression of p27kip1 in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27kip1; and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing’s disease. PMID:23667519

Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

NASA Technical Reports Server (NTRS)

Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.;

In vitro modulation of the interaction between HA95 and LAP2beta by cAMP signaling.

PubMed

Martins, Sandra B; Marstad, Anne; Collas, Philippe

2003-09-09

The nuclear envelope mediates key functions by interacting with chromatin. We recently reported an interaction between the chromatin- and nuclear matrix-associated protein HA95 and the inner nuclear membrane integral protein LAP2beta, implicated in initiation of DNA replication (Martins et al. (2003) J. Cell Biol. 160, 177-188). Here, we show that in vitro, interaction between HA95 and LAP2beta is modulated by cAMP signaling via PKA. Exposure of an anti-HA95 immune precipitate from interphase HeLa cells to a mitotic extract promotes ATP-dependent release of LAP2beta from the HA95 complex. This coincides with Ser and Thr phosphorylation of HA95 and LAP2beta. Inhibition of PKA with PKI abolishes phosphorylation of HA95 and dissociation of LAP2beta from HA95, although LAPbeta remains phosphorylated. Antagonizing cAMP signaling in mitotic extract also abolishes the release of LAP2beta from HA95; however, disrupting PKA anchoring to A-kinase anchoring proteins has no effect. Inhibition of CDK activity in the extract greatly reduces LAP2beta phosphorylation but does not prevent LAP2beta release from HA95. Inhibition of PKC, MAP kinase, or CaM kinase II does not affect mitotic extract-induced dissociation of LAP2beta from HA95. PKA phosphorylates HA95 but not LAP2beta in vitro and elicits a release of LAP2beta from HA95. CDK1 or PKC phosphorylates LAP2beta within the HA95 complex, but neither kinase induces LAP2beta release. Our results indicate that in vitro, the interaction between HA95 and LAP2beta is influenced by a PKA-mediated phosphorylation of HA95 rather than by CDK1- or PKC-mediated phosphorylation of LAP2beta. This suggests an additional level of regulation of a chromatin-nuclear envelope interaction in dividing cells.

Redox-mediated activation of latent transforming growth factor-beta 1

NASA Technical Reports Server (NTRS)

Barcellos-Hoff, M. H.; Dix, T. A.; Chatterjee, A. (Principal Investigator)

1996-01-01

Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during disease mechanisms involving chronic ROS production.

Inhibin beta E is upregulated by drug-induced endoplasmic reticulum stress as a transcriptional target gene of ATF4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brüning, Ansgar, E-mail: ansgar.bruening@med.uni-muenchen.de; Matsingou, Christina; Brem, German Johannes

2012-10-15

Inhibins and activins are gonadal peptide hormones of the transforming growth factor-β super family with important functions in the reproductive system. By contrast, the recently identified inhibin βE subunit, primarily expressed in liver cells, appears to exert functions unrelated to the reproductive system. Previously shown downregulation of inhibin βE in hepatoma cells and anti-proliferative effects of ectopic inhibin βE overexpression indicated growth-regulatory effects of inhibin βE. We observed a selective re-expression of the inhibin βE subunit in HepG2 hepatoblastoma cells, MCF7 breast cancer cells, and HeLa cervical cancer cells under endoplasmic reticulum stress conditions induced by tunicamycin, thapsigargin, and nelfinavir.more » Analysis of XPB1 splicing and ATF4 activation revealed that inhibin βE re-expression was associated with induction of the endoplasmic reticulum stress reaction by these drugs. Transfection of an ATF4 expression plasmid specifically induced inhibin βE expression in HeLa cells and indicates inhibin βE as a hitherto unidentified target gene of ATF4, a key transcription factor of the endoplasmic reticulum stress response. Therefore, the inhibin βE subunit defines not only a new player but also a possible new marker for drug-induced endoplasmic reticulum stress. -- Highlights: ► Endoplasmic reticulum stress induces inhibin beta E expression. ► Inhibin beta E is regulated by the transcription factor ATF4. ► Inhibin beta E expression can be used as a marker for drug-induced ER stress.« less

Coordinate regulation of estrogen-mediated fibronectin matrix assembly and epidermal growth factor receptor transactivation by the G protein-coupled receptor, GPR30.

PubMed

Quinn, Jeffrey A; Graeber, C Thomas; Frackelton, A Raymond; Kim, Minsoo; Schwarzbauer, Jean E; Filardo, Edward J

2009-07-01

Estrogen promotes changes in cytoskeletal architecture not easily attributed to the biological action of estrogen receptors, ERalpha and ERbeta. The Gs protein-coupled transmembrane receptor, GPR30, is linked to specific estrogen binding and rapid estrogen-mediated release of heparin-bound epidermal growth factor. Using marker rescue and dominant interfering mutant strategies, we show that estrogen action via GPR30 promotes fibronectin (FN) matrix assembly by human breast cancer cells. Stimulation with 17beta-estradiol or the ER antagonist, ICI 182, 780, results in the recruitment of FN-engaged integrin alpha5beta1 conformers to fibrillar adhesions and the synthesis of FN fibrils. Concurrent with this cellular response, GPR30 promotes the formation of Src-dependent, Shc-integrin alpha5beta1 complexes. Function-blocking antibodies directed against integrin alpha5beta1 or soluble Arg-Gly-Asp peptide fragments derived from FN specifically inhibited GPR30-mediated epidermal growth factor receptor transactivation. Estrogen-mediated FN matrix assembly and epidermal growth factor receptor transactivation were similarly disrupted in integrin beta1-deficient GE11 cells, whereas reintroduction of integrin beta1 into GE11 cells restored these responses. Mutant Shc (317Y/F) blocked GPR30-induced FN matrix assembly and tyrosyl phosphorylation of erbB1. Interestingly, relative to recombinant wild-type Shc, 317Y/F Shc was more readily retained in GPR30-induced integrin alpha5beta1 complexes, yet this mutant did not prevent endogenous Shc-integrin alpha5beta1 complex formation. Our results suggest that GPR30 coordinates estrogen-mediated FN matrix assembly and growth factor release in human breast cancer cells via a Shc-dependent signaling mechanism that activates integrin alpha5beta1.

Pathogenic prions deviate PrPC signaling in neuronal cells and impair A-beta clearance

PubMed Central

Pradines, E; Hernandez-Rapp, J; Villa-Diaz, A; Dakowski, C; Ardila-Osorio, H; Haik, S; Schneider, B; Launay, J-M; Kellermann, O; Torres, J-M; Mouillet-Richard, S

2013-01-01

The subversion of the normal function exerted by the cellular prion protein (PrPC) in neurons by pathogenic prions is assumed to have a central role in the pathogenesis of transmissible spongiform encephalopathies. Using two murine models of prion infection, the 1C11 neuronal cell line and neurospheres, we document that prion infection is associated with the constitutive activation of signaling targets normally coupled with PrPC, including the Fyn kinase, the mitogen-associated protein kinases ERK1/2 and the CREB transcription factor. PrPC-dependent signaling overactivation in infected cells is associated with the recruitment of p38 and JNK stress-associated kinases. Downstream from CREB, prion-infected cells exhibit reduced activity of the matrix metalloprotease (MMP)-9. As MMP-9 catalyzes the degradation of the amyloid A-beta peptide, the decrease in MMP-9 activity in prion-infected cells causes a significant impairment of the clearance of A-beta, leading to its accumulation. By exploiting two 1C11-infected clones accumulating high or moderate levels of prions, we show that the prion-induced changes are correlated with the level of infectivity. Of note, a dose-dependent increase in A-beta levels was also found in the cerebrospinal fluid of mice inoculated with these infected clones. By demonstrating that pathogenic prions trigger increases in A-beta levels through the deviation of PrPC signaling, our data argue that A-beta may exacerbate prion-induced toxicity. PMID:23303130

Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

PubMed Central

Vachon, P H; Xu, H; Liu, L; Loechel, F; Hayashi, Y; Arahata, K; Reed, J C; Wewer, U M; Engvall, E

1997-01-01

Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders. PMID:9312189

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

PubMed Central

Jhappan, C; Geiser, A G; Kordon, E C; Bagheri, D; Hennighausen, L; Roberts, A B; Smith, G H; Merlino, G

1993-01-01

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland. Images PMID:8491177

The amyloid precursor protein and postnatal neurogenesis/neuroregeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Yanan; Tang, Bor Luen

2006-03-03

The amyloid precursor protein (APP) is the source of amyloid-beta (A{beta}) peptide, produced via its sequential cleavage {beta}- and {gamma}-secretases. Various biophysical forms of A{beta} (and the mutations of APP which results in their elevated levels) have been implicated in the etiology and early onset of Alzheimer's disease. APP's evolutionary conservation and the existence of APP-like isoforms (APLP1 and APLP2) which lack the A{beta} sequence, however, suggest that these might have important physiological functions that are unrelated to A{beta} production. Soluble N-terminal fragments of APP have been known to be neuroprotective, and the interaction of its cytoplasmic C-terminus with amore » myriad of proteins associates it with diverse processes such as axonal transport and transcriptional regulation. The notion for an essential postnatal function of APP has been demonstrated genetically, as mice deficient in both APP and APLP2 or all three APP isoforms exhibit early postnatal lethality and neuroanatomical abnormalities. Recent findings have also brought to light two possible functions of the APP family in Brain-regulation of neural progenitor cell proliferation and axonal outgrowth after injury. Interestingly, these two apparently related neurogenic/neuroregenerative functions of APP involve two separate domains of the molecule.« less

Long-term AICAR administration and exercise prevents diabetes in ZDF rats.

PubMed

Pold, Rasmus; Jensen, Lasse S; Jessen, Niels; Buhl, Esben S; Schmitz, Ole; Flyvbjerg, Allan; Fujii, Nobuharu; Goodyear, Laurie J; Gotfredsen, Carsten F; Brand, Christian L; Lund, Sten

2005-04-01

Lifestyle interventions including exercise programs are cornerstones in the prevention of obesity-related diabetes. The AMP-activated protein kinase (AMPK) has been proposed to be responsible for many of the beneficial effects of exercise on glucose and lipid metabolism. The effects of long-term exercise training or 5-aminoimidazole-4-carboxamide-1-beta-d-riboruranoside (AICAR) treatment, both known AMPK activators, on the development of diabetes in male Zucker diabetic fatty (ZDF) rats were examined. Five-week-old, pre-diabetic ZDF rats underwent daily treadmill running or AICAR treatment over an 8-week period and were compared with an untreated group. In contrast to the untreated, both the exercised and AICAR-treated rats did not develop hyperglycemia during the intervention period. Whole-body insulin sensitivity, as assessed by a hyperinsulinemic-euglycemic clamp at the end of the intervention period, was markedly increased in the exercised and AICAR-treated animals compared with the untreated ZDF rats (P < 0.01). In addition, pancreatic beta-cell morphology was almost normal in the exercised and AICAR-treated animals, indicating that chronic AMPK activation in vivo might preserve beta-cell function. Our results suggest that activation of AMPK may represent a therapeutic approach to improve insulin action and prevent a decrease in beta-cell function associated with type 2 diabetes.

P2X receptor characterization and IL-1/IL-1Ra release from human endothelial cells.

PubMed

Wilson, H L; Varcoe, R W; Stokes, L; Holland, K L; Francis, S E; Dower, S K; Surprenant, A; Crossman, D C

2007-05-01

The pro-inflammatory cytokine, interleukin-1beta (IL-1beta), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL-1beta in response to inflammatory stimuli, but the demonstration and mechanism of release of IL-1 from ECs remains unclear. In activated monocytes, efficient release of bioactive IL-1beta occurred via activation of ATP-gated P2X(7) receptors (P2X(7)Rs). Activation of P2X(7)R in ECs from human umbilical vein (HUVECs) released IL-1 receptor antagonist (IL-1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL-1beta and IL-1Ra synthesis, processing and release, in HUVECs under pro-inflammatory conditions. Quantitative RT-PCR, immunoblotting, ELISA, flow cytometry, and whole-cell patch clamp recordings were used to determine protein expression and receptor function. IL-8-luciferase-reporter was used as an IL-1 sensitive bioassay. HUVECs expressed P2X(4)R and P2X(7)R subtypes and both were significantly up-regulated under inflammatory conditions. P2X(7)R currents were increased 3-fold by inflammatory stimuli, whereas no P2X(4)R-mediated currents were detected. Caspase-1, but not IL-1beta, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro-IL-1beta and increased caspase-1 levels. Activation of P2X(7)Rs resulted in low-level release of bioactive IL-1beta and simultaneous release of IL-1Ra. The net biological effect of release was anti-inflammatory. Endothelial P2X(7)Rs induced secretion of both pro- and anti-inflammatory IL-1 receptor ligands, the balance of which may provide a means for altering the inflammatory state of the arterial vessel wall.

Transforming growth factor beta1 gene variation Leu10Pro affects secretion and function in hepatic cells.

PubMed

Gu, Xing; Ji, Xin; Shi, Le-Hua; Yi, Chang-Hong; Zhao, Yun-Peng; Wang, Ai-Hua; Lu, Lun-Gen; Yu, Wen-Bo; Gao, Chun-Fang

2012-11-01

Our previous work revealed transforming growth factor beta1 (TGFβ1) gene polymorphisms are associated with susceptibility to hepatocellular carcinoma and liver cirrhosis. However, no further study of functional substitution in hepatic cells has yet been reported. This study was designed to uncover the functional mechanisms of TGFβ1 gene polymorphisms in the pathogenesis of liver diseases. Two recombinant TGFβ1 expression plasmids containing TGFβ1 codon 10 Leu/Pro variation were constructed with CMV promoter and transfected into human hepatoma cell lines (HepG2 and SMMU 7721), hepatic stellate cells (LX-2), and immortalized hepatocytes (L02). The secretion capacities of TGFβ1 protein in the transfected cells were determined by ELISA. Apoptosis, proliferative activity, and expression of CD 105, CD83, and CD80 were also measured by use of flow cytometry. The ELISA results showed that cells transfected with CMV-Pro10 were more capable of TGFβ1 secretion than those transfected with CMV-Leu10. Functionally, CMV-Pro10 was more apoptosis-protective and induced more proliferation than CMV-Leu10 in transfected hepatic cells. Pro10 up-regulated expression of CD105 and down-regulated expression of CD83. TGFβ1 gene Leu10Pro variation in signal peptide has significant effects on TGFβ1 secretion and functions in hepatic cells.

In vivo IL-10 and TGF-beta production by PBMC from long-term kidney transplant recipients with excellent graft function: a possible feedback mechanism participating in immunological stability.

PubMed

Alberú, Josefina; Richaud-Patin, Yvonne; Vázquez-Lavista, Luis Gabriel; de Leo, Claudia; Guzmán-Rodríguez, Hugo; Mancilla, Eduardo; Correa-Rotter, Ricardo; Chew-Wong, Alfredo; Llorente, Luis

2004-04-01

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) are Th2-derived multifunctional cytokines that exhibit potent immunoregulatory and anti-inflammatory properties which might prolong graft survival. The aim of this study was to explore whether spontaneous production of IL-10 and TGF-beta by blood mononuclear cells correlates with excellent long-term graft function. A cross-sectional study was carried out in 32 kidney transplant recipients, without albuminuria, treated with azathioprine and prednisone. Spontaneous IL-10 and TGF-beta were measured by enzyme-linked immunosorbent assay in supernatants from 24 h cultured unstimulated peripheral blood mononuclear cells. Both cytokines were also determined in 10 healthy kidney donors. There was no correlation between IL-10 or TGF-beta with any variable tested, namely age, SCr, histocompatibility, and post-transplant follow-up. In vivo IL-10 production displayed a statistical trend to be higher in transplant recipients than in controls (362.3 +/- 465, range 12.5-1929.3 pg/ml, and 189 +/- 170, range 4.17-485.7 pg/ml, respectively; p = 0.08), whereas no difference was observed in TGF-beta among the same groups (134.7 +/- 79.2, range 68-421 pg/ml, and 121.4 +/- 25.8, range 75-151 pg/ml, respectively). Interestingly, a statistically significant inverse correlation was observed between IL-10 and TGF-beta in kidney transplant recipients (p = 0.03). The higher IL-10 production observed in long-term kidney transplant recipients supports the notion that this cytokine contributes in decreasing allogenic immune responses and allows prolongation of allograft survival. The balance between TGF-beta and IL-10 may be of paramount importance in graft acceptance.

Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6⁺CD4⁺ T Cells.

PubMed

Lafferty, Mark K; Sun, Lingling; Christensen-Quick, Aaron; Lu, Wuyuan; Garzino-Demo, Alfredo

2017-05-16

Chemokine receptor type 6 (CCR6)⁺CD4⁺ T cells are preferentially infected and depleted during HIV disease progression, but are preserved in non-progressors. CCR6 is expressed on a heterogeneous population of memory CD4⁺ T cells that are critical to mucosal immunity. Preferential infection of these cells is associated, in part, with high surface expression of CCR5, CXCR4, and α4β7. In addition, CCR6⁺CD4⁺ T cells harbor elevated levels of integrated viral DNA and high levels of proliferation markers. We have previously shown that the CCR6 ligands MIP-3α and human beta defensins inhibit HIV replication. The inhibition required CCR6 and the induction of APOBEC3G. Here, we further characterize the induction of apolipoprotein B mRNA editing enzyme (APOBEC3G) by human beta defensin 2. Human beta defensin 2 rapidly induces transcriptional induction of APOBEC3G that involves extracellular signal-regulated kinases 1/2 (ERK1/2) activation and the transcription factors NFATc2, NFATc1, and IRF4. We demonstrate that human beta defensin 2 selectively protects primary CCR6⁺CD4⁺ T cells infected with HIV-1. The selective protection of CCR6⁺CD4⁺ T cell subsets may be critical in maintaining mucosal immune function and preventing disease progression.

Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules.

PubMed

Yang, Shao-Nian; Wenna, Nancy Dekki; Yu, Jia; Yang, Guang; Qiu, Hua; Yu, Lina; Juntti-Berggren, Lisa; Köhler, Martin; Berggren, Per-Olof

2007-09-01

beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.

PubMed

Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J H; Okkenhaug, Klaus; Vanhaesebroeck, Bart

2008-06-17

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

Regulatory T cells (CD4(+)CD25(bright)FoxP3(+)) expansion in systemic sclerosis correlates with disease activity and severity.

PubMed

Slobodin, Gleb; Ahmad, Mohammad Sheikh; Rosner, Itzhak; Peri, Regina; Rozenbaum, Michael; Kessel, Aharon; Toubi, Elias; Odeh, Majed

2010-01-01

The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc). Ten patients with SSc donated 20ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4(+) cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-beta and IL-10 by activated CD4(+) cells was measured by ELISA in culture supernatants. The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices (r=0.71, p=0.034 and r=0.67, p=0.044, respectively). ELISA-measured production of TGF-beta and IL-10 by CD4(+) cells was similar in patients and controls. Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-beta or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed. 2010 Elsevier Inc. All rights reserved.

Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES).

PubMed

Han, Eun Hee; Gorman, Amanda A; Singh, Puja; Chi, Young-In

HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional Hydrogel Materials Inspired by Amyloid

NASA Astrophysics Data System (ADS)

Schneider, Joel

2012-02-01

Protein assembly resulting in the formation of amyloid fibrils, assemblies rich in cross beta-sheet structure, is normally thought of as a deleterious event associated with disease. However, amyloid formation is also involved in a diverse array of normal biological functions such as cell adhesion, melanin synthesis, insect defense mechanism and modulation of water surface tension by fungi and bacteria. These findings indicate that Nature has evolved to take advantage of large, proteinaceous fibrillar assemblies to elicit function. We are designing functional materials, namely hydrogels, from peptides that self-assembled into fibrillar networks, rich in cross beta-sheet structure. These gels can be used for the direct encapsulation and delivery of small molecule-, protein- and cell-based therapeutics. Loaded gels exhibit shear-thinning/self-healing mechanical properties enabling their delivery via syringe. In addition to their use for delivery, we have found that some of these gels display antibacterial activity. Although cytocompatible towards mammalian cells, the hydrogels can kill a broad spectrum of bacteria on contact.

Physical and functional interaction between integrins and hERG potassium channels.

PubMed

Arcangeli, A; Becchetti, A; Cherubini, A; Crociani, O; Defilippi, P; Guasti, L; Hofmann, G; Pillozzi, S; Olivotto, M; Wanke, E

2004-11-01

Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the beta1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that beta1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.

Restoration of normal phenotype in cancer cells

DOEpatents

Bissell, M.J.; Weaver, V.M.

1998-12-08

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying {beta}{sub 1} integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive. 14 figs.

Method for restoration of normal phenotype in cancer cells

DOEpatents

Bissell, Mina J.; Weaver, Valerie M.

2000-01-01

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying .beta..sub.1 integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive.

Restoration of normal phenotype in cancer cells

DOEpatents

Bissell, Mina J.; Weaver, Valerie M.

1998-01-01

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying .beta..sub.1 integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive.

Analysis of molecular assemblies by flow cytometry: determinants of Gi1 and by binding

NASA Astrophysics Data System (ADS)

Sarvazyan, Noune A.; Neubig, Richard R.

1998-05-01

We report here a novel application of flow cytometry for the quantitative analysis of the high affinity interaction between membrane proteins both in detergent solutions and when reconstituted into lipid vesicles. The approach is further advanced to permit the analysis of binding to expressed protein complexes in native cell membranes. The G protein heterotrimer signal transduction function links the extracellularly activated transmembrane receptors and intracellular effectors. Upon activation, (alpha) and (beta) (gamma) subunits of G protein undergo a dissociation/association cycle on the cell membrane interface. The binding parameters of solubilized G protein (alpha) and (beta) (gamma) subunits have been defined but little is known quantitatively about their interactions in the membrane. Using a novel flow cytometry approach, the binding of low nanomolar concentrations of fluorescein-labeled G(alpha) i1 (F- (alpha) ) to (beta) (gamma) both in detergent solution and in a lipid environment was quantitatively compared. Unlabeled (beta) $gama reconstituted in biotinylated phospholipid vesicles bound F-(alpha) tightly (Kd 6 - 12 nM) while the affinity for biotinylated-(beta) (gamma) in Lubrol was even higher (Kd of 2.9 nM). The application of this approach to proteins expressed in native cell membranes will advance our understanding of G protein function in context of receptor and effector interaction. More generally, this approach can be applied to study the interaction of any fluorescently labeled protein with a membrane protein which can be expressed in Sf9 plasma membranes.

Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling.

PubMed

Guo, Xing; Ramirez, Alejandro; Waddell, David S; Li, Zhizhong; Liu, Xuedong; Wang, Xiao-Fan

2008-01-01

The broad range of biological responses elicited by transforming growth factor-beta (TGF-beta) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-beta (GSK3-beta). Smad3 physically interacts with Axin and GSK3-beta only in the absence of TGF-beta. Reduction in the expression or activity of Axin/GSK3-beta leads to increased Smad3 stability and transcriptional activity without affecting TGF-beta receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-beta. Mechanistically, Axin facilitates GSK3-beta-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-beta, and suggest a new function of the Axin/GSK3-beta complex in modulating critical TGF-beta/Smad3-regulated processes during development and tumor progression.

Combination cancer therapy by hapten-targeted prodrug-activating enzymes and cytokines.

PubMed

Chuang, Kuo-Hsiang; Cheng, Chiu-Min; Roffler, Steve R; Lu, Yu-Lin; Lin, Shiu-Ru; Wang, Jaw-Yuan; Tzou, Wen-Shyong; Su, Yu-Cheng; Chen, Bing-Mae; Cheng, Tian-Lu

2006-01-01

Combination therapy can help overcome limitations in the treatment of heterogeneous tumors. In the current study, we examined whether multiple therapeutic agents could be targeted to anti-dansyl single-chain antibodies (DNS scFv) that were anchored on the plasma membrane of cancer cells. Functional DNS scFv could be stably expressed on CT-26 colon cancer cells both in vitro and in vivo. Dansyl moieties were covalently attached to recombinant beta-glucuronidase (betaG) and interleukin 2 (IL-2) via a flexible poly(ethylene glycol) linker to form DNS-PEG-betaG and DNS-PEG-IL-2 conjugates. The conjugates displayed enzymatic and splenocyte-stimulatory activities, respectively, that were similar to those of the unmodified proteins. The conjugates selectively bound CT-26 cells that expressed anti-DNS scFv (CT-26/DNS cells) but not CT-26 cells that expressed control scFv (CT-26/phOx cells). DNS-PEG-betaG preferentially activated a glucuronide prodrug (BHAMG) of p-hydroxy aniline mustard at CT-26/DNS cells in culture and accumulated in subcutaneous CT-26/DNS tumors after intravenous administration. Systemic administration of DNS-PEG-IL-2 or DNS-PEG-betaG and BHAMG significantly delayed the growth of CT-26/DNS but not control CT-26/phOx tumors. Combination treatment with DNS-PEG-betaG and BHAMG followed by DNS-PEG-IL-2 therapy significantly suppressed the growth of CT-26/DNS tumors as compared to either single-agent regimen. These results show that at least two DNS-modified therapeutic agents can be selectively delivered to DNS scFv receptors in vitro and in vivo, allowing combination therapy of DNS scFv-modified tumors.

[Neuro-skeletal biology and its importance for clinical osteology].

PubMed

Zofková, I

2012-01-01

Bone remodeling is determined by function of two basic cell forms--bone resorbing osteoclasts and bone formation activating osteoblasts. Both cells are under control of a variety of endogenic and environmental factors, which ensure balance between bone resorption and bone formation. This article reviews the neuro-hormonal factors with osteoanabolic (central isoform of serotonin, melatonin, cannabinoids, beta 1 adrenergic system, oxytocin, ACTH and TSH) or osteocatabolic effects (neuropeptide Y, neuromedin U, beta2 adrenergic system). The dual effects of the beta-adrenergic system, serotonin and leptin are also discussed. The goal of studies focused on neuro-skeletal interaction is to synthesize new molecules, which can modify osteo-anabolic or osteo-catabolic pathways.

Adaptive response of rat pancreatic β-cells to insulin resistance induced by monocrotophos: Biochemical evidence.

PubMed

Nagaraju, Raju; Rajini, Padmanabhan Sharda

2016-11-01

Our previous findings clearly suggested the role of duration of exposure to monocrotophos (MCP) in the development of insulin resistance. Rats exposed chronically to MCP developed insulin resistance with hyperinsulinemia without overt diabetes. In continuation of this vital observation, we sought to delineate the biochemical mechanisms that mediate heightened pancreatic β-cell response in the wake of MCP-induced insulin resistance in rats. Adult rats were orally administered (0.9 and 1.8mg/kgb.w/d) MCP for 180days. Terminally, MCP-treated rats exhibited glucose intolerance, hyperinsulinemia, and potentiation of glucose-induced insulin secretion along with elevated levels of circulating IGF1, free fatty acids, corticosterone, and paraoxonase activity. Biochemical analysis of islet extracts revealed increased levels of insulin, malate, pyruvate and ATP with a concomitant increase in activities of cytosolic and mitochondrial enzymes that are known to facilitate insulin secretion and enhanced shuttle activities. Interestingly, islets from MCP-treated rats exhibited increased insulin secretory potential ex vivo compared to those isolated from control rats. Further, MCP-induced islet hypertrophy was associated with increased insulin-positive cells. Our study demonstrates the impact of the biological interaction between MCP and components of metabolic homeostasis on pancreatic beta cell function/s. We speculate that the heightened pancreatic beta cell function evidenced may be mediated by increased IGF1 and paraoxonase activity, which effectively counters insulin resistance induced by chronic exposure to MCP. Our findings emphasize the need for focused research to understand the confounding environmental risk factors which may modulate heightened beta cell functions in the case of organophosphorus insecticide-induced insulin resistance. Such an approach may help us to explain the sharp increase in the prevalence of type II diabetes worldwide. Copyright © 2016 Elsevier B.V. All rights reserved.

No effect of the altered peptide ligand NBI-6024 on beta-cell residual function and insulin needs in new-onset type 1 diabetes.

PubMed

Walter, Markus; Philotheou, Areti; Bonnici, François; Ziegler, Anette-G; Jimenez, Roland

2009-11-01

This randomized, four-arm, placebo-controlled, dose-ranging phase 2 trial was conducted to determine whether repeated subcutaneous injections of the altered peptide ligand, NBI-6024, designed to inhibit autoreactive T-cells, improves beta-cell function in patients with recently diagnosed type 1 diabetes. A total of 188 patients, aged 10-35 years, with recently diagnosed type 1 diabetes were randomly assigned for a treatment consisting of the subcutaneous administration of placebo or 1, 0.5, or 0.1 mg NBI-6024 at baseline, weeks 2 and 4, and then monthly until month 24. Fasting, peak, and area under the curve (AUC) C-peptide concentrations during a 2-h mixed-meal tolerance test were measured at 3-month intervals during treatment. Immune function parameters (islet antibodies and CD4 and CD8 T-cells) were also studied. The mean peak C-peptide concentration at 24 months after study entry showed no significant difference between the groups treated with 0.1 mg (0.59 pmol/ml), 0.5 mg (0.57 pmol/ml), and 1.0 mg NBI-6024 (0.48 pmol/ml) and the placebo group (0.54 pmol/ml). Fasting, stimulated peak, and AUC C-peptide concentrations declined linearly in all groups by approximately 60% over the 24-month treatment period. The average daily insulin needs at month 24 were also comparable between the four groups. No treatment-related changes in islet antibodies and T cell numbers were observed. Treatment with altered peptide ligand NBI-6024 at repeated doses of 0.1, 0.5, or 1.0 mg did not improve or maintain beta-cell function.

Type 1 Diabetes TrialNet--an international collaborative clinical trials network.

PubMed

Skyler, Jay S; Greenbaum, Carla J; Lachin, John M; Leschek, Ellen; Rafkin-Mervis, Lisa; Savage, Peter; Spain, Lisa

2008-12-01

Type 1 Diabetes TrialNet is an international consortium of clinical research centers aimed at the prevention or delay of type 1 diabetes (T1D). The fundamental goal of TrialNet is to counter the T1D disease process by immune modulation and/or enhancement of beta cell proliferation and regeneration. To achieve this goal, TrialNet researchers are working to better understand the natural history of the disease, to identify persons at risk, and to clinically evaluate novel therapies that balance potential risks and benefits. The particular focus is on studies of preventive measures. In addition, TrialNet evaluates therapies in individuals with newly diagnosed T1D with preserved beta cell function to help determine the risk/benefit profile and gain an initial assessment of potential efficacy in preservation of beta cell function, so that promising agents can be studied in prevention trials. In addition, TrialNet evaluates methodologies that enhance the conduct of its clinical trials, which includes tests of outcome assessment methodology, the evaluation of surrogate markers, and mechanistic studies laying the foundation for future clinical trials.

Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

PubMed

Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

2016-01-01

Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors.

PubMed Central

Paria, B C; Dey, S K

1990-01-01

We have established a model that shows cooperative interaction among preimplantation embryos and the role of growth factors on their development and growth. Two-cell mouse embryos cultured singly in 25-microliters microdrops had inferior development to blastocysts and lower cell numbers per blastocyst compared with those cultured in groups of 5 or 10. The inferior development of singly cultured embryos was markedly improved by addition of epidermal growth factor (EGF) or transforming growth factor alpha or beta 1 (TGF-alpha or TGF-beta 1) to the culture medium. The stage of embryonic development, primarily affected by these treatments, was between eight-cell/morula and blastocyst. Furthermore, blastocysts developed from eight-cell embryos cultured in groups or singly in the presence of EGF showed a higher incidence of zona hatching compared with those cultured singly in the absence of EGF. Detection of EGF receptors on the embryonic cell surface at eight-cell/morula and blastocyst stages suggests beneficial effects of EGF or TGF-alpha on preimplantation embryo development and blastocyst functions. Insulin-like growth factor I (IGF-I) had no influence on embryo development. To further document the cooperative interactions among embryos, the volume of the culture medium was doubled to 50 microliters. This increase in culture volume was even more detrimental to the development of singly cultured embryos. However, this detrimental effect was significantly reversed by EGF and reversed even more markedly by a combination of EGF and TGF-beta 1 but not by TGF-beta 1 alone. Although TGF-beta 1 plus IGF-I caused a modest improvement of embryo development, the response was not as great as shown by EGF alone. Furthermore, IGF-I had no additive effect on EGF-induced embryonic development. The study presents clear evidence that specific growth factors of embryonic and/or reproductive tract origin participate in preimplantation embryo development and blastocyst functions in an autocrine/paracrine manner. Images PMID:2352946

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus.

PubMed

Kanasaki, Keizo; Kanda, Yoshiko; Palmsten, Kristin; Tanjore, Harikrishna; Lee, Soo Bong; Lebleu, Valerie S; Gattone, Vincent H; Kalluri, Raghu

2008-01-15

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

Antibodies against glutamic acid decarboxylase and indices of insulin resistance and insulin secretion in nondiabetic adults: a cross-sectional study

PubMed Central

Mendivil, Carlos O; Toloza, Freddy JK; Ricardo-Silgado, Maria L; Morales-Álvarez, Martha C; Mantilla-Rivas, Jose O; Pinzón-Cortés, Jairo A; Lemus, Hernán N

2017-01-01

Background Autoimmunity against insulin-producing beta cells from pancreatic islets is a common phenomenon in type 1 diabetes and latent autoimmune diabetes in adults. Some reports have also related beta-cell autoimmunity to insulin resistance (IR) in type 2 diabetes. However, the extent to which autoimmunity against components of beta cells is present and relates to IR and insulin secretion in nondiabetic adults is uncertain. Aim To explore the association between antibodies against glutamic acid decarboxylase (GADA), a major antigen from beta cells, and indices of whole-body IR and beta-cell capacity/insulin secretion in adults who do not have diabetes. Methods We studied 81 adults of both sexes aged 30–70, without known diabetes or any autoimmune disease. Participants underwent an oral glucose tolerance test (OGTT) with determination of plasma glucose and insulin at 0, 30, 60, 90, and 120 minutes. From these results we calculated indices of insulin resistance (homeostasis model assessment of insulin resistance [HOMA-IR] and incremental area under the insulin curve [iAUCins]) and insulin secretion (corrected insulin response at 30 minutes and HOMA beta-cell%). GADAs were measured in fasting plasma using immunoenzymatic methods. Results We found an overall prevalence of GADA positivity of 21.3%, without differences by sex and no correlation with age. GADA titers did not change monotonically across quartiles of any of the IR or insulin secretion indices studies. GADA did not correlate linearly with fasting IR expressed as HOMA-IR (Spearman’s r=−0.18, p=0.10) or postabsorptive IR expressed as iAUCins (r=−0.15, p=0.18), but did show a trend toward a negative correlation with insulin secretory capacity expressed by the HOMA-beta cell% index (r=−0.20, p=0.07). Hemoglobin A1c, body mass index, and waist circumference were not associated with GADA titers. Conclusion GADA positivity is frequent and likely related to impaired beta-cell function among adults without known diabetes. PMID:28507444

Role of T cell TGF beta signaling in intestinal cytokine responses and helminthic immune modulation

USDA-ARS?s Scientific Manuscript database

Colonization with helminthic parasites down-regulates inflammation in murine colitis and improves activity scores in human inflammatory bowel disease. Helminths induce mucosal regulatory T cells, which are important for intestinal immunologic homeostasis. Regulatory T cell function involves cytoki...

Vimentin affects localization and activity of sodium-glucose cotransporter SGLT1 in membrane rafts.

PubMed

Runembert, Isabelle; Queffeulou, Guillaume; Federici, Pierre; Vrtovsnik, François; Colucci-Guyon, Emma; Babinet, Charles; Briand, Pascale; Trugnan, Germain; Friedlander, Gérard; Terzi, Fabiola

2002-02-15

It has been reported that vimentin, a cytoskeleton filament that is expressed only in mesenchymal cells after birth, is re-expressed in epithelial cells in vivo under pathological conditions and in vitro in primary culture. Whether vimentin re-expression is only a marker of cellular dedifferentiation or is instrumental in the maintenance of cell structure and/or function is a matter of debate. To address this issue, we used renal proximal tubular cells in primary culture from vimentin-null mice (Vim(-/-)) and from wild-type littermates (Vim(+/+)). The absence of vimentin did not affect cell morphology, proliferation and activity of hydrolases, but dramatically decreased Na-glucose cotransport activity. This phenotype was associated with a specific reduction of SGLT1 protein in the detergent-resistant membrane microdomains (DRM). In Vim(+/+) cells, disruption of these microdomains by methyl-beta-cyclodextrin decreased SGLT1 protein abundance in DRM, a change that was paralleled by a decrease of Na-glucose transport activity. Importantly, we showed that vimentin is located to DRM, but it disappeared after methyl-beta-cyclodextrin treatment. In Vim(-/-) cells, supplementation of cholesterol with cholesterol-methyl-beta-cyclodextrin complexes completely restored Na-glucose transport activity. Interestingly, neither cholesterol content nor cholesterol metabolism changed in Vim(-/-) cells. Our results are consistent with the view that re-expression of vimentin in epithelial cells could be instrumental to maintain the physical state of rafts and, thus, the function of DRM-associated proteins.

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function.

PubMed

Steeghs, Liana; van Vliet, Sandra J; Uronen-Hansson, Heli; van Mourik, Andries; Engering, Anneke; Sanchez-Hernandez, Martha; Klein, Nigel; Callard, Robin; van Putten, Jos P M; van der Ley, Peter; van Kooyk, Yvette; van de Winkel, Jan G J

2006-02-01

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto-N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(beta1-3)-Gal(beta1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.

Increased neuronal beta-amyloid precursor protein expression in human temporal lobe epilepsy: association with interleukin-1 alpha immunoreactivity.

PubMed

Sheng, J G; Boop, F A; Mrak, R E; Griffin, W S

1994-11-01

Levels of immunoreactive beta-amyloid precursor protein and interleukin-1 alpha were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa beta-amyloid precursor protein isoform were elevated to fourfold (p < 0.05) those of controls and those of the 130-kDa isoform to threefold (p < 0.05), whereas those of the 120-kDa isoform (p > 0.05) were not different from control values. beta-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 +/- 12 vs. 8 +/- 3/mm2; p < 0.001). However, neither beta-amyloid precursor protein-immunoreactive dystrophic neurites nor beta-amyloid deposits were found in this tissue. Interleukin-1 alpha-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 +/- 8 vs. 25 +/- 5/mm2; p < 0.001), and these cells were often found adjacent to beta-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of beta-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

Bridging the gap between protein carboxyl methylation and phospholipid methylation to understand glucose-stimulated insulin secretion from the pancreatic beta cell.

PubMed

Kowluru, Anjaneyulu

2008-01-15

Recent findings have implicated post-translational modifications at C-terminal cysteines [e.g., methylation] of specific proteins [e.g., G-proteins] in glucose-stimulated insulin secretion [GSIS]. Furthermore, methylation at the C-terminal leucine of the catalytic subunit of protein phosphatase 2A [PP2Ac] has also been shown to be relevant for GSIS. In addition to these two classes of protein methyl transferases, a novel class of glucose-activated phospholipid methyl transferases have also been identified in the beta cell. These enzymes catalyze three successive methylations of phosphatidylethanolamine to yield phosphatidylcholine. The "newly formed" phosphatidylcholine is felt to induce alterations in the membrane fluidity, which might favor vesicular fusion with the plasma membrane for the exocytosis of insulin. The objectives of this commentary are to: (i) review the existing evidence on the regulation, by glucose and other insulin secretagogues, of post-translational carboxylmethylation [CML] of specific proteins in the beta cell; (ii) discuss the experimental evidence, which implicates regulation, by glucose and other insulin secretagogues, of phosphatidylethanolamine methylation in the islet beta cell; (iii) propose a model for potential cross-talk between the protein and lipid methylation pathways in the regulation of GSIS and (iv) highlight potential avenues for future research, including the development of specific pharmacological inhibitors to further decipher regulatory roles for these methylation reactions in islet beta cell function.

Thermogelling chitosan and collagen composite hydrogels initiated with beta-glycerophosphate for bone tissue engineering.

PubMed

Wang, Limin; Stegemann, Jan P

2010-05-01

Chitosan and collagen type I are naturally derived materials used as cell carriers because of their ability to mimic the extracellular environment and direct cell function. In this study beta-glycerophosphate (beta-GP), an osteogenic medium supplement and a weak base, was used to simultaneously initiate gelation of pure chitosan, pure collagen, and chitosan-collagen composite materials at physiological pH and temperature. Adult human bone marrow-derived stem cells (hBMSC) encapsulated in such hydrogels at chitosan/collagen ratios of 100/0, 65/35, 25/75, and 0/100 wt% exhibited high viability at day 1 after encapsulation, but DNA content dropped by about half over 12 days in pure chitosan materials while it increased twofold in materials containing collagen. Collagen-containing materials compacted more strongly and were significantly stiffer than pure chitosan gels. In monolayer culture, exposure of hBMSC to beta-GP resulted in decreased cell metabolic activity that varied with concentration and exposure time, but washing effectively removed excess beta-GP from hydrogels. The presence of chitosan in materials resulted in higher expression of osterix and bone sialoprotein genes in medium with and without osteogenic supplements. Chitosan also increased alkaline phosphatase activity and calcium deposition in osteogenic medium. Chitosan-collagen composite materials have potential as matrices for cell encapsulation and delivery, or as in situ gel-forming materials for tissue repair. Copyright 2010 Elsevier Ltd. All rights reserved.

Changes in beta cell function during the proximate post-diagnosis period in persons with type 1 diabetes.

PubMed

DiMeglio, Linda A; Cheng, Peiyao; Beck, Roy W; Kollman, Craig; Ruedy, Katrina J; Slover, Robert; Aye, Tandy; Weinzimer, Stuart A; Bremer, Andrew A; Buckingham, Bruce

2016-06-01

Prior studies examining beta-cell preservation in type 1 diabetes have predominantly assessed stimulated C-peptide concentrations approximately 10 wk after diagnosis. We examined whether earlier assessments might aid in prediction of beta cell function over time. Using data from a multi-center randomized trial assessing the effect of intensive diabetes management initiated within 1 wk of diagnosis, we assessed which clinical factors predicted 90-min mixed-meal tolerance test (MMTT) stimulated C-peptide values obtained 2 and 6 wk after diagnosis. We also studied associations of these factors with C-peptide values at 1- and 2-year post-diagnosis. Data from intervention and control groups were pooled. Among 67 study participants (mean age 13.3 ± 5.7 yr, range 7.8-45.7 yr) in multivariable analyses, C-peptide increased from baseline to 2 wks and then 6 wk. C-peptide levels at these times were significantly correlated with 1- and 2-yr C-peptide concentrations (all p < 0.001), with the strongest observed associations between 6-wk C-peptide and the 1- and 2-yr values (r = 0.66 and r = 0.61, respectively). In multivariable analyses, greater baseline and 6-wk C-peptide, and older age independently predicted greater 1- and 2-yr C-peptide concentrations. C-peptide assessments close to diagnosis were predictive of subsequent C-peptide production. Our data demonstrate a clear increase in C-peptide over the initial 6 wk after diabetes diagnosis followed by a plateau. Our data do not suggest that MMTT assessments performed closer to diagnosis than 6 wk would improve prediction of subsequent residual beta cell function. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Metabolic Stress and Compromised Identity of Pancreatic Beta Cells

PubMed Central

Swisa, Avital; Glaser, Benjamin; Dor, Yuval

2017-01-01

Beta cell failure is a central feature of type 2 diabetes (T2D), but the molecular underpinnings of the process remain only partly understood. It has been suggested that beta cell failure in T2D involves massive cell death. Other studies ascribe beta cell failure to cell exhaustion, due to chronic oxidative or endoplasmic reticulum stress leading to cellular dysfunction. More recently it was proposed that beta cells in T2D may lose their differentiated identity, possibly even gaining features of other islet cell types. The loss of beta cell identity appears to be driven by glucotoxicity inhibiting the activity of key beta cell transcription factors including Pdx1, Nkx6.1, MafA and Pax6, thereby silencing beta cell genes and derepressing alternative islet cell genes. The loss of beta cell identity is at least partly reversible upon normalization of glycemia, with implications for the reversibility of T2D, although it is not known if beta cell failure reaches eventually a point of no return. In this review we discuss current evidence for metabolism-driven compromised beta cell identity, key knowledge gaps and opportunities for utility in the treatment of T2D. PMID:28270834

Metabolic Stress and Compromised Identity of Pancreatic Beta Cells.

PubMed

Swisa, Avital; Glaser, Benjamin; Dor, Yuval

2017-01-01

Beta cell failure is a central feature of type 2 diabetes (T2D), but the molecular underpinnings of the process remain only partly understood. It has been suggested that beta cell failure in T2D involves massive cell death. Other studies ascribe beta cell failure to cell exhaustion, due to chronic oxidative or endoplasmic reticulum stress leading to cellular dysfunction. More recently it was proposed that beta cells in T2D may lose their differentiated identity, possibly even gaining features of other islet cell types. The loss of beta cell identity appears to be driven by glucotoxicity inhibiting the activity of key beta cell transcription factors including Pdx1, Nkx6.1, MafA and Pax6, thereby silencing beta cell genes and derepressing alternative islet cell genes. The loss of beta cell identity is at least partly reversible upon normalization of glycemia, with implications for the reversibility of T2D, although it is not known if beta cell failure reaches eventually a point of no return. In this review we discuss current evidence for metabolism-driven compromised beta cell identity, key knowledge gaps and opportunities for utility in the treatment of T2D.

Specific beta1-adrenergic receptor silencing with small interfering RNA lowers high blood pressure and improves cardiac function in myocardial ischemia.

PubMed

Arnold, Anne-Sophie; Tang, Yao Liang; Qian, Keping; Shen, Leping; Valencia, Valery; Phillips, Michael Ian; Zhang, Yuan Clare

2007-01-01

Beta-blockers are widely used and effective for treating hypertension, acute myocardial infarction (MI) and heart failure, but they present side-effects mainly due to antagonism of beta2-adrenergic receptor (AR). Currently available beta-blockers are at best selective but not specific for beta1 or beta2-AR. To specifically inhibit the expression of the beta1-AR, we developed a small interfering RNA (siRNA) targeted to beta1-AR. Three different sequences of beta1 siRNA were delivered into C6-2B cells with 90% efficiency. One of the three sequences reduced the level of beta1-AR mRNA by 70%. The siRNA was highly specific for beta1-AR inhibition with no overlap with beta2-AR. To test this in vivo, systemic injection of beta1 siRNA complexed with liposomes resulted in efficient delivery into the heart, lung, kidney and liver, and effectively reduced beta1-AR expression in the heart without altering beta2-AR. beta1 siRNA significantly lowered blood pressure of spontaneously hypertensive rats (SHR) for at least 12 days and reduced cardiac hypertrophy following a single injection. Pretreatment with beta1 siRNA 3 days before induction of MI in Wistar rats significantly improved cardiac function, as demonstrated by dP/dt and electrocardiogram following the MI. The protective mechanism involved reduction of cardiomyocyte apoptosis in the beta1 siRNA-treated hearts. The present study demonstrates the possibility of using siRNA for treating cardiovascular diseases and may represent a novel beta-blocker specific for beta1-AR.

Modulation of lens cell adhesion molecules by particle beams

NASA Technical Reports Server (NTRS)

McNamara, M. P.; Bjornstad, K. A.; Chang, P. Y.; Chou, W.; Lockett, S. J.; Blakely, E. A.

2001-01-01

Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast, there was a dose-dependent increase in ICAM-1 immunofluorescence in confluent, but not exponentially-growing cells. These results suggest that proton irradiation downregulates beta 1-integrin and upregulates ICAM-1, potentially contributing to cell death or to aberrant differentiation via modulation of anchorage and/or signal transduction functions. Quantification of the expression levels of the CAMs by Western analysis is in progress.

Abdominal {gamma}-Radiation Induces an Accumulation of Function-Impaired Regulatory T Cells in the Small Intestine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Billiard, Fabienne; Buard, Valerie; Benderitter, Marc

Purpose: To assess the frequency and the functional characteristics of one major component of immune tolerance, the CD4{sup +}FoxP3{sup +} regulatory T cells (Tregs) in a mouse model of abdominal irradiation. Methods and Materials: Mice were exposed to a single abdominal dose of {gamma}-radiation (10 Gy). We evaluated small intestine Treg infiltration by Foxp3 immunostaining and the functional suppressive activity of Tregs isolated from mesenteric lymph nodes. Results: Foxp3 immunostaining showed that radiation induced a long-term infiltration of the intestine by Tregs (levels 5.5 times greater than in controls). Co-culture of Tregs from mesenteric lymph nodes with CD4{sup +} effectormore » cells showed that the Tregs had lost their suppressive function. This loss was associated with a significant decrease in the levels of Foxp3, TGF-{beta}, and CTLA-4 mRNA, all required for optimal Treg function. At Day 90 after irradiation, Tregs regained their suppressive activity as forkhead box P3 (Foxp3), transforming growth factor beta (TGF-{beta}), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression returned to normal. Analysis of the secretory function of mesenteric lymph node Tregs, activated in vitro with anti-CD3/anti-CD28 Abs, showed that this dysfunction was independent of a defect in interleukin-10 secretion. Conclusion: Radiation caused a long-term accumulation of function-impaired Foxp3{sup +}CD4{sup +} Tregs in the intestine. Our study provides new insights into how radiation affects the immune tolerance in peripheral tissues.« less

Structural and biological mimicry of protein surface recognition by [alpha/beta]-peptide foldamers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horne, W. Seth; Johnson, Lisa M.; Ketas, Thomas J.

Unnatural oligomers that can mimic protein surfaces offer a potentially useful strategy for blocking biomedically important protein-protein interactions. Here we evaluate an approach based on combining {alpha}- and {beta}-amino acid residues in the context of a polypeptide sequence from the HIV protein gp41, which represents an excellent testbed because of the wealth of available structural and biological information. We show that {alpha}/{beta}-peptides can mimic structural and functional properties of a critical gp41 subunit. Physical studies in solution, crystallographic data, and results from cell-fusion and virus-infectivity assays collectively indicate that the gp41-mimetic {alpha}/{beta}-peptides effectively block HIV-cell fusion via a mechanism comparablemore » to that of gp41-derived {alpha}-peptides. An optimized {alpha}/{beta}-peptide is far less susceptible to proteolytic degradation than is an analogous {alpha}-peptide. Our findings show how a two-stage design approach, in which sequence-based {alpha} {yields} {beta} replacements are followed by site-specific backbone rigidification, can lead to physical and biological mimicry of a natural biorecognition process.« less

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

PubMed

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

Unraveling the contribution of pancreatic beta-cell suicide in autoimmune type 1 diabetes✩

PubMed Central

Jaberi-Douraki, Majid; Schnell, Santiago; Pietropaolo, Massimo; Khadra, Anmar

2014-01-01

In type 1 diabetes, an autoimmune disease mediated by autoreactive T-cells that attack insulin-secreting pancreatic beta-cells, it has been suggested that disease progression may additionally require protective mechanisms in the target tissue to impede such auto-destructive mechanisms. We hypothesize that the autoimmune attack against beta-cells causes endoplasmic reticulum stress by forcing the remaining beta-cells to synthesize and secrete defective insulin. To rescue beta-cell from the endoplasmic reticulum stress, beta-cells activate the unfolded protein response to restore protein homeostasis and normal insulin synthesis. Here we investigate the compensatory role of unfolded protein response by developing a multi-state model of type 1 diabetes that takes into account beta-cell destruction caused by pathogenic autoreactive T-cells and apoptosis triggered by endoplasmic reticulum stress. We discuss the mechanism of unfolded protein response activation and how it counters beta-cell extinction caused by an autoimmune attack and/or irreversible damage by endoplasmic reticulum stress. Our results reveal important insights about the balance between beta-cell destruction by autoimmune attack (beta-cell homicide) and beta-cell apoptosis by endoplasmic reticulum stress (beta-cell suicide). It also provides an explanation as to why the unfolded protein response may not be a successful therapeutic target to treat type 1 diabetes. PMID:24831415

The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by beta-amyloid peptide.

PubMed

Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, W E

2010-05-01

beta-Amyloid peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Abeta-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Abeta and on neurite outgrowth in PC12 cells were investigated. Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Abeta(1-42). Similar protective effects against Abeta(1-42) were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Abeta load was markedly diminished in the brain of those animals after treatment with piracetam. Abeta production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Abeta-induced mitochondrial dysfunction and Abeta-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Abeta on brain function.

Adaptation and impairment of DNA repair function in pollen of Betula verrucosa and seeds of Oenothera biennis from differently radionuclide-contaminated sites of Chernobyl.

PubMed

Boubriak, I I; Grodzinsky, D M; Polischuk, V P; Naumenko, V D; Gushcha, N P; Micheev, A N; McCready, S J; Osborne, D J

2008-01-01

The plants that have remained in the contaminated areas around Chernobyl since 1986 encapsulate the effects of radiation. Such plants are chronically exposed to radionuclides that they have accumulated internally as well as to alpha-, beta- and gamma-emitting radionuclides from external sources and from the soil. This radiation leads to genetic damage that can be countered by DNA repair systems. The objective of this study is to follow DNA repair and adaptation in haploid cells (birch pollen) and diploid cells (seed embryos of the evening primrose) from plants that have been growing in situ in different radionuclide fall-out sites in monitored regions surrounding the Chernobyl explosion of 1986. Radionuclide levels in soil were detected using gamma-spectroscopy and radiochemistry. DNA repair assays included measurement of unscheduled DNA synthesis, electrophoretic determination of single-strand DNA breaks and image analysis of rDNA repeats after repair intervals. Nucleosome levels were established using an ELISA kit. Birch pollen collected in 1987 failed to perform unscheduled DNA synthesis, but pollen at gamma/beta-emitter sites has now recovered this ability. At a site with high levels of combined alpha- and gamma/beta-emitters, pollen still exhibits hidden damage, as shown by reduced unscheduled DNA synthesis and failure to repair lesions in rDNA repeats properly. Evening primrose seed embryos generated on plants at the same gamma/beta-emitter sites now show an improved DNA repair capacity and ability to germinate under abiotic stresses (salinity and accelerated ageing). Again those from combined alpha- and gamma/beta-contaminated site do not show this improvement. Chronic irradiation at gamma/beta-emitter sites has provided opportunities for plant cells (both pollen and embryo cells) to adapt to ionizing irradiation and other environmental stresses. This may be explained by facilitation of DNA repair function.

Effect of beta-agonists on LAM progression and treatment.

PubMed

Le, Kang; Steagall, Wendy K; Stylianou, Mario; Pacheco-Rodriguez, Gustavo; Darling, Thomas N; Vaughan, Martha; Moss, Joel

2018-01-30

Lymphangioleiomyomatosis (LAM), a rare disease of women, is associated with cystic lung destruction resulting from the proliferation of abnormal smooth muscle-like LAM cells with mutations in the tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2 The mutant genes and encoded proteins are responsible for activation of the mechanistic target of rapamycin (mTOR), which is inhibited by sirolimus (rapamycin), a drug used to treat LAM. Patients who have LAM may also be treated with bronchodilators for asthma-like symptoms due to LAM. We observed stabilization of forced expiratory volume in 1 s over time in patients receiving sirolimus and long-acting beta-agonists with short-acting rescue inhalers compared with patients receiving only sirolimus. Because beta-agonists increase cAMP and PKA activity, we investigated effects of PKA activation on the mTOR pathway. Human skin TSC2 +/- fibroblasts or LAM lung cells incubated short-term with isoproterenol (beta-agonist) showed a sirolimus-independent increase in phosphorylation of S6, a downstream effector of the mTOR pathway, and increased cell growth. Cells incubated long-term with isoproterenol, which may lead to beta-adrenergic receptor desensitization, did not show increased S6 phosphorylation. Inhibition of PKA blocked the isoproterenol effect on S6 phosphorylation. Thus, activation of PKA by beta-agonists increased phospho-S6 independent of mTOR, an effect abrogated by beta-agonist-driven receptor desensitization. In agreement, retrospective clinical data from patients with LAM suggested that a combination of bronchodilators in conjunction with sirolimus may be preferable to sirolimus alone for stabilization of pulmonary function.

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.

PubMed

Wu, Kongming; Yang, Ying; Wang, Chenguang; Davoli, Maria A; D'Amico, Mark; Li, Anping; Cveklova, Kveta; Kozmik, Zbynek; Lisanti, Michael P; Russell, Robert G; Cvekl, Ales; Pestell, Richard G

2003-12-19

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

Molecular Mechanisms of Toxicity and Cell Damage by Chemicals in a Human Pancreatic Beta Cell Line, 1.1B4.

PubMed

Vasu, Srividya; McClenaghan, Neville H; Flatt, Peter R

2016-10-01

Mechanisms of toxicity and cell damage were investigated in novel clonal human pancreatic beta cell line, 1.1B4, after exposure to streptozotocin, alloxan, ninhydrin, and hydrogen peroxide. Viability, DNA damage, insulin secretion/content, [Ca]i, and glucokinase/hexokinase, mRNA expression were measured by MTT assay, comet assay, radioimmunoassay, fluorometric imaging plate reader, enzyme-coupled photometry, and real-time polymerase chain reaction, respectively. Chemicals significantly reduced 1.1B4 cell viability in a time/concentration-dependent manner. Chronic 18-hour exposure decreased cellular insulin, glucokinase, and hexokinase activities. Chemicals decreased transcription of INS, GCK, PCSK1, PCSK2, and GJA1 (involved in secretory function). Insulin release and [Ca]i responses to nutrients and membrane-depolarizing agents were impaired. Streptozotocin and alloxan up-regulated transcription of genes, SOD1 and SOD2 (antioxidant enzymes). Ninhydrin and hydrogen peroxide up-regulated SOD2 transcription, whereas alloxan and hydrogen peroxide increased CAT transcription. Chemicals induced DNA damage, apoptosis, and increased caspase 3/7 activity. Streptozotocin and alloxan decreased transcription of BCL2 while increasing transcription of BAX. Chemicals did not affect transcription of HSPA4 and HSPA5 and nitrite production. 1.1B4 cells represent a useful model of human beta cells. Chemicals impaired 1.1B4 cell secretory function and activated antioxidant defense and apoptotic pathways without activating endoplasmic reticulum stress response/nitrosative stress.

A DCM study of spectral asymmetries in feedforward and feedback connections between visual areas V1 and V4 in the monkey.

PubMed

Bastos, A M; Litvak, V; Moran, R; Bosman, C A; Fries, P; Friston, K J

2015-03-01

This paper reports a dynamic causal modeling study of electrocorticographic (ECoG) data that addresses functional asymmetries between forward and backward connections in the visual cortical hierarchy. Specifically, we ask whether forward connections employ gamma-band frequencies, while backward connections preferentially use lower (beta-band) frequencies. We addressed this question by modeling empirical cross spectra using a neural mass model equipped with superficial and deep pyramidal cell populations-that model the source of forward and backward connections, respectively. This enabled us to reconstruct the transfer functions and associated spectra of specific subpopulations within cortical sources. We first established that Bayesian model comparison was able to discriminate between forward and backward connections, defined in terms of their cells of origin. We then confirmed that model selection was able to identify extrastriate (V4) sources as being hierarchically higher than early visual (V1) sources. Finally, an examination of the auto spectra and transfer functions associated with superficial and deep pyramidal cells confirmed that forward connections employed predominantly higher (gamma) frequencies, while backward connections were mediated by lower (alpha/beta) frequencies. We discuss these findings in relation to current views about alpha, beta, and gamma oscillations and predictive coding in the brain. Copyright © 2015. Published by Elsevier Inc.

CRISPR/Cas9n-Mediated Deletion of the Snail 1Gene (SNAI1) Reveals Its Role in Regulating Cell Morphology, Cell-Cell Interactions, and Gene Expression in Ovarian Cancer (RMG-1) Cells.

PubMed

Haraguchi, Misako; Sato, Masahiro; Ozawa, Masayuki

2015-01-01

Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E-cadherin transcription, its precise roles in each of the phenomena of EMT are not completely understood, particularly in cytoskeletal changes. Previous studies have employed gene knockdown systems to determine the functions of Snail1. However, incomplete protein knockdown is often associated with these systems, which may cause incorrect interpretation of the data. To more precisely evaluate the functions of Snail1, we generated a stable cell line with a targeted ablation of Snail1 (Snail1 KO) by using the CRISPR/Cas9n system. Snail1 KO cells show increased cell-cell adhesion, decreased cell-substrate adhesion and cell migration, changes to their cytoskeletal organization that include few stress fibers and abundant cortical actin, and upregulation of epithelial marker genes such as E-cadherin, occludin, and claudin-1. However, morphological changes were induced by treatment of Snail1 KO cells with TGF-beta. Other transcription factors that induce EMT were also induced by treatment with TGF-beta. The precise deletion of Snail1 by the CRISPR/Cas9n system provides clear evidence that loss of Snail1 causes changes in the actin cytoskeleton, decreases cell-substrate adhesion, and increases cell-cell adhesion. Treatment of RMG1 cells with TGF-beta suggests redundancy among the transcription factors that induce EMT.

A special repertoire of alpha:beta T cells in neonatal mice.

PubMed Central

Bogue, M; Candéias, S; Benoist, C; Mathis, D

1991-01-01

According to several functional criteria, the mature thymocytes of neonatal and adult mice are distinctly different. We wondered whether these differences in function might have a structural correlate: do neonates have a distinct repertoire of alpha:beta T cells? In this study, we have exploited the power of polymerase chain reaction technology to generate large numbers of T cell receptor sequences from sorted thymocyte populations from newborn and adult mice. The newborn-derived sequences show very few N nucleotide additions, usually the major source of diversity in T cell receptors. Most interestingly, the paucity of N insertions appears to be exaggerated by selection events that operate during T cell differentiation in the thymus. The significance of these results is largely: (i) that they parallel recent findings on the B cell repertoire in neonates, raising questions about the reactivities specified by such a special repertoire; and (ii) that they suggest a means to 'tag' T cells exported perinatally, allowing one to test the premise that autoreactive T cells derive preferentially from the newborn repertoire. Images PMID:1834457

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp; Fujiwara, Yuki; Yamaguchi, Hideki

2011-05-20

Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however,more » its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.« less

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

PubMed

Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

2015-10-01

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. Copyright © 2015 Elsevier Inc. All rights reserved.

TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah

2011-11-18

Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology inmore » pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.« less

Residual beta cell function in newly diagnosed type 1 diabetes after treatment with atorvastatin: the Randomized DIATOR Trial.

PubMed

Martin, Stephan; Herder, Christian; Schloot, Nanette C; Koenig, Wolfgang; Heise, Tim; Heinemann, Lutz; Kolb, Hubert

2011-03-11

Recent evidence suggests that the lipid-lowering agent atorvastatin is also a potent immunomodulator. The aim of this study was to investigate the possible effect of atorvastatin on the decline of residual beta cell function in recent-onset type 1 diabetes. The randomised placebo-controlled Diabetes and Atorvastatin (DIATOR) Trial included 89 patients with newly diagnosed type 1 diabetes and islet autoantibodies (mean age 30 years, 40% females), in 12 centres in Germany. Patients received placebo or 80 mg/d atorvastatin for 18 months. As primary outcome stimulated serum C-peptide levels were determined 90 min after a standardized liquid mixed meal. An intent-to-treat analysis was performed. Fasting and stimulated C-peptide levels were not significantly different between groups at 18 months. However, median fasting serum C-peptide levels dropped from baseline to 12 and 18 months in the placebo group (from 0. 34 to 0.23 and 0.20 nmol/l, p<0.001) versus a nonsignificant decline in the atorvastatin group (from 0.34 to 0.27 and 0.30 nmol/l, ns). Median stimulated C-peptide concentrations declined between baseline and 12 months (placebo from 0.89 to 0.71 nmol/l, atorvastatin from 0.88 to 0.73 nmol/l, p<0.01 each) followed by a major loss by month 18 in the placebo group (to 0.48 nmol/l, p = 0.047) but not in the atorvastatin group (to 0.71 nmol/l, ns). Median levels of total cholesterol and C-reactive protein decreased in the atorvastatin group only (p<0.001 and p = 0.04). Metabolic control was similar between groups. Atorvastatin treatment did not significantly preserve beta cell function although there may have been a slower decline of beta-cell function which merits further study. ClinicalTrials.gov NCT00974740.

Insulin sensitivity and beta-cell function after carbohydrate oral loading in hip replacement surgery: a double-blind, randomised controlled clinical trial.

PubMed

Ljunggren, Stefan; Hahn, Robert G; Nyström, Thomas

2014-06-01

Surgery initiates a series of physiological stress processes in the body, inducing transient insulin resistance. Preoperative carbohydrate treatment can reduce the latter phenomenon. We investigated the effects of carbohydrate loading on insulin sensitivity and beta-cell function after elective hip replacement. Twenty-three nondiabetic patients (mean age of 68 years) who underwent elective hip replacement surgery participated in this double-blind controlled study. The patients were randomised to a nutrition group, which ingested a carbohydrate-rich fluid (50 kcal/100 ml) (Preop(®)), or a control group (tap water flavoured with lemon) 800 ml + 400 ml before the surgery. The insulin response (beta-cell function) and the insulin sensitivity were measured with an intravenous glucose tolerance test (IVGTT) and a hyperinsulinaemic euglycaemic glucose clamp, respectively, one day before and two days after the surgery. Insulin sensitivity decreased by 51% (median; 25-75th percentiles 35-61) after ingesting Preop(®) and by 39% (21-51) after ingesting in the control group (n.s.). The postoperative IVGTT in the nutrition group was followed by a significantly larger area under the curve (AUC) for plasma insulin (+54% versus the preoperative IVGTT) compared to the control group (+7%). This difference was already apparent during the first phase (0-10 min) of insulin secretion (+20 and -21%, respectively; P < 0.05). The patients randomised to the carbohydrate oral fluid or the water prior to the surgery demonstrated a significant but similar decrease in insulin sensitivity. The carbohydrates increased the beta-cell function as a compensatory response to the disposition index, resulting in a smaller reduction in surgery-induced insulin resistance compared to the tap water. The study was registered at http://www.clinicaltrials.gov (NCT01774084). Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Norrin mediates neuroprotective effects on retinal ganglion cells via activation of the Wnt/beta-catenin signaling pathway and the induction of neuroprotective growth factors in Muller cells.

PubMed

Seitz, Roswitha; Hackl, Simon; Seibuchner, Thomas; Tamm, Ernst R; Ohlmann, Andreas

2010-04-28

Norrin is a secreted protein that binds to frizzled 4 and controls development of capillaries in retina and inner ear. We provide evidence that Norrin has distinct neuroprotective properties that are independent from its effects on vascular development. The function of Norrin was investigated in a mouse model of excitotoxic retinal ganglion cell (RGC) damage after intravitreal injection of NMDA, and in cultured Müller glia or immortalized RGC-5 cells. Intravitreal injection of Norrin significantly increased the number of surviving RGC axons in the optic nerve and decreased apoptotic death of retinal neurons following NMDA-mediated damage. This effect could be blocked by adding dickkopf (DKK)-1, an inhibitor of the Wnt/beta-catenin signaling pathway. Treatment of eyes with combined Norrin/NMDA activated Wnt/beta-catenin signaling and increased the retinal expression of leukemia inhibitory factor and endothelin-2, as well as that of neurotrophic growth factors such as fibroblast growth factor-2, brain-derived neurotrophic factor, lens epithelium-derived growth factor, and ciliary neurotrophic factor. A similar activation of Wnt/beta-catenin signaling and an increased expression of neurotrophic factors was observed in cultured Müller cells after treatment with Norrin, effects that again could be blocked by adding DKK-1. In addition, conditioned cell culture medium of Norrin-treated Müller cells increased survival of differentiated RGC-5 cells. We conclude that Norrin has pronounced neuroprotective properties on retinal neurons with the distinct potential to decrease the damaging effects of NMDA-induced RGC loss. The effects of Norrin involve activation of Wnt/beta-catenin signaling and subsequent induction of neurotrophic growth factors in Müller cells.

Expression of alpha and beta subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the Wv or Sld loci.

PubMed

Schulte, B A; Steel, K P

1994-07-01

Mice homozygous for mutations at the viable dominant spotting (Wv) and Steel-dickie (Sld) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of Wv/Wv and Sld/Sld mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the alpha 1 and beta 1 subunits. Vestibular dark cells showed no reactivity for beta 1 but expressed abundant beta 2, whereas, strial marginal cells stained strongly for both beta isoforms. The only qualitative difference between mutant and wild-type mice was the absence of beta 1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the beta 1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K+ leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K+ normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final link in the conduit for moving K+ from perilymph to the intrastrial compartment.

Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium.

PubMed

Campbell, Shirley; Otis, Melissa; Côté, Mylène; Gallo-Payet, Nicole; Payet, Marcel Daniel

2003-04-01

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta.

PubMed

Coffinier, Catherine; Gresh, Lionel; Fiette, Laurence; Tronche, François; Schütz, Günther; Babinet, Charles; Pontoglio, Marco; Yaniv, Moshe; Barra, Jacqueline

2002-04-01

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). The paucity of small IHBD was linked to a failure in the organization of duct structures during liver organogenesis, suggesting an essential function of Hnf1b in bile duct morphogenesis. Mutant mice also lacked interlobular arteries. As HNF1beta is not expressed in these cells, it further emphasizes the link between arterial and biliary formation. Hepatocyte metabolism was also affected and we identified hepatocyte-specific HNF1beta target genes involved in bile acids sensing and in fatty acid oxidation.

Whole organism high content screening identifies stimulators of pancreatic beta-cell proliferation.

PubMed

Tsuji, Naoki; Ninov, Nikolay; Delawary, Mina; Osman, Sahar; Roh, Alex S; Gut, Philipp; Stainier, Didier Y R

2014-01-01

Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.

Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

PubMed

Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Integrin Beta 1 Suppresses Multilayering of a Simple Epithelium

PubMed Central

Chen, Jichao; Krasnow, Mark A.

2012-01-01

Epithelia are classified as either simple, a single cell layer thick, or stratified (multilayered). Stratified epithelia arise from simple epithelia during development, and transcription factor p63 functions as a key positive regulator of epidermal stratification. Here we show that deletion of integrin beta 1 (Itgb1) in the developing mouse airway epithelium abrogates airway branching and converts this monolayer epithelium into a multilayer epithelium with more than 10 extra layers. Mutant lung epithelial cells change mitotic spindle orientation to seed outer layers, and cells in different layers become molecularly and functionally distinct, hallmarks of normal stratification. However, mutant lung epithelial cells do not activate p63 and do not switch to the stratified keratin profile of epidermal cells. These data, together with previous data implicating Itgb1 in regulation of epidermal stratification, suggest that the simple-versus-stratified developmental decision may involve not only stratification inducers like p63 but suppressors like Itgb1 that prevent simple epithelia from inappropriately activating key steps in the stratification program. PMID:23285215

Cell kill by megavoltage protons with high LET.

PubMed

Kuperman, Vadim Y

2016-07-21

The aim of the current study is to develop a radiobiological model which describes the effect of linear energy transfer (LET) on cell survival and relative biological effectiveness (RBE) of megavoltage protons. By assuming the existence of critical sites within a cell, analytical expression for cell survival S as a function of LET is derived. The obtained results indicate that in cases where dose per fraction is small, [Formula: see text] is a linear-quadratic (LQ) function of dose while both alpha and beta radio-sensitivities are non-linearly dependent on LET. In particular, in the current model alpha increases with increasing LET while beta decreases. Conversely, in the case of large dose per fraction, the LQ dependence of [Formula: see text] on dose is invalid. The proposed radiobiological model predicts cell survival probability and RBE which, in general, deviate from the results obtained by using conventional LQ formalism. The differences between the LQ model and that described in the current study are reflected in the calculated RBE of protons.

Mechanical unloading of the failing human heart fails to activate the protein kinase B/Akt/glycogen synthase kinase-3beta survival pathway.

PubMed

Razeghi, Peter; Bruckner, Brian A; Sharma, Saumya; Youker, Keith A; Frazier, O H; Taegtmeyer, Heinrich

2003-01-01

Left ventricular assist device (LVAD) support of the failing human heart improves myocyte function and increases cell survival. One potential mechanism underlying this phenomenon is activation of the protein kinase B (PKB)/Akt/glycogen synthase kinase-3beta (GSK-3beta) survival pathway. Left ventricular tissue was obtained both at the time of implantation and explantation of the LVAD (n = 11). Six patients were diagnosed with idiopathic dilated cardiomyopathy, 4 patients with ischemic cardiomyopathy and 1 patient with peripartum cardiomyopathy. The mean duration of LVAD support was 205 +/- 35 days. Myocyte diameter and phosphorylation of ERK were used as indices for reverse remodeling. Transcript levels of genes required for the activation of PKB/Akt (insulin-like growth factor-1, insulin receptor substrate-1) were measured by quantitative RT-PCR. In addition, we measured the relative activity of PKB/Akt and GSK-3beta, and assayed for molecular and histological indices of PKB/Akt activation (cyclooxygenase mRNA levels and glycogen levels). Myocyte diameter and phosphorylation of ERK decreased with LVAD support. In contrast, none of the components of the PKB/Akt/GSK-3beta pathway changed significantly with mechanical unloading. The PKB/Akt/GSK-3beta pathway is not activated during LVAD support. Other signaling pathways must be responsible for the improvement of cellular function and cell survival during LVAD support. Copyright 2003 S. Karger AG, Basel

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion.

PubMed

Ramaswamy, S; Grace, C; Mattei, A A; Siemienowicz, K; Brownlee, W; MacCallum, J; McNeilly, A S; Duncan, W C; Rae, M T

2016-06-06

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P = 0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P = 0.001), sustained into adolescence (P = 0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P = 0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P = 0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P = 0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion

PubMed Central

Ramaswamy, S.; Grace, C.; Mattei, A. A.; Siemienowicz, K.; Brownlee, W.; MacCallum, J.; McNeilly, A. S.; Duncan, W. C.; Rae, M. T.

2016-01-01

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P = 0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P = 0.001), sustained into adolescence (P = 0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P = 0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P = 0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P = 0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells. PMID:27265420

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.

PubMed

Okuyama, H; Shimahara, Y; Kawada, N; Seki, S; Kristensen, D B; Yoshizato, K; Uyama, N; Yamaoka, Y

2001-07-27

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.

Repression of TGF-beta signaling by the oncogenic protein SKI in human melanomas: consequences for proliferation, survival, and metastasis.

PubMed

Medrano, Estela E

2003-05-19

Transforming growth factor-beta (TGF-beta ) has dual and paradoxical functions as a tumor suppressor and promoter of tumor progression and metastasis. TGF-Ji-mediated growth inhibition is gradually lost during melanoma tumor progression, but there are no measurable defects at the receptor level. Furthermore, melanoma cells release high levels of TGF-beta to the microenvironment, which upon activation induces matrix deposition, angiogenesis, survival, and transition to more aggressive phenotypes. The SKI and SnoN protein family associate with and repress the activity of Smad2, Smad3, and Smad4, three members of the TGF-fl signaling pathway. SKI also facilitates cell-cycle progression by targeting the RB pathway by at least two ways: it directly associates with RB and represses its activity when expressed at high levels, and indirectly, it represses Smad-mediated induction of p21(Waf-1) This results in increased CDK2 activity, RB phosphorylation,and inactivation. Therefore, high levels of SKI result in lesions to the RB pathway in a manner similar to p16 (INK4a) loss. SKI mRNA and protein levels dramatically increase during human melanoma tumor progression. In addition,the SKI protein shifts from nuclear localization in intraepidermal melanoma cells to nuclear and cytoplasmic in invasive and metastatic melanomas. Here, I discuss the basis for repression of intracellular TGF-beta signaling by SKI, some additional activities of this protein, and propose that by disrupting multiple tumor suppressor pathways, SKI functions as a melanoma oncogene.

TGF-{beta} receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Droguett, Rebeca; Cabello-Verrugio, Claudio; Santander, Cristian

2010-09-10

Skeletal muscle differentiation is strongly inhibited by transforming growth factor type {beta} (TGF-{beta}), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-{beta}-receptors (TGF-{beta}-Rs) during skeletal muscle differentiation. We found a decrease of TGF-{beta} signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-{beta}. No changemore » in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-{beta}-R type I (TGF-{beta}-RI) and type II (TGF-{beta}-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-{beta}-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-{beta}-RII lacking the cytoplasmic domain. The TGF-{beta}-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation; although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-{beta}-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-{beta} receptors independent of Smad proteins are essential for skeletal muscle differentiation.« less

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

PubMed

Soncin, Francesca; Mohamet, Lisa; Eckardt, Dominik; Ritson, Sarah; Eastham, Angela M; Bobola, Nicoletta; Russell, Angela; Davies, Steve; Kemler, Rolf; Merry, Catherine L R; Ward, Christopher M

2009-09-01

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad(-/-)), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad(-/-) ES cells is dependent on the beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad(-/-) ES cells is unlikely to be via beta-catenin signaling. Exposure of Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor beta family signaling in the self-renewal of Ecad(-/-) ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1.

PubMed

Kida, Hiroshi; Sugano, Yuri; Iizuka, Ryo; Fujihashi, Masahiro; Yohda, Masafumi; Miki, Kunio

2008-11-14

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two alpha and two beta subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of alpha (alpha1 and alpha2) and beta subunits (beta1 and beta2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD beta1 subunit at 1.9 A resolution and its functional analysis. TsPFD beta1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The beta hairpin linkers of beta1 subunits assemble to form a beta barrel "body" around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the beta1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric beta1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD beta1 subunits act as molecular chaperones in living cells of some archaea.

Glycogen synthase kinase-3beta and the p25 activator of cyclin dependent kinase 5 increase pausing of mitochondria in neurons.

PubMed

Morel, M; Authelet, M; Dedecker, R; Brion, J P

2010-06-02

The complex bi-directional axoplasmic transport of mitochondria is essential for proper metabolic functioning of neurons and is controlled by phosphorylation. We have investigated by time-lapse imaging the effects of increased expression of glycogen synthase kinase-3beta (GSK-3beta) and of the p25 activator of cyclin dependent kinase 5 on mitochondria movements in mammalian cortical neurons and in PC12 cells. Both GSK-3beta and p25 increased the stationary behaviour of mitochondria in PC12 and in neurons, decreased their anterograde transport but did not affect the intrinsic velocities of mitochondria. The microtubule-associated tau proteins were more phosphorylated in GSK-3beta and p25 transfected neurons, but ultrastructural observation showed that these cells still contained microtubules and nocodazole treatment further reduced residual mitochondria movements in GSK-3beta or p25 transfected neurons, indicating that microtubule disruption was not the primary cause of increased mitochondrial stationary behaviour in GSK-3beta or p25 transfected neurons. Our results suggest that increased expression of GSK-3beta and p25 acted rather by decreasing the frequency of mitochondrial movements driven by molecular motors and that GSK-3beta and p25 might regulate these transports by controlling the time that mitochondria spend pausing, rather than their velocities. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Creep function of a single living cell.

PubMed

Desprat, Nicolas; Richert, Alain; Simeon, Jacqueline; Asnacios, Atef

2005-03-01

We used a novel uniaxial stretching rheometer to measure the creep function J(t) of an isolated living cell. We show, for the first time at the scale of the whole cell, that J(t) behaves as a power-law J(t) = At(alpha). For N = 43 mice myoblasts (C2-7), we find alpha = 0.24 +/- 0.01 and A = (2.4 +/- 0.3) 10(-3) Pa(-1) s(-alpha). Using Laplace Transforms, we compare A and alpha to the parameters G(0) and beta of the complex modulus G*(omega) = G(0)omega(beta) measured by other authors using magnetic twisting cytometry and atomic force microscopy. Excellent agreement between A and G(0) on the one hand, and between alpha and beta on the other hand, indicated that the power-law is an intrinsic feature of cell mechanics and not the signature of a particular technique. Moreover, the agreement between measurements at very different size scales, going from a few tens of nanometers to the scale of the whole cell, suggests that self-similarity could be a central feature of cell mechanical structure. Finally, we show that the power-law behavior could explain previous results first interpreted as instantaneous elasticity. Thus, we think that the living cell must definitely be thought of as a material with a large and continuous distribution of relaxation time constants which cannot be described by models with a finite number of springs and dash-pots.

Inhibitory effect of tributyltin on expression of steroidogenic enzymes in mouse testis.

PubMed

Kim, Suel-Kee; Kim, Jong-Hoon; Han, Jung Ho; Yoon, Yong-Dal

2008-01-01

Tributyltin (TBT) is known to disrupt the development of reproductive organs, thereby reducing fertility. The aim of this study was to evaluate the acute toxicity of TBT on the testicular development and steroid hormone production. Immature (3-week-old) male mice were given a single administration of 25, 50, or 100 mg/kg of TBT by oral gavage. Lumen formation in seminiferous tubule was remarkably delayed, and the number of apoptotic germ cells found inside the tubules was increased in the TBT-exposed animals, whereas no apoptotic signal was observed in interstitial Leydig cells. Reduced serum testosterone concentration and down-regulated expressions of the mRNAs for cholesterol side-chain cleavage enzyme (P450scc), 17alpha -hydroxylase/C(17-20) lyase (P450(17alpha)), 3beta -hydroxysteroid-dehydrogenase (3beta -HSD), and 17beta -hydroxysteroid-dehydrogenase (17beta -HSD) were also observed after TBT exposure. Altogether, these findings demonstrate that exposure to TBT is associated with induced apoptosis of testicular germ cells and inhibition of steroidogenesis by reduction in the expression of steroidogenic enzymes in interstitial Leydig cells. These adverse effects of TBT would cause serious defects in testicular development and function.

Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions.

PubMed

Sugita, Sunao; Horie, Shintaro; Nakamura, Orie; Maruyama, Kazuichi; Takase, Hiroshi; Usui, Yoshihiko; Takeuchi, Masaru; Ishidoh, Kazumi; Koike, Masato; Uchiyama, Yasuo; Peters, Christoph; Yamamoto, Yoshimi; Mochizuki, Manabu

2009-10-15

Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

Amyloid precursor protein and Presenilin 1 interaction studied by FRET in human H4 cells.

PubMed

Nizzari, Mario; Venezia, Valentina; Bianchini, Paolo; Caorsi, Valentina; Diaspro, Alberto; Repetto, Emanuela; Thellung, Stefano; Corsaro, Alessandro; Carlo, Pia; Schettini, Gennaro; Florio, Tullio; Russo, Claudio

2007-01-01

The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.

HES6 reverses nuclear reprogramming of insulin-producing cells following cell fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ball, Andrew J.; Abrahamsson, Annelie E.; Tyrberg, Bjoern

2007-04-06

To examine the mechanism by which growth-stimulated pancreatic {beta}-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and {beta}lox5, a cell line derived from human {beta}-cells which progressively dedifferentiated in culture. MIN6/{beta}lox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that {beta}lox5 expresses a dominant transacting factor(s) that represses {beta}-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary {beta}-cells as well as MIN6, but wasmore » repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in {beta}-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the {beta}-cell differentiated state.« less

What couples glycolysis to mitochondrial signal generation in glucose-stimulated insulin secretion?

PubMed

Ishihara, H; Wollheim, C B

2000-05-01

Pancreatic islet beta-cells are poised to generate metabolic messengers in the mitochondria that link glucose metabolism to insulin exocytosis. This is accomplished through the tight coupling of glycolysis to mitochondrial activation. The messenger molecules ATP and glutamate are produced after the metabolism of glycolysis-derived pyruvate in the mitochondria. The entry of monocarboxylates such as pyruvate into the beta cell is limited, explaining why overexpression of monocarboxylate transporters unravels pyruvate-stimulated insulin secretion. NADH generated by glycolysis is efficiently reoxidized by highly active mitochondrial shuttles rather than by lactate dehydrogenase. Overexpression of this enzyme does not alter glucose-stimulated insulin secretion, suggesting that NADH availability restricts the conversion of pyruvate to lactate in the beta cell. These metabolic features permit the fuel function of glucose to be extended to the generation of signaling molecules, which increases cytosolic Ca2+ and promotes insulin exocytosis.

Transport of beta-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis.

PubMed

Kunji, E R; Hagting, A; De Vries, C J; Juillard, V; Haandrikman, A J; Poolman, B; Konings, W N

1995-01-27

In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed. Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein). The wild type strain and the DtpT- mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy. The amino acids are not accumulated significantly inside the cells by the Opp- and DtpT- Opp- mutants. When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains. Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system. These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L. lactis.

New discoveries on the biology and detection of human chorionic gonadotropin

PubMed Central

Cole, Laurence A

2009-01-01

Human chorionic gonadotropin (hCG) is a glycoprotein hormone comprising 2 subunits, alpha and beta joined non covalently. While similar in structure to luteinizing hormone (LH), hCG exists in multiple hormonal and non-endocrine agents, rather than as a single molecule like LH and the other glycoprotein hormones. These are regular hCG, hyperglycosylated hCG and the free beta-subunit of hyperglycosylated hCG. For 88 years regular hCG has been known as a promoter of corpus luteal progesterone production, even though this function only explains 3 weeks of a full gestations production of regular hCG. Research in recent years has explained the full gestational production by demonstration of critical functions in trophoblast differentiation and in fetal nutrition through myometrial spiral artery angiogenesis. While regular hCG is made by fused villous syncytiotrophoblast cells, extravillous invasive cytotrophoblast cells make the variant hyperglycosylated hCG. This variant is an autocrine factor, acting on extravillous invasive cytotrophoblast cells to initiate and control invasion as occurs at implantation of pregnancy and the establishment of hemochorial placentation, and malignancy as occurs in invasive hydatidiform mole and choriocarcinoma. Hyperglycosylated hCG inhibits apoptosis in extravillous invasive cytotrophoblast cells promoting cell invasion, growth and malignancy. Other non-trophoblastic malignancies retro-differentiate and produce a hyperglycosylated free beta-subunit of hCG (hCG free beta). This has been shown to be an autocrine factor antagonizing apoptosis furthering cancer cell growth and malignancy. New applications have been demonstrated for total hCG measurements and detection of the 3 hCG variants in pregnancy detection, monitoring pregnancy outcome, determining risk for Down syndrome fetus, predicting preeclampsia, detecting pituitary hCG, detecting and managing gestational trophoblastic diseases, diagnosing quiescent gestational trophoblastic disease, diagnosing placental site trophoblastic tumor, managing testicular germ cell malignancies, and monitoring other human malignancies. There are very few molecules with such wide and varying functions as regular hCG and its variants, and very few tests with such a wide spectrum of clinical applications as total hCG. PMID:19171054

Smad3 phosphoisoform-mediated signaling during sporadic human colorectal carcinogenesis.

PubMed

Matsuzaki, K

2006-06-01

Transforming growth factor-beta (TGF-beta) signaling occurring during human colorectal carcinogenesis involves a shift in TGF-beta function, reducing the cytokine's antiproliferative effect, while increasing actions that promote invasion and metastasis. TGF-beta signaling involves phosphorylation of Smad3 at serine residues 208 and 213 in the linker region and serine residues 423 and 425 in the C-terminal region. Exogenous TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). Either pSmad3C or pSmad3L oligomerizes with Smad4, and translocates into nuclei. While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells in vivo, JNK/pSmad3L-mediated signaling promotes tumor cell invasion and extracellular matrix synthesis by activated mesenchymal cells. Furthermore, hepatocyte growth factor signaling interacts with TGF-beta to activate the JNK/pSmad3L pathway, accelerating nuclear transport of cytoplasmic pSmad3L. This reduces accessibility of unphosphorylated Smad3 to membrane-anchored TbetaRI, preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and antiproliferative effects of TGF-beta on epithelial cells. As neoplasia progresses from normal colorectal epithelium through adenoma to invasive adenocarcinoma with distant metastasis, nuclear pSmad3L gradually increases while pSmad3C decreases. The shift from TbetaRI/pSmad3C-mediated to JNK/pSmad3L-mediated signaling is a major mechanism orchestrating a complex transition of TGF-beta signaling during sporadic human colorectal carcinogenesis. This review summarizes the recent understanding of Smad3 phosphoisoform-mediated signaling, particularly 'cross-talk' between Smad3 and JNK pathways that cooperatively promote oncogenic activities. Understanding of these actions should help to develop more effective therapy against human colorectal cancer, involving inhibition of JNK/pSmad3L pathway.

Progressive deterioration of beta-cell function in obese youth with type 2 diabetes

USDA-ARS?s Scientific Manuscript database

In adults, type 2 diabetes (T2DM) is characterized with progressive deterioration in insulin secretion. Data are scanty in youth. We investigated prospectively the change in ß-cell function and in insulin sensitivity in youth with T2DM. Six adolescents with T2DM [hemoglobin A1c (HbA1c) 6.6 +/- 1.0%]...

Fibronectin regulates the activation of THP-1 cells by TGF-beta1.

PubMed

Wang, A C; Fu, L

2001-03-01

To determine how fibronectin regulates the immunomodulatory effects of transforming growth factor (TGF)-beta on THP-1 cells. THP-1 monocytic cell line. THP-1 cells were primed for 48 h in the presence or absence of 250 pM TGF-beta1. Assays or assessments carried out, together with statistical test applied. We found that adherence to fibronectin dramatically modulates the effects of TGF-beta1 on the human monocytic cell line THP-1. TGF-beta did not significantly affect constitutive interleukin (IL)-8 secretion or IL-1beta-induced IL-8 secretion from suspended cells. In contrast, TGF-beta stimulated IL-8 secretion as well as augmented IL-1beta-induced IL-8 secretion from adherent cells. The differential effects of TGF-beta1 on IL-8 secretion from suspended and adherent cells could not be explained by differences in IL-1 receptor antagonist production. The effects of fibronectin on TGF-beta1 induced IL-8 secretion from THP-1 cells were mimicked by adhesion to immobilized anti-a4beta1 integrin antibody and to a fibronectin fragment containing the CS-1 domain. These results indicate that alpha4beta1-mediated adhesion to fibronectin may play a key role during inflammation by profoundly influencing the effects of TGF-beta1 on monocytes.

SKI knockdown inhibits human melanoma tumor growth in vivo.

PubMed

Chen, Dahu; Lin, Qiushi; Box, Neil; Roop, Dennis; Ishii, Shunsuke; Matsuzaki, Koichi; Fan, Tao; Hornyak, Thomas J; Reed, Jon A; Stavnezer, Ed; Timchenko, Nikolai A; Medrano, Estela E

2009-12-01

The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

Infection of human T lymphotropic virus-I-specific immune T cell clones by human T lymphotropic virus-I.

PubMed Central

Mitsuya, H; Jarrett, R F; Cossman, J; Cohen, O J; Kao, C S; Guo, H G; Reitz, M S; Broder, S

1986-01-01

Human T lymphotropic virus-I (HTLV-I)-specific T cell lines were established and cloned. K5, an OKT8+ clone bearing multiple proviral integration sites, retained its HTLV-I-specific cytotoxicity and a normal dependence on interleukin 2 (IL-2), indicating that there is a finite number of transforming integration sites. R2, an OKT4+ HTLV-I-infected clone, initially mounted a proliferative response to HTLV-I; but then its IL-2-independent proliferation increased and the antigen specificity was lost. All HTLV-I-infected clones tested including K7, another OKT8+ transformed cytotoxic clone that had lost its reactivity, expressed comparable levels of T cell receptor beta-chain (TCR-beta) messenger (m)RNA. Although clones K5 and K7 had different functional properties, they had the same rearrangement of the TCR-beta gene, suggesting that they had the same clonal origin. These data indicate that HTLV-I-specific T cells retain their immune reactivity for variable periods of time following infection, but then usually lose it; in some cases, however, no alteration in function can be detected. The data also suggest that different consequences can take place in the same clone depending on the pattern of retroviral infection. Images PMID:2877011

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

PubMed Central

van der Meulen, Talitha; Xie, Ruiyu; Kelly, Olivia G.; Vale, Wylie W.; Sander, Maike; Huising, Mark O.

2012-01-01

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. PMID:23251699

Hyperoxia reduces insulin release and induces mitochondrial dysfunction with possible implications for hyperoxic treatment of neonates.

PubMed

Hals, Ingrid; Ohki, Tsuyoshi; Singh, Rinku; Ma, Zuheng; Björklund, Anneli; Balasuriya, Chandima; Scholz, Hanne; Grill, Valdemar

2017-10-01

We previously showed that hyperoxia in vitro negatively affects beta cells of the rat. Here, we tested for possible clinical significance as well as mitochondrial interactions by hyperoxia, using human islets (function and viability), INS-1 832/13 cells (mitochondrial metabolism), and mouse neonates (effects in vivo). Lastly, we assessed relevant parameters in a cohort of individuals born preterm and then exposed to hyperoxia. Human islets and INS-1 832/13 cells were exposed to 24 h of hyperoxia (90-92% oxygen). Mouse neonates were subjected to 5 days of continuous hyperoxia. Individuals born preterm were evaluated in terms of glucose homeostasis and beta cell function by HbA1c and the HOMA2 formula. In human islets, hyperoxia significantly reduced glucose-stimulated insulin secretion by 42.2 ± 5.3% and viability assessed by MTT by 22.5 ± 5.4%. Hyperoxia down-regulated mitochondrial complex II by 21 ± 5% and upregulated complex III by 26 ± 10.1% and complex IV by 37 ± 10.6%. Partly similar effects on mitochondrial complexes were found in hyperoxia-exposed INS-1 832/13 cells. Exposure to hyperoxia swiftly reduced oxygen consumption in these cells and increased mitochondrial uncoupling. Hyperoxia transiently but significantly reduced insulin release in mouse neonates. Individuals born preterm displayed higher HbA1c versus controls, as well as insulin resistance. Thus, hyperoxia exerts negative effects in vitro on human beta cells and results indicate inhibitory effects on insulin secretion in vivo in mouse neonates. Negative effects may be lessened by the demonstrated swift and profound mitochondrial adaptability. Our findings open the possibility that hyperoxia could negatively affect beta cells of preterm human neonates. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

PubMed Central

Engl, Tobias; Makarević, Jasmina; Relja, Borna; Natsheh, Iyad; Müller, Iris; Beecken, Wolf-Dietrich; Jonas, Dietger; Blaheta, Roman A

2005-01-01

Background Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype. PMID:15644133

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

PubMed

Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

2010-08-01

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

Improvement of macrophage dysfunction by administration of anti-transforming growth factor-beta antibody in EL4-bearing hosts.

PubMed

Maeda, H; Tsuru, S; Shiraishi, A

1994-11-01

An experimental therapy for improvement of macrophage dysfunction caused by transforming growth factor-beta (TGF-beta) was tried in EL4 tumor-bearing mice. TGF-beta was detected in cell-free ascitic fluid from EL4-bearers, but not in that from normal mice, by western blot analysis. The ascites also showed growth-suppressive activity against Mv1Lu cells, and the suppressive activity was potentiated by transient acidification. To investigate whether the functions of peritoneal macrophages were suppressed in EL4-bearers, the abilities to produce nitric oxide and tumor necrosis factor-alpha (TNF-alpha) upon lipopolysaccharide (LPS) stimulation were measured. Both abilities of macrophages in EL4-bearing mice were suppressed remarkably on day 9, and decreased further by day 14, compared with non-tumor-bearing controls. TGF-beta activity was abrogated by administration of anti-TGF-beta antibody to EL4-bearing mice. While a large amount of TGF-beta was detected in ascitic fluid from control EL4-bearers, little TGF-beta was detectable in ascites from EL4-bearers given anti-TGF-beta antibody. Furthermore, while control macrophages exhibited little or no production of nitric oxide and TNF-alpha on LPS stimulation in vitro, macrophages from EL4-bearers administered with anti-TGF-beta antibody showed the same ability as normal macrophages. These results clearly indicate that TGF-beta contributes to macrophage dysfunction and that the administration of specific antibody for TGF-beta reverses macrophage dysfunction in EL4-bearing hosts.

Silencing of Smed-betacatenin1 generates radial-like hypercephalized planarians.

PubMed

Iglesias, Marta; Gomez-Skarmeta, Jose Luis; Saló, Emili; Adell, Teresa

2008-04-01

Little is known about the molecular mechanisms responsible for axis establishment during non-embryonic processes such as regeneration and homeostasis. To address this issue, we set out to analyze the role of the canonical Wnt pathway in planarians, flatworms renowned for their extraordinary morphological plasticity. Canonical Wnt signalling is an evolutionarily conserved mechanism to confer polarity during embryonic development, specifying the anteroposterior (AP) axis in most bilaterians and the dorsoventral (DV) axis in early vertebrate embryos. beta-Catenin is a key element in this pathway, although it is a bifunctional protein that is also involved in cell-cell adhesion. Here, we report the characterization of two beta-catenin homologs from Schmidtea mediterranea (Smed-betacatenin1/2). Loss of function of Smed-betacatenin1, but not Smed-betacatenin2, in both regenerating and intact planarians, generates radial-like hypercephalized planarians in which the AP axis disappears but the DV axis remains unaffected, representing a unique example of a striking body symmetry transformation. The radial-like hypercephalized phenotype demonstrates the requirement for Smed-betacatenin1 in AP axis re-establishment and maintenance, and supports a conserved role for canonical Wnt signalling in AP axis specification, whereas the role of beta-catenin in DV axis establishment would be a vertebrate innovation. When considered alongside the protein domains present in each S. mediterranea beta-catenin and the results of functional assays in Xenopus embryos demonstrating nuclear accumulation and axis induction with Smed-betacatenin1, but not Smed-betacatenin2, these data suggest that S. mediterranea beta-catenins could be functionally specialized and that only Smed-betacatenin1 is involved in Wnt signalling.

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

PubMed Central

Gewirtz, A M; Calabretta, B; Rucinski, B; Niewiarowski, S; Xu, W Y

1989-01-01

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis. Images PMID:2523411

Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

NASA Technical Reports Server (NTRS)

Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

2002-01-01

Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

PubMed Central

Bolisetty, Mohan; Kursawe, Romy; Sun, Lili; Sivakamasundari, V.; Kycia, Ina

2017-01-01

Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type–specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis. PMID:27864352

Characterization of T cell repertoire changes in acute Kawasaki disease

PubMed Central

1993-01-01

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology that is associated with marked activation of T cells and monocyte/macrophages. Using a quantitative polymerase chain reaction (PCR) technique, we recently found that the acute phase of KD is associated with the expansion of T cells expressing the V beta 2 and V beta 8.1 gene segments. In the present work, we used a newly developed anti-V beta 2 monoclonal antibody (mAb) and studied a new group of KD patients to extend our previous PCR results. Immunofluorescence analysis confirmed that V beta 2-bearing T cells are selectively increased in patients with acute KD. The increase occurred primarily in the CD4 T cell subset. The percentages of V beta 2+ T cells as determined by mAb reactivity and flow cytometry correlated linearly with V beta expression as quantitated by PCR. However, T cells from acute KD patients appeared to express proportionately higher levels of V beta 2 transcripts per cell as compared with healthy controls or convalescent KD patients. Sequence analysis of T cell receptor beta chain genes of V beta 2 and V beta 8.1 expressing T cells from acute KD patients showed extensive junctional region diversity. These data showing polyclonal expansion of V beta 2+ and V beta 8+ T cells in acute KD provide additional insight into the immunopathogenesis of this disease. PMID:8094737

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

PubMed

Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

2015-10-13

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu

2005-06-17

Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1more » proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physiopathology.« less

Advanced glycation end products and the progressive course of renal disease.

PubMed

Heidland, A; Sebekova, K; Schinzel, R

2001-10-01

In experimental and human diabetic nephropathy (DN), it has been shown that advanced glycation end products (AGEs), in particular, carboxymethyl-lysine and pentosidine, accumulate with malondialdehyde in glomerular lesions in relation to disease severity and in the presence of an upregulated receptor for AGE (RAGE) in podocytes. Toxic effects of AGEs result from structural and functional alterations in plasma and extracellular matrix (ECM) proteins, in particular, from cross-linking of proteins and interaction of AGEs with their receptors and/or binding proteins. In mesangial and endothelial cells, the AGE-RAGE interaction caused enhanced formation of oxygen radicals with subsequent activation of nuclear factor-kappaB and release of pro-inflammatory cytokines (interleukin-6, tumor necrosis factor-alpha), growth factors (transforming growth factor-beta1 [TGF-beta1], insulin-like growth factor-1), and adhesion molecules (vascular cell adhesion molecule-1, intercellular adhesion molecule-1). In tubular cells, incubation with AGE albumin was followed by stimulation of the mitogen-activating protein (MAP) kinase pathway and its downstream target, the activating protien-1 (AP-1) complex, TGF-beta1 overexpression, enhanced protein kinase C activity, decreased cell proliferation, and impaired protein degradation rate, in part caused by decreased cathepsin activities. The pathogenic relevance of AGEs was further verified by in vivo experiments in euglycemic rats and mice by the parenteral administration of AGE albumin, leading in the glomeruli to TGF-beta1 overproduction, enhanced gene expression of ECM proteins, and morphological lesions similar to those of DN. Evidence for the pathogenic relevance of AGEs in DN also comes from experimental studies in which the formation and/or action of AGEs was modulated by aminoguanidine, OPB-9195, pyridoxamine, soluble RAGEs, serine protease trypsin, and antioxidants, resulting in improved cell and/or renal function.

Integrin αvβ6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis.

PubMed

Peng, Zhen-Wei; Ikenaga, Naoki; Liu, Susan B; Sverdlov, Deanna Y; Vaid, Kahini A; Dixit, Richa; Weinreb, Paul H; Violette, Shelia; Sheppard, Dean; Schuppan, Detlef; Popov, Yury

2016-01-01

Integrin αvβ6 is rapidly up-regulated on cells of epithelial lineage during tissue injury, where one of its primary functions is activation of latent transforming growth factor beta 1 (TGFβ1). In human liver cirrhosis, αvβ6 is overexpressed by cells comprising the ductular reaction, and its inhibition suppresses experimental biliary fibrosis in rodents. Here, we show that αvβ6 is expressed on the actively proliferating subset of hepatic progenitor cells and is required for their progenitor function in vivo and in vitro through integrin αvβ6-dependent TGFβ1 activation. Freshly isolated αvβ6(+) liver cells demonstrate clonogenic potential and differentiate into cholangiocytes and functional hepatocytes in vitro, whereas colony formation by epithelial cell adhesion molecule-positive progenitor cells is blocked by αvβ6-neutralizing antibody and in integrin beta 6-deficient cells. Inhibition of progenitors by anti-αvβ6 antibody is recapitulated by TGFβ1 neutralization and rescued by addition of bioactive TGFβ1. Genetic disruption or selective targeting of αvβ6 with 3G9 antibody potently inhibits progenitor cell responses in mouse models of chronic biliary injury and protects from liver fibrosis and tumorigenesis, two conditions clinically associated with exacerbated ductular reaction. These results suggest that αvβ6 is a promising target for chronic fibrotic liver diseases and associated cancers. © 2015 by the American Association for the Study of Liver Diseases.

Alpha-SNAP functions in insulin exocytosis from mature, but not immature secretory granules in pancreatic beta cells.

PubMed

Nakamichi, Y; Nagamatsu, S

1999-06-24

To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells. Copyright 1999 Academic Press.

Mitochondria-targeted antioxidants protect against amyloid-beta toxicity in Alzheimer's disease neurons.

PubMed

Manczak, Maria; Mao, Peizhong; Calkins, Marcus J; Cornea, Anda; Reddy, Arubala P; Murphy, Michael P; Szeto, Hazel H; Park, Byung; Reddy, P Hemachandra

2010-01-01

The purpose of our study was to investigate the effects of the mitochondria-targeted antioxidants, MitoQ and SS31, and the anti-aging agent resveratrol on neurons from a mouse model (Tg2576 line) of Alzheimer's disease (AD) and on mouse neuroblastoma (N2a) cells incubated with the amyloid-beta (Abeta) peptide. Using electron and confocal microscopy, gene expression analysis, and biochemical methods, we studied mitochondrial structure and function and neurite outgrowth in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta. In N2a cells only incubated with the Abeta, we found increased expressions of mitochondrial fission genes and decreased expression of fusion genes and also decreased expression of peroxiredoxins. Electron microscopy of the N2a cells incubated with Abeta revealed a significantly increased number of mitochondria, indicating that Abeta fragments mitochondria. Biochemical analysis revealed that function is defective in mitochondria. Neurite outgrowth was significantly decreased in Abeta-incubated N2a cells, indicating that Abeta affects neurite outgrowth. However, in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta, abnormal expression of peroxiredoxins and mitochondrial structural genes were prevented and mitochondrial function was normal; intact mitochondria were present and neurite outgrowth was significantly increased. In primary neurons from amyloid-beta precursor protein transgenic mice that were treated with MitoQ and SS31, neurite outgrowth was significantly increased and cyclophilin D expression was significantly decreased. These findings suggest that MitoQ and SS31 prevent Abeta toxicity, which would warrant the study of MitoQ and SS31 as potential drugs to treat patients with AD.

[Exploration of relationship between the expression level of DNA polymerase beta and 60Co gamma-ray radiosensitivity].

PubMed

Cui, Jie; Xu, Xin; Yang, Mo; Chen, Chen; Zhao, Wei; Wu, Mei; Zhang, Zun-zhen

2011-11-01

To explore the relationship between the expression level of DNA polymerase beta (pol beta) and 60Co gamma-ray radiosensitivity and provide a basis on improving the efficiency of radiotherapy theoretically. pol beta wild-type cells (pol beta +/+), pol beta null cells (pol beta -/-) and pol beta overexpressed cells (polp beta oe) were applied as a model system. The radiosensitivity of 60Co gamma-ray on the cell was detected by MTT assay and clone formation assay. The DCFH-DA fluorescent probe was used to examine the cellular ROS after 60Co gamma-rays radiation. MTT assay showed that after radiation by 60Co gamma-rays followed with 72 h incubation, the cell viabilities in the three kinds of cells decreased significantly with a dose-response relationship (r-/+ = -0.976, r-/- = -0.977, r(oe) = -0.982, P<0.05). In addition, the viability of pol beta -/- cell was lower than those of other two kinds of cells at the same dose (P<0.05). Likewise, the colony number and colony formation rate in all tested cells also decreased after exposure to 60Co gamma-rays. The ROS level in the three kinds of cells was enhanced after treatment with 60Co gamma-ray, and the ROS level in pol beta -/- cells was much higher than that in the other two kinds of cells (P<0.05). Cell death caused by 60Co gamma-ray may associated with the DNA oxidative damage mediated by ROS; Overexpression of pol beta could protect against oxidative DNA damage, thus the cell apoptosis/death, thereby leading to reducing the radiosensitivity of 60Co gamma-rays, while null of DNA pol beta could increase radiosensitivity of 60Co gamma-rays by compromising the DNA repair.

Inorganic mercury causes pancreatic beta-cell death via the oxidative stress-induced apoptotic and necrotic pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Yawen; Huang Chunfa; Yang Chingyao

2010-03-15

Mercury is a well-known highly toxic metal. In this study, we characterize and investigate the cytotoxicity and its possible mechanisms of inorganic mercury in pancreatic beta-cells. Mercury chloride (HgCl{sub 2}) dose-dependently decreased the function of insulin secretion and cell viability in pancreatic beta-cell-derived HIT-T15 cells and isolated mouse pancreatic islets. HgCl{sub 2} significantly increased ROS formation in HIT-T15 cells. Antioxidant N-acetylcysteine effectively reversed HgCl{sub 2}-induced insulin secretion dysfunction in HIT-T15 cells and isolated mouse pancreatic islets. Moreover, HgCl{sub 2} increased sub-G1 hypodiploids and annexin-V binding in HIT-T15 cells, indicating that HgCl{sub 2} possessed ability in apoptosis induction. HgCl{sub 2} alsomore » displayed several features of mitochondria-dependent apoptotic signals including disruption of the mitochondrial membrane potential, increase of mitochondrial cytochrome c release and activations of poly (ADP-ribose) polymerase (PARP) and caspase 3. Exposure of HIT-T15 cells to HgCl{sub 2} could significantly increase both apoptotic and necrotic cell populations by acridine orange/ethidium bromide dual staining. Meanwhile, HgCl{sub 2} could also trigger the depletion of intracellular ATP levels and increase the LDH release from HIT-T15 cells. These HgCl{sub 2}-induced cell death-related signals could be significantly reversed by N-acetylcysteine. The intracellular mercury levels were markedly elevated in HgCl{sub 2}-treated HIT-T15 cells. Taken together, these results suggest that HgCl{sub 2}-induced oxidative stress causes pancreatic beta-cell dysfunction and cytotoxicity involved the co-existence of apoptotic and necrotic cell death.« less

Beta-cell function in individuals carrying the mitochondrial tRNA leu (UUR) mutation.

PubMed

Salles, João Eduardo; Kasamatsu, Teresa S; Dib, Sérgio A; Moisés, Regina S

2007-01-01

To assess the beta-cell function in individuals with mitochondrial DNA A3243G mutation with normal glucose tolerance (NGT) or diabetes mellitus (DM). Furthermore, in diabetic individuals, we evaluated the effect of coenzyme Q10 supplementation on insulin secretory response. Eight mutation-positive individuals with NGT (n = 4) or DM (n = 4) were studied. beta-Cell function was evaluated by C-peptide levels before and after a mixed liquid meal (Sustacal) challenge and by first-phase insulin response. Fasting and Sustacal-stimulated C-peptide levels were significantly lower in diabetic patients than that in controls (area under the curve: 104.1 +/- 75.7 vs 520.8 +/- 173.8, P = 0.001), whereas in individuals with NGT, this response was preserved (area under the curve: 537.8 +/- 74.3 vs 520.8 +/- 179.8, P = 0.87). The duration of diabetes was negatively correlated with fasting C-peptide levels (r = -0.961, P = 0.038). Among the 3 patients with residual insulin secretion, the short-term treatment with coenzyme Q10 (3 months) improved C-peptide levels in 2 of them. The first-phase insulin response was diminished in 2 individuals with NGT, the oldest ones. We showed an impaired insulin secretory capacity in individuals carrying the A3243G mutation, this possibly being the primary defect contributing to the development of DM. In addition, our data suggest that this could be a functional defect.

E-cadherin and cell adhesion: a role in architecture and function in the pancreatic islet.

PubMed

Rogers, Gareth J; Hodgkin, Matthew N; Squires, Paul E

2007-01-01

The efficient secretion of insulin from beta-cells requires extensive intra-islet communication. The cell surface adhesion protein epithelial (E)-cadherin (ECAD) establishes and maintains epithelial tissues such as the islets of Langerhans. In this study, the role of ECAD in regulating insulin secretion from pseudoislets was investigated. The effect of an immuno-neutralising ECAD on gross morphology, cytosolic calcium signalling, direct cell-to-cell communication and insulin secretion was assessed by fura-2 microfluorimetry, Lucifer Yellow dye injection and insulin ELISA in an insulin-secreting model system. Antibody blockade of ECAD reduces glucose-evoked changes in [Ca(2+)](i) and insulin secretion. Neutralisation of ECAD causes a breakdown in the glucose-stimulated synchronicity of calcium oscillations between discrete regions within the pseudoislet, and the transfer of dye from an individual cell within a cell cluster is attenuated in the absence of ECAD ligation, demonstrating that gap junction communication is disrupted. The functional consequence of neutralising ECAD is a significant reduction in insulin secretion. Cell adhesion via ECAD has distinct roles in the regulation of intercellular communication between beta-cells within islets, with potential repercussions for insulin secretion.

Leupaxin acts as a mediator in prostate carcinoma progression through deregulation of p120catenin expression.

PubMed

Kaulfuss, S; von Hardenberg, S; Schweyer, S; Herr, A M; Laccone, F; Wolf, S; Burfeind, P

2009-11-12

Recently, we could show that the focal adhesion protein leupaxin (LPXN) is expressed in human prostate carcinomas (PCa) and induces invasiveness of androgen-independent PCa cells. In this study we show that LPXN enhanced the progression of existing PCa in vivo by breeding transgenic mice with prostate-specific LPXN expression and TRAMP mice (transgenic adenocarcinoma of mouse prostate). Double transgenic LPXN/TRAMP mice showed a significant increase in poorly differentiated PCa and distant metastases as compared with control TRAMP mice. Additional studies on primary PCa cells generated from both transgenic backgrounds confirmed the connection regarding LPXN overexpression and increased motility and invasiveness of PCa cells. One mediator of LPXN-induced invasion was found to be the cell-cell adhesion protein p120catenin (p120CTN). Both in vitro and in vivo experiments revealed that p120CTN expression negatively correlates with LPXN expression, followed by a redistribution of beta-catenin. Downregulation of LPXN using small interfering RNAs (siRNAs) resulted in a membranous localization of beta-catenin, whereas strong nuclear accumulation of beta-catenin was observed in p120CTN knockdown cells leading to enhanced transcription of the beta-catenin target gene matrix metalloprotease-7. In conclusion, the present results indicate that LPXN enhances the progression of PCa through downregulation of p120CTN expression and that LPXN could function as a marker for aggressive PCa in the future.

Differential expression of islet glutaredoxin 1 and 5 with high reactive oxygen species production in a mouse model of diabesity.

PubMed

Petry, Sebastian Friedrich; Sharifpanah, Fatemeh; Sauer, Heinrich; Linn, Thomas

2017-01-01

The onset and progression of diabetes mellitus type 2 is highly contingent on the amount of functional beta-cell mass. An underlying cause of beta-cell decay in diabetes is oxidative stress, which markedly affects the insulin producing pancreatic cells due to their poor antioxidant defence capacity. Consequently, disturbances of cellular redox signaling have been implicated to play a major role in beta-cell loss in diabetes mellitus type 2. There is evidence suggesting that the glutaredoxin (Grx) system exerts a protective role for pancreatic islets, but the exact mechanisms have not yet been elucidated. In this study, a mouse model for diabetes mellitus type 2 was used to gain further insight into the significance of Grx for the islets of Langerhans in the diabetic metabolism. We have observed distinct differences in the expression levels of Grx in pancreatic islets between obese, diabetic db mice and lean, non-diabetic controls. This finding is the first report about a decrease of Grx expression levels in pancreatic islets of diabetic mice which was accompanied by declining insulin secretion, increase of reactive oxygen species (ROS) production level, and cell cycle alterations. These data demonstrate the essential role of the Grx system for the beta-cell during metabolic stress which may provide a new target for diabetes mellitus type 2 treatment.

A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells

PubMed Central

Johnsson, Per; Ackley, Amanda; Vidarsdottir, Linda; Lui, Weng-Onn; Corcoran, Martin; Grandér, Dan; Morris, Kevin V.

2013-01-01

PTEN is a tumor suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1 encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, alpha and beta. The alpha isoform functions in trans, localizes to the PTEN promoter, and epigenetically modulates PTEN transcription by the recruitment of DNMT3a and EZH2. In contrast, the beta isoform interacts with PTENpg1 through an RNA:RNA pairing interaction, which affects PTEN protein output via changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell cycle arrest and sensitizes cells to doxorubicin, suggesting a biological function for the respective PTENpg1 expressed asRNAs. PMID:23435381

MTNR1B rs10830963 is associated with fasting plasma glucose, HbA1C and impaired beta-cell function in Chinese Hans from Shanghai

PubMed Central

2010-01-01

Background Genome-wide association studies (GWAS) in White Europeans have shown that genetic variation rs10830963 in melatonin receptor 1B gene (MTNR1B) is associated with fasting glucose and type 2 diabetes, which has also been replicated in several Asian populations. As a variant in the gene involved in the regulation of circadian rhythms, the effect of the variant on sleep status remains unknown. This study aimed to investigate the effects of MTNR1B rs10830963 on fasting glucose, type 2 diabetes and sleep status in Chinese Hans. Methods MTNR1B rs10830963 was genotyped in a population-based cohort including 3,210 unrelated Chinese Hans from Beijing and Shanghai, and tested for associations with risk of type 2 diabetes, diabetes-related traits and sleep status. Results We confirmed the associations of MTNR1B rs10830963 with fasting glucose (beta = 0.11 mmol/l, 95%CI [0.03, 0.18], P = 0.005), glycated hemoglobin (HbA1c) (beta = 0.07%, 95%CI [0.02,0.12], P = 0.004) and homeostasis model assessment of beta-cell function (HOMA-B) (beta = -5.01%, 95%CI [-8.24,-1.77], P = 0.003) in the Shanghai, but not in the Beijing subpopulation (P ≥ 0.58). The effect size of MTNR1B rs10830963 on fasting glucose in Shanghai Chinese Hans was comparable to that reported from other Asian populations. We found no evidence of associations with type 2 diabetes (OR 1.05 [0.90-1.23], P = 0.54), homeostasis model assessment of insulin sensitivity (HOMA-S) (P = 0.86) or sleep status (P ≥ 0.44). Conclusions A common variant in MTNR1B was associated with fasting glucose, HbA1C and HOMA-B but not with sleep status in Chinese Hans from Shanghai, strengthening the role of MTNR1B rs10830963 in fasting glycemia and impaired beta-cell function. PMID:20398260

Atypical properties of a conventional calcium channel beta subunit from the platyhelminth Schistosoma mansoni.

PubMed

Salvador-Recatalà, Vicenta; Schneider, Toni; Greenberg, Robert M

2008-03-26

The function of voltage-gated calcium (Cav) channels greatly depends on coupling to cytoplasmic accessory beta subunits, which not only promote surface expression, but also modulate gating and kinetic properties of the alpha1 subunit. Schistosomes, parasitic platyhelminths that cause schistosomiasis, express two beta subunit subtypes: a structurally conventional beta subunit and a variant beta subunit with unusual functional properties. We have previously characterized the functional properties of the variant Cavbeta subunit. Here, we focus on the modulatory phenotype of the conventional Cavbeta subunit (SmCavbeta) using the human Cav2.3 channel as the substrate for SmCavbeta and the whole-cell patch-clamp technique. The conventional Schistosoma mansoni Cavbeta subunit markedly increases Cav2.3 currents, slows macroscopic inactivation and shifts steady state inactivation in the hyperpolarizing direction. However, currents produced by Cav2.3 in the presence of SmCavbeta run-down to approximately 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration. This suppressive effect was independent of Ca2+, but dependent on intracellular Mg2+-ATP. Additional experiments revealed that SmCavbeta lends the Cav2.3/SmCavbeta complex sensitivity to Na+ ions. A mutant version of the Cavbeta subunit lacking the first forty-six amino acids, including a string of twenty-two acidic residues, no longer conferred sensitivity to intracellular Mg2+-ATP and Na+ ions, while continuing to show wild type modulation of current amplitude and inactivation of Cav2.3. The data presented in this article provide insights into novel mechanisms employed by platyhelminth Cavbeta subunits to modulate voltage-gated Ca2+ currents that indicate interactions between the Ca2+ channel complex and chelated forms of ATP as well as Na+ ions. These results have potentially important implications for understanding previously unknown mechanisms by which platyhelminths and perhaps other organisms modulate Ca2+ currents in excitable cells.

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay.

PubMed

Zheng, Xiaofen; De Paiva, Cintia S; Rao, Kavita; Li, De-Quan; Farley, William J; Stern, Michael; Pflugfelder, Stephen C

2010-09-01

To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.

Studies on the bioavailability of the provitamin A carotenoid, beta-carotene, using human exfoliated colonic epithelial cells.

PubMed

Gireesh, T; Nair, P P; Sudhakaran, P R

2004-08-01

The possibility of using exfoliated colonic epithelial cells for assessing the bioavailability of beta-carotene was examined. Analysis of exfoliated colonic epithelial cells showed the presence of beta-carotene and vitamin A. The beta-carotene content was significantly lower in cells from stool samples of subjects on a beta-carotene-poor diet than those receiving a single dose of a beta-carotene supplement. Colonic epithelial cells isolated from stool samples collected daily during a wash-out period while the subjects were on a beta-carotene-poor diet showed a steady decrease in beta-carotene content, reaching the lowest value on day 7. Kinetic analysis showed that a single dose of a beta-carotene supplement in the form of spirulina (Spirulina platensis) or agathi (Sesbania grandiflora) after the wash-out period caused an increase in the beta-carotene content after a lag period of 5-7 d, but the vitamin A levels during these periods were not significantly affected. Analysis of plasma beta-carotene concentration also showed similar changes, which correlated with those of exfoliated colonic cells. A relationship between the beta-carotene content of the diet and that of the colonic epithelial cells suggests that analysis of the beta-carotene content in exfoliated human colonic epithelial cells is a useful non-invasive method to assess the bioavailability of provitamin A beta-carotene.

p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex.

PubMed

Pardossi-Piquard, Raphaëlle; Dunys, Julie; Giaime, Emilie; Guillot-Sestier, Marie-Victoire; St George-Hyslop, Peter; Checler, Frédéric; Alves da Costa, Cristine

2009-04-01

Nicastrin (NCT) is a component of the presenilin (PS)-dependent gamma-secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent gamma-secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the gamma-secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes.

Characterizing components of the Saw Palmetto Berry Extract (SPBE) on prostate cancer cell growth and traction.

PubMed

Scholtysek, Carina; Krukiewicz, Aleksandra A; Alonso, José-Luis; Sharma, Karan P; Sharma, Pal C; Goldmann, Wolfgang H

2009-02-13

Saw Palmetto Berry Extract (SPBE) is applied for prostate health and treatment of urinary tract infections, nonbacterial prostitis and Benign Prostatic Hyperplasia (BPH) in man. An assumption is that SPBE affects tumor cell progression and migration in breast and prostate tissue. In this work, DU-145 cells were used to demonstrate that SPBE and its sterol components, beta-sitosterol and stigmasterol, inhibit prostate cancer growth by increasing p53 protein expression and also inhibit carcinoma development by decreasing p21 and p27 protein expression. In the presence of cholesterol, these features are not only reversed but increased significantly. The results show for the first time the potential of SPBE, beta-sitosterol and stigmasterol as potential anti-tumor agents. Since the protein p53 is also regarded as nuclear matrix protein facilitating actin cytoskeletal binding, 2D tractions were measured. The cell adhesion strength in the presence of SPBE, beta-sitosterol and cholesterol and the observation was that the increase in p53 expression triggered an increase in the intracellular force generation. The results suggest a dual function of p53 in cells.

Hyperglycaemia attenuates in vivo reprogramming of pancreatic exocrine cells to beta cells in mice

PubMed Central

Cavelti-Weder, Claudia; Li, Weida; Zumsteg, Adrian; Stemann-Andersen, Marianne; Zhang, Yuemei; Yamada, Takatsugu; Wang, Max; Lu, Jiaqi; Jermendy, Agnes; Bee, Yong Mong; Bonner-Weir, Susan; Weir, Gordon C.; Zhou, Qiao

2016-01-01

Aims/hypothesis Reprogramming of pancreatic exocrine to insulin-producing cells by viral delivery of the genes encoding transcription factors neurogenin-3 (Ngn3), pancreas/duodenum homeobox protein 1 (Pdx1) and MafA is an efficient method for reversing diabetes in murine models. The variables that modulate reprogramming success are currently ill-defined. Methods Here, we assess the impact of glycaemia on in vivo reprogramming in a mouse model of streptozotocin-induced beta cell ablation, using subsequent islet transplantation or insulin pellet implantation for creation of groups with differing levels of glycaemia before viral delivery of transcription factors. Results We observed that hyperglycaemia significantly impaired reprogramming of exocrine to insulin-producing cells in their quantity, differentiation status and function. With hyperglycaemia, the reprogramming of acinar towards beta cells was less complete. Moreover, inflammatory tissue changes within the exocrine pancreas including macrophage accumulation were found, which may represent the tissue’s response to clear the pancreas from insufficiently reprogrammed cells. Conclusions/interpretation Our findings shed light on normoglycaemia as a prerequisite for optimal reprogramming success in a diabetes model, which might be important in other tissue engineering approaches and disease models, potentially facilitating their translational applications. PMID:26693711

Hyperglycaemia attenuates in vivo reprogramming of pancreatic exocrine cells to beta cells in mice.

PubMed

Cavelti-Weder, Claudia; Li, Weida; Zumsteg, Adrian; Stemann-Andersen, Marianne; Zhang, Yuemei; Yamada, Takatsugu; Wang, Max; Lu, Jiaqi; Jermendy, Agnes; Bee, Yong Mong; Bonner-Weir, Susan; Weir, Gordon C; Zhou, Qiao

2016-03-01

Reprogramming of pancreatic exocrine to insulin-producing cells by viral delivery of the genes encoding transcription factors neurogenin-3 (Ngn3), pancreas/duodenum homeobox protein 1 (Pdx1) and MafA is an efficient method for reversing diabetes in murine models. The variables that modulate reprogramming success are currently ill-defined. Here, we assess the impact of glycaemia on in vivo reprogramming in a mouse model of streptozotocin-induced beta cell ablation, using subsequent islet transplantation or insulin pellet implantation for creation of groups with differing levels of glycaemia before viral delivery of transcription factors. We observed that hyperglycaemia significantly impaired reprogramming of exocrine to insulin-producing cells in their quantity, differentiation status and function. With hyperglycaemia, the reprogramming of acinar towards beta cells was less complete. Moreover, inflammatory tissue changes within the exocrine pancreas including macrophage accumulation were found, which may represent the tissue's response to clear the pancreas from insufficiently reprogrammed cells. Our findings shed light on normoglycaemia as a prerequisite for optimal reprogramming success in a diabetes model, which might be important in other tissue engineering approaches and disease models, potentially facilitating their translational applications.

SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of {beta}-catenin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Il-Rae; Koh, Sang Seok; Department of Functional Genomics, University of Science and Technology, Daejeon 305-333

2012-06-29

Highlights: Black-Right-Pointing-Pointer SIRT1 inhibits protein levels of {beta}-catenin and its transcriptional activity. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for the decrease of {beta}-catenin expression. Black-Right-Pointing-Pointer SIRT1-mediated degradation of {beta}-catenin is not required for GSK-3{beta} and Siah-1 but for proteosome. Black-Right-Pointing-Pointer SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of {beta}-catenin, we postulated that {beta}-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, knownmore » to target {beta}-catenin in a colon cancer model, suppresses {beta}-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of {beta}-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced {beta}-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of {beta}-catenin. Treatment with MG132, a proteasomal inhibitor, restored {beta}-catenin protein levels, suggesting that SIRT1-mediated degradation of {beta}-catenin requires proteasomal activity. It was reported that inhibition of GSK-3{beta} or Siah-1 stabilizes {beta}-catenin in colon cancer cells, but suppression of GSK-3{beta} or Siah-1 using siRNA in the presence of resveratrol instead diminished {beta}-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3{beta} and Siah-1 are not involved in SIRT1-mediated degradation of {beta}-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target molecule of {beta}-catenin. These results suggest that SIRT1 activation may be a therapeutic strategy for treatment of pancreatic cancer cells that express PAUF via the down-regulation of {beta}-catenin.« less

PED/PEA-15 inhibits hydrogen peroxide-induced apoptosis in Ins-1E pancreatic beta-cells via PLD-1.

PubMed

Fiory, Francesca; Parrillo, Luca; Raciti, Gregory Alexander; Zatterale, Federica; Nigro, Cecilia; Mirra, Paola; Falco, Roberta; Ulianich, Luca; Di Jeso, Bruno; Formisano, Pietro; Miele, Claudia; Beguinot, Francesco

2014-01-01

The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg(PED/PEA-15) mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E(PED/PEA-15)). In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E(PED/PEA-15) cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E(PED/PEA-15). These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

Upregulation of CD94 on CD8+T Cells in Anterior Chamber-Associated Immune Deviation

PubMed Central

He, Hao; Yang, Peizeng; Jiang, Liqiong; Zhang, Junfeng; Zhao, Changlin; Chen, Lina; Lin, Xiaomin; Zhou, Hongyan; Kijlstra, Aize

2008-01-01

Background CD8+ regulatory T cells (Treg) have been considered to be involved in a model of ocular-induced tolerance, known as anterior chamber-associated immune deviation (ACAID). The phenotype and characteristics of CD8+Treg in ACAID remain only poorly understood. Recent studies have reported that the CD94-Qa-1 system is implicated in the induction of ACAID CD8+Treg, but the functions and characteristics of CD8+CD94+T cells remain unclear. Results Both mRNA and protein of CD94 and NKG2A were markedly up-regulated on splenic CD8+T cells of ACAID mice compared with controls. Flow cytometric analysis showed that very few CD8+CD94+T cells express granzyme B, perforin and Foxp3. CD8+CD94+T cells, but not CD8+CD94-T cells, magnetically isolated from the spleens of ACAID mice, produced large amounts of TGF-beta1 and exhibited suppressive activity in vitro. Neutralization of TGF-beta1 caused reversal of suppression mediated by CD8+CD94+T cells. Conclusion CD8+CD94+T cells from ACAID mice exhibited suppressive activity in association with enhanced expression of TGF-beta1, suggesting that CD8+Treg are mainly distributed in CD94+T cell subpopulations. PMID:18816417

Platelet-derived growth factor-DD targeting arrests pathological angiogenesis by modulating glycogen synthase kinase-3beta phosphorylation.

PubMed

Kumar, Anil; Hou, Xu; Lee, Chunsik; Li, Yang; Maminishkis, Arvydas; Tang, Zhongshu; Zhang, Fan; Langer, Harald F; Arjunan, Pachiappan; Dong, Lijin; Wu, Zhijian; Zhu, Linda Y; Wang, Lianchun; Min, Wang; Colosi, Peter; Chavakis, Triantafyllos; Li, Xuri

2010-05-14

Platelet-derived growth factor-DD (PDGF-DD) is a recently discovered member of the PDGF family. The role of PDGF-DD in pathological angiogenesis and the underlying cellular and molecular mechanisms remain largely unexplored. In this study, using different animal models, we showed that PDGF-DD expression was up-regulated during pathological angiogenesis, and inhibition of PDGF-DD suppressed both choroidal and retinal neovascularization. We also demonstrated a novel mechanism mediating the function of PDGF-DD. PDGF-DD induced glycogen synthase kinase-3beta (GSK3beta) Ser(9) phosphorylation and Tyr(216) dephosphorylation in vitro and in vivo, leading to increased cell survival. Consistently, GSK3beta activity was required for the antiangiogenic effect of PDGF-DD targeting. Moreover, PDGF-DD regulated the expression of GSK3beta and many other genes important for angiogenesis and apoptosis. Thus, we identified PDGF-DD as an important target gene for antiangiogenic therapy due to its pleiotropic effects on vascular and non-vascular cells. PDGF-DD inhibition may offer new therapeutic options to treat neovascular diseases.

miR-324-3p promotes gastric cancer development by activating Smad4-mediated Wnt/beta-catenin signaling pathway.

PubMed

Sun, Guang-Li; Li, Zheng; Wang, Wei-Zhi; Chen, Zheng; Zhang, Lei; Li, Qing; Wei, Song; Li, Bo-Wen; Xu, Jiang-Hao; Chen, Liang; He, Zhong-Yuan; Ying, Kai; Zhang, Xuan; Xu, Hao; Zhang, Dian-Cai; Xu, Ze-Kuan

2018-06-01

Emerging evidence suggested that miRNAs can function as oncogenes or tumor suppressors by regulating downstream target genes. miR-324-3p has been reported to function in several carcinomas, but its role in gastric cancer (GC) is still unknown. This study aims to explore the effects of miR-324-3p on the development of GC. Expression of miR-324-3p was examined in GC cells and tissues by qRT-PCR. Effects of miR-324-3p on GC cells were evaluated by cell vitality assay, colony formation assay, cell migration assay, and flow cytometric assay. The dual luciferase assay was used to verify whether miR-324-3p could interact with the potential target genes. Western blot was used to assess the expression level of Smad4 and beta-catenin. Intracellular ATP level was also examined. The tumor xenografts were established using nude mice. A gastric organoid model was made from fresh stomach tissue. miR-324-3p was expressed at higher levels in the tumor tissues compared with adjacent normal tissues. Overexpression of miR-324-3p promoted cell growth, migration, and decreased apoptosis. miR-324-3p repressed the expression of Smad4, and loss of Smad4 activated the Wnt/beta-catenin signaling pathway. Overexpression of Smad4 rescued the effects of miR-324-3p on GC cells. The intracellular ATP level was upregulated with overexpression of miR-324-3p. miR-324-3p facilitated tumor cell colonization and growth in vivo and contributed to the growth of gastric organoids. The results suggested that miR-324-3p promoted GC through activating the Smad4-mediated Wnt/beta-catenin signaling pathway. The miR-324-3p/Smad4/Wnt signaling axis may be a potential therapeutic target to prevent GC progression.

The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells.

PubMed

Kanamori, Mutsumi; Sandy, Peter; Marzinotto, Stefania; Benetti, Roberta; Kai, Chikatoshi; Hayashizaki, Yoshihide; Schneider, Claudio; Suzuki, Harukazu

2003-10-03

Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is beta-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of beta-catenin that may control its transcriptional activity, we performed a mammalian two-hybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between beta-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of beta-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of beta-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous beta-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorage-independent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/beta-catenin signaling pathway.

Relationship of Soluble RAGE with Insulin Resistance and Beta Cell Function during Development of Type 2 Diabetes Mellitus

PubMed Central

Biswas, Subrata Kumar; Mohtarin, Sabreena; Mudi, Sonchita Rani; Anwar, Taznuva; Banu, Laila Anjuman; Alam, Sheikh Md. Khorshed; Fariduddin, Md.; Arslan, M. Iqbal

2015-01-01

This study examined whether circulating levels of soluble receptor for advanced glycation end products (sRAGE) alter in prediabetes and correlate with insulin resistance (IR) and beta cell function in prediabetes and newly diagnosed type 2 diabetes mellitus (T2DM). Subjects without previous history of diabetes were recruited and grouped as control, prediabetes, and newly diagnosed T2DM. The control subjects (n = 40) and people with prediabetes (n = 52) and diabetes (n = 66) were similar in terms of age, sex, BMI, systolic and diastolic BP, and fasting insulin level. HOMA-IR was found significantly higher in people with diabetes than control subjects (p < 0.001) and people with prediabetes (p = 0.005); and HOMA-%B was found significantly deteriorated in people with diabetes (p < 0.001) compared to control subjects and people with prediabetes. However, serum sRAGE levels did not show any significant alteration in people with prediabetes compared to control subjects. Moreover, univariate and multivariate analyses did not identify any significant correlation and statistical association of sRAGE with HOMA-IR and HOMA-%B in people with prediabetes and newly diagnosed T2DM. Our data suggest that serum sRAGE levels do not alter in people with prediabetes compared to control subjects and do not correlate or associate with IR and beta cell function during development of T2DM. PMID:26078977

Relationship of Soluble RAGE with Insulin Resistance and Beta Cell Function during Development of Type 2 Diabetes Mellitus.

PubMed

Biswas, Subrata Kumar; Mohtarin, Sabreena; Mudi, Sonchita Rani; Anwar, Taznuva; Banu, Laila Anjuman; Alam, Sheikh Md Khorshed; Fariduddin, Md; Arslan, M Iqbal

2015-01-01

This study examined whether circulating levels of soluble receptor for advanced glycation end products (sRAGE) alter in prediabetes and correlate with insulin resistance (IR) and beta cell function in prediabetes and newly diagnosed type 2 diabetes mellitus (T2DM). Subjects without previous history of diabetes were recruited and grouped as control, prediabetes, and newly diagnosed T2DM. The control subjects (n = 40) and people with prediabetes (n = 52) and diabetes (n = 66) were similar in terms of age, sex, BMI, systolic and diastolic BP, and fasting insulin level. HOMA-IR was found significantly higher in people with diabetes than control subjects (p < 0.001) and people with prediabetes (p = 0.005); and HOMA-%B was found significantly deteriorated in people with diabetes (p < 0.001) compared to control subjects and people with prediabetes. However, serum sRAGE levels did not show any significant alteration in people with prediabetes compared to control subjects. Moreover, univariate and multivariate analyses did not identify any significant correlation and statistical association of sRAGE with HOMA-IR and HOMA-%B in people with prediabetes and newly diagnosed T2DM. Our data suggest that serum sRAGE levels do not alter in people with prediabetes compared to control subjects and do not correlate or associate with IR and beta cell function during development of T2DM.

Mixed-meal tolerance test versus glucagon stimulation test for the assessment of beta-cell function in therapeutic trials in type 1 diabetes.

PubMed

Greenbaum, Carla J; Mandrup-Poulsen, Thomas; McGee, Paula Friedenberg; Battelino, Tadej; Haastert, Burkhard; Ludvigsson, Johnny; Pozzilli, Paolo; Lachin, John M; Kolb, Hubert

2008-10-01

Beta-cell function in type 1 diabetes clinical trials is commonly measured by C-peptide response to a secretagogue in either a mixed-meal tolerance test (MMTT) or a glucagon stimulation test (GST). The Type 1 Diabetes TrialNet Research Group and the European C-peptide Trial (ECPT) Study Group conducted parallel randomized studies to compare the sensitivity, reproducibility, and tolerability of these procedures. In randomized sequences, 148 TrialNet subjects completed 549 tests with up to 2 MMTT and 2 GST tests on separate days, and 118 ECPT subjects completed 348 tests (up to 3 each) with either two MMTTs or two GSTs. Among individuals with up to 4 years' duration of type 1 diabetes, >85% had measurable stimulated C-peptide values. The MMTT stimulus produced significantly higher concentrations of C-peptide than the GST. Whereas both tests were highly reproducible, the MMTT was significantly more so (R(2) = 0.96 for peak C-peptide response). Overall, the majority of subjects preferred the MMTT, and there were few adverse events. Some older subjects preferred the shorter duration of the GST. Nausea was reported in the majority of GST studies, particularly in the young age-group. The MMTT is preferred for the assessment of beta-cell function in therapeutic trials in type 1 diabetes.

Recombineering Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

From the rat to the beta cell: a fast and effective technique of separation of Langerhans islets and direct purification of pancreatic beta cells.

PubMed

Tamagno, Gianluca; Vigolo, Simonetta; Olivieri, Massimiliano; Martini, Chiara; De Carlo, Eugenio

2014-01-01

Isolated Langerhans islets represent a useful model for the study of the endocrine pancreas. The possibility to purify pancreatic beta cells from a mixed Langerhans islet cell population may lead towards a dedicated focus on beta cell research. We describe an effective and rapid immunomagnetic technique for the direct purification of beta cells from isolated Langerhans islets of rat. After the sacrifice of the rat, the Langerhans islets were separated by ductal injection of the pancreas with collagenase, altered to a mixed Langerhans islet cell population and incubated with conditioned immunomagnetic beads targeted to the beta cell surface. The beads were previously coated with a specific antibody against the surface of the beta cell, namely K14D10. The suspension of mixed Langerhans islet cells and immunomagnetic K14D10-conditioned beads was pelleted by a magnetic particle concentrator to isolate the bead-bound cells, which were finally suspended in a culture medium. The purified cells were immunoreactive for insulin and no glucagon-positive cells were detected at immunocytochemistry. Real Time PCR confirmed the purification of the pancreatic beta cells. This immunomagnetic technique allows a rapid, effective and consistent purification of beta cells from isolated Langerhans islets in a direct manner by conditioning the immunomagnetic beads only. This technique is easy, fast and reproducible. It promises to be a reliable method for providing purified beta cells for in vitro research.

Targeting dysfunctional beta-cell signaling for the potential treatment of type 1 diabetes mellitus.

PubMed

Fenske, Rachel J; Kimple, Michelle E

2018-03-01

Since its discovery and purification by Frederick Banting in 1921, exogenous insulin has remained almost the sole therapy for type 1 diabetes mellitus. While insulin alleviates the primary dysfunction of the disease, many other aspects of the pathophysiology of type 1 diabetes mellitus are unaffected. Research aimed towards the discovery of novel type 1 diabetes mellitus therapeutics targeting different cell signaling pathways is gaining momentum. The focus of these efforts has been almost entirely on the impact of immunomodulatory drugs, particularly those that have already received FDA-approval for other autoimmune diseases. However, these drugs can often have severe side effects, while also putting already immunocompromised individuals at an increased risk for other infections. Potential therapeutic targets in the insulin-producing beta-cell have been largely ignored by the type 1 diabetes mellitus field, save the glucagon-like peptide 1 receptor. While there is preliminary evidence to support the clinical exploration of glucagon-like peptide 1 receptor-based drugs as type 1 diabetes mellitus adjuvant therapeutics, there is a vast space for other putative therapeutic targets to be explored. The alpha subunit of the heterotrimeric G z protein (Gα z ) has been shown to promote beta-cell inflammation, dysfunction, death, and failure to replicate in the context of diabetes in a number of mouse models. Genetic loss of Gα z or inhibition of the Gα z signaling pathway through dietary interventions is protective against the development of insulitis and hyperglycemia. The multifaceted effects of Gα z in regards to beta-cell health in the context of diabetes make it an ideal therapeutic target for further study. It is our belief that a low-risk, effective therapy for type 1 diabetes mellitus will involve a multidimensional approach targeting a number of regulatory systems, not the least of which is the insulin-producing beta-cell. Impact statement The expanding investigation of beta-cell therapeutic targets for the treatment and prevention of type 1 diabetes mellitus is fundamentally relevant and timely. This review summarizes the overall scope of research into novel type 1 diabetes mellitus therapeutics, highlighting weaknesses or caveats in current clinical trials as well as describing potential new targets to pursue. More specifically, signaling proteins that act as modulators of beta-cell function, survival, and replication, as well as immune infiltration may need to be targeted to develop the most efficient pharmaceutical interventions for type 1 diabetes mellitus. One such beta-cell signaling pathway, mediated by the alpha subunit of the heterotrimeric G z protein (Gα z ), is discussed in more detail. The work described here will be critical in moving the field forward as it emphasizes the central role of the beta-cell in type 1 diabetes mellitus disease pathology.

Cell surface expression of beta 2-microglobulin (beta 2m) correlates with stages of differentiation in B cell tumours.

PubMed Central

Jones, R A; Scott, C S; Norfolk, D R; Stark, A N; Child, J A

1987-01-01

Cell surface beta 2-microglobulin (beta 2m) densities of malignant B cells were determined by enzyme immunoassay in 97 cases of immunologically defined lymphoproliferative disease. Absolute beta 2m densities were found to depend on disease category with the lowest levels found on cells from chronic lymphocytic leukaemia (mean = 5.6 ng/10(6) cells, n = 27); atypical chronic lymphocytic leukaemia (mean = 5.9 ng/10(6) cells, n = 8); and prolymphocytoid chronic lymphocytic leukaemia variant (mean = 6.0 ng/10(6) cells, n = 16). beta 2m densities for B non-Hodgkin's lymphoma (n = 14) and B prolymphocytic leukaemia (n = 17) cases were 8.1 and 10.0 ng/10(6) cells, respectively, and the highest densities were found on cells from "late-B cell" tumours (mean = 14.3 ng/10(6) cells). Plasma cells from cases of Ig secreting tumours expressed unexpectedly low beta 2m densities (mean = 9.3 ng/10(6) cells; n = 6). PMID:3108331

Inhibitory activity of fenoterol on Dermatophagoides-, Parietaria-, tetanus-toxoid-, and Candida albicans-stimulated blood mononuclear cells: differences in beta2-adrenoreceptor stimulation but not in cell apoptosis.

PubMed

Silvestri, M; Oddera, S; Scarso, L; Pistoia, V; Tasso, P; Rossi, G A

2000-05-01

beta2-adrenoreceptor agonists have the ability to downregulate in vitro the proliferative response of peripheral blood mononuclear cells (BMCs). This activity could be related to a variety of beta2-adrenoreceptor-mediated functions, including induction of cell apoptosis in activated T-cells. To test this hypothesis, BMCs from atopic subjects, sensitized to house dust mites (Dermatophagoides [Der p]) and/or to Parietaria were incubated with fenoterol (10(-8)-10(-5) M) in the presence of (a) purified allergen extracts (Der p [5 microg/mL] or Parietaria [5 microg/mL]) or (b) antigens (tetanus toxoid [1 microg/mL] or Candida albicans [5 x 10(5) bodies/mL]). The BMC proliferation was assessed by [3H] thymidine incorporation and cell apoptosis was assessed by evaluating DNA fragmentation by a fluorescence technique, using propidium iodide. In cultures stimulated with Der p or with Parietaria, fenoterol induced a dose-dependent inhibition of BMC proliferation, significant also at the lowest concentration tested (10(-8) M) (p < 0.05, each comparison). In contrast, the inhibitory activity of the drug on tetanus-toxoid-stimulated BMCs was significant only at the highest dose tested (10(-5)M) (p < 0.05), whereas no effect was seen when BMCs were stimulated with C. albicans extract (p > 0.05). The different inhibitory efficacy of fenoterol appeared to be related to the degree of activation of beta2-adrenoreceptors on the different BMC populations that responded to the different stimuli. Indeed, in the presence of fenoterol (10(-6) and 10(-5)M), a significant increase in cyclic adenosine monophosphate (cAMP) levels was seen in Der p- or Parietaria-stimulated cells (p < 0.05; each comparison), but not in cell cultures stimulated with tetanus toxoid or with C. albicans extracts (p > 0.05; each comparison). Finally, the percentage of cells with fragmented DNA was lower in cultures stimulated with Der p or Parietaria than in those stimulated with tetanus toxoid or C. albicans, and the presence of fenoterol did not modify cell apoptosis (p > 0.05; each comparison). Thus, the different inhibitory activity of fenoterol on BMCs activated by allergens (Der p or Parietaria) or by antigens (tetanus toxoid or C. albicans) seems to be related to differences in beta2-adrenoreceptor expression and/or function in the different antigen-specific T-cell subsets, but it is not influenced by changes in cell apoptosis.

Pancreatic beta cell function following liraglutide-augmented weight loss in individuals with prediabetes: analysis of a randomised, placebo-controlled study

PubMed Central

Liu, Alice; Ariel, Danit; Abbasi, Fahim; Lamendola, Cindy; Grove, Kaylene; Tomasso, Vanessa; Reaven, Gerald

2016-01-01

Aims/hypothesis Liraglutide can modulate insulin secretion by directly stimulating beta cells or indirectly through weight loss and enhanced insulin sensitivity. Recently, we showed that liraglutide treatment in overweight individuals with prediabetes (impaired fasting glucose and/or impaired glucose tolerance) led to greater weight loss (−7.7% vs −3.9%) and improvement in insulin resistance compared with placebo. The current study evaluates the effects on beta cell function of weight loss augmented by liraglutide compared with weight loss alone. Methods This was a parallel, randomised study conducted in a single academic centre. Both participants and study administrators were blinded to treatment assignment. Individuals who were 40–70 years old, overweight (BMI 27–40 kg/m2) and with prediabetes were randomised (via a computerised system) to receive liraglutide (n = 35) or matching placebo (n = 33), and 49 participants were analysed. All were instructed to follow an energy-restricted diet. Primary outcome was insulin secretory function, which was evaluated in response to graded infusions of glucose and day-long mixed meals. Results Liraglutide treatment (n = 24) significantly (p ≤0.03) increased the insulin secretion rate (% mean change [95% CI]; 21% [12, 31] vs −4% [−11, 3]) and pancreatic beta cell sensitivity to intravenous glucose (229% [161, 276] vs −0.5% (−15, 14]), and decreased insulin clearance rate (−3.5% [−11, 4] vs 8.2 [0.2, 16]) as compared with placebo (n = 25). The liraglutide-treated group also had significantly (p ≤0.03) lower day-long glucose (−8.2% [−11, −6] vs −0.1 [−3, 2]) and NEFA concentrations (−14 [−20, −8] vs −2.1 [−10, 6]) following mixed meals, whereas day-long insulin concentrations did not significantly differ as compared with placebo. In a multivariate regression analysis, weight loss was associated with a decrease in insulin secretion rate and day-long glucose and insulin concentrations in the placebo group (p ≤0.05), but there was no association with weight loss in the liraglutide group. The most common side effect of liraglutide was nausea. Conclusions/interpretation A direct stimulatory effect on beta cell function was the predominant change in liraglutide-augmented weight loss. These changes appear to be independent of weight loss. Trial registration ClinicalTrials.gov NCT01784965 PMID:24326527

The transforming activity of Ski and SnoN is dependent on their ability to repress the activity of Smad proteins.

PubMed

He, Jun; Tegen, Sarah B; Krawitz, Ariel R; Martin, G Steven; Luo, Kunxin

2003-08-15

The regulation of cell growth and differentiation by transforming growth factor-beta (TGF-beta) is mediated by the Smad proteins. In the nucleus, the Smad proteins are negatively regulated by two closely related nuclear proto-oncoproteins, Ski and SnoN. When overexpressed, Ski and SnoN induce oncogenic transformation of chicken embryo fibroblasts. However, the mechanism of transformation by Ski and SnoN has not been defined. We have previously reported that Ski and SnoN interact directly with Smad2, Smad3, and Smad4 and repress their ability to activate TGF-beta target genes through multiple mechanisms. Because Smad proteins are tumor suppressors, we hypothesized that the ability of Ski and SnoN to inactivate Smad function may be responsible for their transforming activity. Here, we show that the receptor regulated Smad proteins (Smad2 and Smad3) and common mediator Smad (Smad4) bind to different regions in Ski and SnoN. Mutation of both regions, but not each region alone, markedly impaired the ability of Ski and SnoN to repress TGF-beta-induced transcriptional activation and cell cycle arrest. Moreover, when expressed in chicken embryo fibroblasts, mutant Ski or SnoN defective in binding to the Smad proteins failed to induce oncogenic transformation. These results suggest that the ability of Ski and SnoN to repress the growth inhibitory function of the Smad proteins is required for their transforming activity. This may account for the resistance to TGF-beta-induced growth arrest in some human cancer cell lines that express high levels of Ski or SnoN.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Hae Jin; Park, Joongkyu; Seo, Su Ryeon

Down syndrome is mainly caused by a trisomy of chromosome 21. The Down syndrome critical region 2 (DSCR2) gene is located within a part of chromosome 21, the Down syndrome critical region (DSCR). To investigate the function of DSCR2, we sought to identify DSCR2-interacting proteins using yeast two-hybrid assays. A human fetal brain cDNA library was screened, and DSCR2 was found to interact with a member of the nuclear receptor superfamily, peroxisome proliferator-activated receptor {beta}, (PPAR{beta}). A co-immunoprecipitation assay demonstrated that DSCR2 physically interacts with PPAR{beta} in mammalian HEK293 cells. DSCR2 also inhibited the ligand-induced transcriptional activity of PPAR{beta}. Furthermore,more » PPAR{beta} also decreased the solubility of DSCR2, which increased levels of insoluble DSCR2.« less

Demonstration of interleukin-1 beta transcripts in acute myeloblastic leukemic cells by in situ hybridization.

PubMed

Nakamura, M; Kanakura, Y; Furukawa, Y; Ernst, T J; Griffin, J D

1990-07-01

The cells from some patients with acute myeloblastic leukemia will secrete autostimulatory cytokines in tissue culture without the addition of stimulators such as phorbol 12-myristate 13-acetate. Production of interleukin-1 beta (IL-1 beta), for example, has been observed in up to 50% of cases. In order to investigate the nature of the cell secreting IL-1 beta in AML, we used an antisense RNA probe to detect specific IL-1 beta transcripts in individual leukemic cells by in situ hybridization. In fresh, uncultured cells, IL-1 beta transcripts were observed in 1-40% of undifferentiated leukemic blast cells in 17 of 19 cases. In situ hybridization was at least as sensitive as Northern blot analysis in detecting IL-1 beta transcripts. No correlation of IL-1 beta transcript expression with FAB classification was observed. Normal blood and bone marrow mononuclear cells did not contain cells expressing IL-1 beta transcripts. These results support the concept that the regulation of cytokine genes in AML cells is aberrant.

Small amounts of tissue preserve pancreatic function: Long-term follow-up study of middle-segment preserving pancreatectomy.

PubMed

Lu, Zipeng; Yin, Jie; Wei, Jishu; Dai, Cuncai; Wu, Junli; Gao, Wentao; Xu, Qing; Dai, Hao; Li, Qiang; Guo, Feng; Chen, Jianmin; Xi, Chunhua; Wu, Pengfei; Zhang, Kai; Jiang, Kuirong; Miao, Yi

2016-11-01

Middle-segment preserving pancreatectomy (MPP) is a novel procedure for treating multifocal lesions of the pancreas while preserving pancreatic function. However, long-term pancreatic function after this procedure remains unclear.The aims of this current study are to investigate short- and long-term outcomes, especially long-term pancreatic endocrine function, after MPP.From September 2011 to December 2015, 7 patients underwent MPP in our institution, and 5 cases with long-term outcomes were further analyzed in a retrospective manner. Percentage of tissue preservation was calculated using computed tomography volumetry. Serum insulin and C-peptide levels after oral glucose challenge were evaluated in 5 patients. Beta-cell secreting function including modified homeostasis model assessment of beta-cell function (HOMA2-beta), area under the curve (AUC) for C-peptide, and C-peptide index were evaluated and compared with those after pancreaticoduodenectomy (PD) and total pancreatectomy. Exocrine function was assessed based on questionnaires.Our case series included 3 women and 2 men, with median age of 50 (37-81) years. Four patients underwent pylorus-preserving PD together with distal pancreatectomy (DP), including 1 with spleen preserved. The remaining patient underwent Beger procedure and spleen-preserving DP. Median operation time and estimated intraoperative blood loss were 330 (250-615) min and 800 (400-5500) mL, respectively. Histological examination revealed 3 cases of metastatic lesion to the pancreas, 1 case of chronic pancreatitis, and 1 neuroendocrine tumor. Major postoperative complications included 3 cases of delayed gastric emptying and 2 cases of postoperative pancreatic fistula. Imaging studies showed that segments representing 18.2% to 39.5% of the pancreas with good blood supply had been preserved. With a median 35.0 months of follow-ups on pancreatic functions, only 1 patient developed new-onset diabetes mellitus of the 4 preoperatively euglycemic patients. Beta-cell function parameters in this group of patients were quite comparable to those after Whipple procedure, and seemed better than those after total pancreatectomy. No symptoms of hypoglycemia were identified in any patient, although half of the patients reported symptoms of exocrine insufficiency.In conclusion, MPP is a feasible and effective procedure for middle-segment sparing multicentric lesions in the pancreas, and patients exhibit satisfied endocrine function after surgery.

Small amounts of tissue preserve pancreatic function

PubMed Central

Lu, Zipeng; Yin, Jie; Wei, Jishu; Dai, Cuncai; Wu, Junli; Gao, Wentao; Xu, Qing; Dai, Hao; Li, Qiang; Guo, Feng; Chen, Jianmin; Xi, Chunhua; Wu, Pengfei; Zhang, Kai; Jiang, Kuirong; Miao, Yi

2016-01-01

Abstract Middle-segment preserving pancreatectomy (MPP) is a novel procedure for treating multifocal lesions of the pancreas while preserving pancreatic function. However, long-term pancreatic function after this procedure remains unclear. The aims of this current study are to investigate short- and long-term outcomes, especially long-term pancreatic endocrine function, after MPP. From September 2011 to December 2015, 7 patients underwent MPP in our institution, and 5 cases with long-term outcomes were further analyzed in a retrospective manner. Percentage of tissue preservation was calculated using computed tomography volumetry. Serum insulin and C-peptide levels after oral glucose challenge were evaluated in 5 patients. Beta-cell secreting function including modified homeostasis model assessment of beta-cell function (HOMA2-beta), area under the curve (AUC) for C-peptide, and C-peptide index were evaluated and compared with those after pancreaticoduodenectomy (PD) and total pancreatectomy. Exocrine function was assessed based on questionnaires. Our case series included 3 women and 2 men, with median age of 50 (37–81) years. Four patients underwent pylorus-preserving PD together with distal pancreatectomy (DP), including 1 with spleen preserved. The remaining patient underwent Beger procedure and spleen-preserving DP. Median operation time and estimated intraoperative blood loss were 330 (250–615) min and 800 (400–5500) mL, respectively. Histological examination revealed 3 cases of metastatic lesion to the pancreas, 1 case of chronic pancreatitis, and 1 neuroendocrine tumor. Major postoperative complications included 3 cases of delayed gastric emptying and 2 cases of postoperative pancreatic fistula. Imaging studies showed that segments representing 18.2% to 39.5% of the pancreas with good blood supply had been preserved. With a median 35.0 months of follow-ups on pancreatic functions, only 1 patient developed new-onset diabetes mellitus of the 4 preoperatively euglycemic patients. Beta-cell function parameters in this group of patients were quite comparable to those after Whipple procedure, and seemed better than those after total pancreatectomy. No symptoms of hypoglycemia were identified in any patient, although half of the patients reported symptoms of exocrine insufficiency. In conclusion, MPP is a feasible and effective procedure for middle-segment sparing multicentric lesions in the pancreas, and patients exhibit satisfied endocrine function after surgery. PMID:27861351

Gamma delta lymphocytes in endocrine autoimmunity: evidence of expansion in Graves' disease but not in type 1 diabetes.

PubMed Central

Roura-Mir, I C; Alcalde, L; Vargas, F; Tolosa, E; Obiols, G; Foz, M; Jaraquemada, D; Pujol-Borrell, R

1993-01-01

Endocrine autoimmune disorders are mediated by T cell-dependent responses to organ-specific antigens, but the mechanisms initiating the process remain unknown. Lymphocytes which use the gamma delta heterodimer as T cell receptor (TCR) for antigen constitute a distinct subset of T cells whose function remains elusive. In order to investigate their possible involvement in endocrine autoimmunity we have determined the proportion of gamma delta T cells in the peripheral blood of 23 patients with type 1 (insulin-dependent) diabetes mellitus (type-1 DM) and 30 patients with autoimmune thyrotoxicosis (Graves' disease). T lymphocyte TCR expression was assessed by fluorescence-activated flow cytometry on peripheral blood mononuclear cells using MoAbs UCHT1 (CD3), TCR delta 1 (gamma delta TCR), WT31 and beta F1 (alpha beta TCR) and both the percentage of T cells expressing gamma delta and the ratio gamma delta/alpha beta were calculated. In the diabetic patients gamma delta cells were not significantly different from the control group (7.7 +/- 54% versus 8.0 +/- 5.5% of T cells, P NS). There was no relation between the proportion of gamma delta lymphocytes and the presence of islet cell antibodies (ICA) in the sera. The Graves' patients showed a tendency towards a higher proportion of gamma delta T lymphocytes than the controls (gamma delta/alpha beta ratios: 0.095 +/- 0.047 versus 0.063 +/- 0.022, P = 0.03). In 14 Graves' patients the number of gamma delta were measured in paired samples of peripheral and intrathyroidal lymphocytes, demonstrating an expansion of gamma delta within the thyroid glands (0.21 +/- 0.3 versus 0.095 +/- 0.047, P = 0.032). Immunohistochemical studies showed that gamma delta cells were scattered among the predominant alpha beta lymphocytes infiltrating the thyroid gland and that they account for 10% of intraepithelial lymphocytes. No relation was found between the increase of gamma delta lymphocytes and any clinical features. PMID:8485915

Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway

USGS Publications Warehouse

Jung, T.W.; Lee, M.W.; Lee, Y.-J.; Kim, S.M.

2012-01-01

Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH(2) terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK-PI3 kinase-JNK pathway.

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

PubMed

Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

2007-11-23

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

Butyrate modulates TGF-beta1 generation and function: potential renal benefit for Acacia(sen) SUPERGUM (gum arabic)?

PubMed

Matsumoto, N; Riley, S; Fraser, D; Al-Assaf, S; Ishimura, E; Wolever, T; Phillips, G O; Phillips, A O

2006-01-01

Anecdotal evidence suggests that high fibre supplementation of dietary intake may have health benefits in renal disease related to alterations in circulating levels of short-chain fatty acids. The aim of the study was to examine the hypothesis that dietary manipulation may increase serum butyrate and thus have potential beneficial effects in renal disease. We examined the effect of dietary supplementation with a gum arabic sample of standardized molecular characteristics, Acacia(sen) SUPERGUM EM2 (SUPERGUM), on systemic levels of butyrate in normal human subjects. In an in vitro study, we also examined the potential role of butyrate in modifying the generation of the profibrotic cytokine transforming growth factor-beta (TGF-beta1) by renal epithelial cells. Following 8 weeks of dietary supplementation with 25 g/day of SUPERGUM, there was a two-fold increase in serum butyrate (n=7, P=0.03). In vitro work demonstrated that exposure of renal epithelial cells to elevated concentrations of butyrate suppressed both basal and stimulated TGF-beta1 synthesis. The action of butyrate was mediated by suppression of the extracellular signal-regulated kinase/mitogen-activated protein kinase signalling pathway. In addition, butyrate exposures reduced the response of renal epithelial cells to TGF-beta1 as assessed by luciferase activity of a TGF-beta-responsive reporter construct. Attenuation of TGF-beta1 signalling was associated with reduced phosphorylation of Smad 3 and decreased trafficking of TGF-beta1 receptors into signalling, non-lipid raft-associated membrane fractions. In conclusion, the data demonstrate that dietary supplementation with SUPERGU increased serum butyrate, which at least in vitro has beneficial effects on renal pro-fibrotic cytokine generation.

Comparative analysis of Homo sapiens and Mus musculus cyclin-dependent kinase (CDK) inhibitor genes p16 (MTS1) and p15 (MTS2).

PubMed

Jiang, P; Stone, S; Wagner, R; Wang, S; Dayananth, P; Kozak, C A; Wold, B; Kamb, A

1995-12-01

Cyclin-dependent kinase inhibitors are a growing family of molecules that regulate important transitions in the cell cycle. At least one of these molecules, p16, has been implicated in human tumorigenesis while its close homolog, p15, is induced by cell contact and transforming growth factor-beta (TGF-beta). To investigate the evolutionary and functional features of p15 and p16, we have isolated mouse (Mus musculus) homologs of each gene. Comparative analysis of these sequences provides evidence that the genes have similar functions in mouse and human. In addition, the comparison suggests that a gene conversion event is part of the evolution of the human p15 and p16 genes.

Meprin A and meprin {alpha} generate biologically functional IL-1{beta} from pro-IL-1{beta}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herzog, Christian; University of Arkansas for Medical Sciences, Department of Medicine, Little Rock, AR 72205; Haun, Randy S.

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin {alpha} are capable of generating biologically active IL-1{beta} from its precursor pro-IL-1{beta}. Amino-acid sequencing analysis reveals that meprin A and meprin {alpha} cleave pro-IL-1{beta} at the His{sup 115}-Asp{sup 116} bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin {beta} site. The biological activity of the pro-IL-1{beta} cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1{beta} product produced by meprin {beta}more » or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1{beta}, meprin inhibitor actinonin significantly reduces levels of serum IL-1{beta}. Meprin A and meprin {alpha} may therefore play a critical role in the production of active IL-1{beta} during inflammation and tissue injury.« less

Subunit assembly of hemoglobin: an important determinant of hematologic phenotype.

PubMed

Bunn, H F

1987-01-01

Hemoglobin's physiologic properties depend on the orderly assembly of its subunits in erythropoietic cells. The biosynthesis of alpha- and beta-globin polypeptide chains is normally balanced. Heme rapidly binds to the globin subunit, either during translation or shortly thereafter. The formation of the alpha beta-dimer is facilitated by electrostatic attraction of a positively charged alpha-subunit to a negatively charged beta-subunit. The alpha beta-dimer dissociates extremely slowly. The difference between the rate of dissociation of alpha beta- and alpha gamma-dimers with increasing pH explains the well-known alkaline resistance of Hb F. Two dimers combine to form the functioning alpha 2 beta 2-tetramer. This model of hemoglobin assembly explains the different levels of positively charged and negatively charged mutant hemoglobins that are encountered in heterozygotes and the effect of alpha-thalassemia and heme deficiency states in modifying the level of the variant hemoglobin as well as Hb A2. Electrostatic interactions also affect the binding of hemoglobin to the cytoplasmic surface of the red cell membrane and may underlie the formation of target cells. Enhanced binding of positively charged variants such as S and C trigger a normally dormant pathway for potassium and water loss. Thus, the positive charge on beta c is responsible for the two major contributors to the pathogenesis of Hb SC disease: increased proportion of Hb S and increased intracellular hemoglobin concentration. It is likely that electrostatic interactions play an important role in the assembly of a number of other multisubunit macromolecules, including membrane receptors, cytoskeletal proteins, and DNA binding proteins.

Neuropilin-1 and neuropilin-2 are differentially expressed in human proteinuric nephropathies and cytokine-stimulated proximal tubular cells.

PubMed

Schramek, Herbert; Sarközi, Rita; Lauterberg, Christina; Kronbichler, Andreas; Pirklbauer, Markus; Albrecht, Rudolf; Noppert, Susie-Jane; Perco, Paul; Rudnicki, Michael; Strutz, Frank M; Mayer, Gert

2009-11-01

Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are transmembrane glycoproteins with large extracellular domains that interact with class 3 semaphorins, vascular endothelial growth factor (VEGF) family members, and ligands, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-beta1 (TGF-beta1), and fibroblast growth factor2 (FGF2). Neuropilins (NRPs) have been implicated in tumor growth and vascularization, as novel mediators of the primary immune response and in regeneration and repair; however, their role in renal pathophysiology is largely unknown. Here, we report upregulation of tubular and interstitial NRP2 protein expression in patients with focal segmental glomerulosclerosis (FSGS). In an additional cohort of patients with minimal change disease (MCD), membranous nephropathy (MN), and FSGS, elevated NRP2 mRNA expression in kidney biopsies inversely correlated with estimated glomerular filtration rate (eGFR) at the time of biopsy. Furthermore, upregulation of NRP2 mRNA correlated with post-bioptic decline of kidney function. Expression of NRP1 and NRP2 in human proximal tubular cells (PTCs) was differentially affected after stimulation with TGF-beta1, interleukin-1beta (IL-1beta), and oncostatin M (OSM). Although the pro-fibrotic mediators, TGF-beta1 and IL-1beta, induced upregulation of NRP2 expression but downregulation of NRP1 expression, OSM stimulated the expression of both NRP1 and NRP2. Basal and OSM-induced NRP1 mRNA expression, as well as TGF-beta1-induced NRP2 mRNA and protein expression were partially mediated by MEK1/2-ERK1/2 signaling. This is the first report suggesting a differential role of NRP1 and NRP2 in renal fibrogenesis, and TGF-beta1, IL-1beta, and OSM represent the first ligands known to stimulate NRP2 expression in mammalian cells.

Id-1 promotes TGF-{beta}1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Di Kaijun; Wong, Y.C.; Wang Xianghong

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-{beta}1 (transforming growth factor {beta}1). Here we demonstrate a novel role of Id-1 in promoting TGF-{beta}1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-{beta}1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-{beta}1-induced cell motility was mediated through activation ofmore » MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-{beta}1-treated cells through down-regulation of E-cadherin, redistribution of {beta}-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-{beta}1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-{beta}1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.« less

Blockade of rat alpha3beta4 nicotinic receptor function by methadone, its metabolites, and structural analogs.

PubMed

Xiao, Y; Smith, R D; Caruso, F S; Kellar, K J

2001-10-01

The opioid agonist properties of (+/-)-methadone are ascribed almost entirely to the (-)-methadone enantiomer. To extend our knowledge of the pharmacological actions of methadone at ligand-gated ion channels, we investigated the effects of the two enantiomers of methadone and its metabolites R-(+)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium perchlorate (EDDP) and R-(+)-2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline hydrochloride (EMDP), as well as structural analogs of methadone, including (-)-alpha-acetylmethadol hydrochloride (LAAM) and (+)-alpha-propoxyphene, on rat alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs) stably expressed in a human embryonic kidney 293 cell line, designated KXalpha3beta4R2. (+/-)-methadone inhibited nicotine-stimulated 86Rb+ efflux from the cells in a concentration-dependent manner with an IC50 value of 1.9 +/- 0.2 microM, indicating that it is a potent nAChR antagonist. The (-)- and (+)-enantiomers of methadone have similar inhibitory potencies on nicotine-stimulated 86Rb+ efflux, with IC50 values of approximately 2 microM. EDDP, the major metabolite of methadone, is even more potent, with an IC50 value of approximately 0.5 microM, making it one of the most potent nicotinic receptor blockers reported. In the presence of (+/-)-methadone, EDDP, or LAAM, the maximum nicotine-stimulated 86Rb+ efflux was markedly decreased, but the EC50 value for nicotine stimulation was altered only slightly, if at all, indicating that these compounds block alpha3beta4 nicotinic receptor function by a noncompetitive mechanism. Consistent with a noncompetitive mechanism, (+/-)-methadone, its metabolites, and structural analogs have very low affinity for nicotinic receptor agonist binding sites in membrane homogenates from KXalpha3beta4R2 cells. We conclude that both enantiomers of methadone and its metabolites as well as LAAM and (+)-alpha-propoxyphene are potent noncompetitive antagonists of alpha3beta4 nAChRs.

Antidiabetic and Beta Cell-Protection Activities of Purple Corn Anthocyanins

PubMed Central

Hong, Su Hee; Heo, Jee-In; Kim, Jeong-Hyeon; Kwon, Sang-Oh; Yeo, Kyung-Mok; Bakowska-Barczak, Anna M.; Kolodziejczyk, Paul; Ryu, Ok-Hyun; Choi, Moon-Ki; Kang, Young-Hee; Lim, Soon Sung; Suh, Hong-Won; Huh, Sung-Oh; Lee, Jae-Yong

2013-01-01

Antidiabetic and beta cell-protection activities of purple corn anthocyanins (PCA) were examined in pancreatic beta cell culture and db/db mice. Only PCA among several plant anthocyanins and polyphenols showed insulin secretion activity in culture of HIT-T15 cells. PCA had excellent antihyperglycemic activity (in terms of blood glucose level and OGTT) and HbA1c-decreasing activity when compared with glimepiride, a sulfonylurea in db/db mice. In addition, PCA showed efficient protection activity of pancreatic beta cell from cell death in HIT-T15 cell culture and db/db mice. The result showed that PCA had antidiabetic and beta cell-protection activities in pancreatic beta cell culture and db/db mice. PMID:24244813

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

PubMed Central

Deressa, Tekalign; Strandt, Helen; Florindo Pinheiro, Douglas; Mittermair, Roberta; Pizarro Pesado, Jennifer; Thalhamer, Josef; Hammerl, Peter; Stoecklinger, Angelika

2015-01-01

The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin+ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin+ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation. PMID:26030383

Human beta-defensin 3 has immunosuppressive activity in vitro and in vivo.

PubMed

Semple, Fiona; Webb, Sheila; Li, Hsin-Ni; Patel, Hetal B; Perretti, Mauro; Jackson, Ian J; Gray, Mohini; Davidson, Donald J; Dorin, Julia R

2010-04-01

Beta-defensins are antimicrobial peptides with an essential role in the innate immune response. In addition beta-defensins can also chemoattract cells involved in adaptive immunity. Until now, based on evidence from dendritic cell stimulation, human beta defensin-3 (hBD3) was considered pro-inflammatory. We present evidence here that hBD3 lacks pro-inflammatory activity in human and mouse primary Mphi. In addition, in the presence of LPS, hBD3 and the murine orthologue Defb14 (but not hBD2), effectively inhibit TNF-alpha and IL-6 accumulation implying an anti-inflammatory function. hBD3 also inhibits CD40/IFN-gamma stimulation of Mphi and in vivo, hBD3 significantly reduces the LPS-induced TNF-alpha level in serum. Recent work has revealed that hBD3 binds melanocortin receptors but we provide evidence that these are not involved in hBD3 immunomodulatory activity. This implies a dual role for hBD3 in antimicrobial activity and resolution of inflammation.

Relationship Between Beta Cell Dysfunction and Severity of Disease Among Critically Ill Children: A STROBE-Compliant Prospective Observational Study.

PubMed

Liu, Ping-Ping; Lu, Xiu-Lan; Xiao, Zheng-Hui; Qiu, Jun; Zhu, Yi-Min

2016-05-01

Although beta cell dysfunction has been proved to predict prognosis among humans and animals, its prediction on severity of disease remains unclear among children. The present study was aimed to examine the relationship between beta cell dysfunction and severity of disease among critically ill children.This prospective study included 1146 critically ill children, who were admitted to Pediatric Intensive Care Unit (PICU) of Hunan Children's Hospital from November 2011 to August 2013. Information on characteristics, laboratory tests, and prognostic outcomes was collected. Homeostasis model assessment (HOMA)-β, evaluating beta cell function, was used to divide all participants into 4 groups: HOMA-β = 100% (group I, n = 339), 80% ≤ HOMA-β < 100% (group II, n = 71), 40% ≤ HOMA-β < 80% (group III, n = 293), and HOMA-β < 40% (group IV, n = 443). Severity of disease was assessed using the worst Sequential Organ Failure Assessment (SOFA) score, Pediatric Risk of Mortality (PRISM) III score, incidence of organ damage, septic shock, multiple organ dysfunction syndrome (MODS), mechanical ventilation (MV) and mortality. Logistic regression analysis was used to evaluate the risk of developing poor outcomes among patients in different HOMA-β groups, with group I as the reference group.Among 1146 children, incidence of HOMA-β < 100% was 70.41%. C-peptide and insulin declined with the decrement of HOMA-β (P < 0.01). C-reactive protein and procalcitonin levels, rather than white blood cell, were significantly different among 4 groups (P < 0.01). In addition, the worst SOFA score and the worst PRISMIII score increased with declined HOMA-β. For example, the worst SOFA score in group I, II, III, and IV was 1.55 ± 1.85, 1.71 ± 1.93, 1.92 ± 1.63, and 2.18 ± 1.77, respectively. Furthermore, patients with declined HOMA-β had higher risk of developing septic shock, MODS, MV, and mortality, even after adjusting age, gender, myocardial injury, and lung injury. For instance, compared with group I, the multivariate-adjusted odds ratio (95% confidence interval) for developing septic shock was 2.17 (0.59, 8.02), 2.94 (2.18, 6.46), and 2.76 (1.18, 6.46) among patients in group II, III, and IV, respectively.Beta cell dysfunction reflected the severity of disease among critically ill children. Therefore, assessment of beta cell function is critically important to reduce incidence of adverse events in PICU.

Extracellular heat shock protein HSP90{beta} secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-{beta}1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Shigeki; Kulkarni, Ashok B., E-mail: ak40m@nih.gov

2010-07-30

Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-{beta} signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understandingmore » of the latent TGF-{beta} activation process. In this study, we have identified heat shock protein 90 {beta} (HSP90{beta}) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90{beta} into extracellular space which inhibits the activation of latent TGF-{beta}1, and that there is a subsequent decrease in cell proliferation. TGF-{beta}1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90{beta}. Thus, extracellular HSP90{beta} is a negative regulator for the activation of latent TGF-{beta}1 modulating TGF-{beta} signaling in the extracellular domain. -- Research highlights: {yields} Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex. {yields} This complex consists of latency-associated peptide (LAP) and the mature ligand. {yields} The release of the mature ligand from LAP is the first step in TGF-{beta} activation. {yields} We identified for the first time a novel mechanism for this activation process. {yields} Heat shock protein 90 {beta} is discovered as a negative regulator for this process.« less

Insulin Sensitivity and Secretion in Obese Type 2 Diabetic Women after Various Bariatric Operations

PubMed Central

Vrbikova, Jana; Kunesova, Marie; Kyrou, Ioannis; Tura, Andrea; Hill, Martin; Grimmichova, Tereza; Dvorakova, Katerina; Sramkova, Petra; Dolezalova, Karin; Lischkova, Olga; Vcelak, Josef; Hainer, Vojtech; Bendlova, Bela; Kumar, Sudhesh; Fried, Martin

2017-01-01

Objective To compare the effects of biliopancreatic diversion (BPD) and laparoscopic gastric banding (LAGB) on insulin sensitivity and secretion with the effects of laparoscopic gastric plication (P). Methods A total of 52 obese women (age 30-66 years) suffering from type 2 diabetes mellitus (T2DM) were prospectively recruited into three study groups: 16 BPD; 16 LAGB, and 20 P. Euglycemic clamps and mixed meal tolerance tests were performed before, at 1 month and at 6 months after bariatric surgery. Beta cell function derived from the meal test parameters was evaluated using mathematical modeling. Results Glucose disposal per kilogram of fat free mass (a marker of peripheral insulin sensitivity) increased significantly in all groups, especially after 1 month. Basal insulin secretion decreased significantly after all three types of operations, with the most marked decrease after BPD compared with P and LAGB. Total insulin secretion decreased significantly only following the BPD. Beta cell glucose sensitivity did not change significantly post-surgery in any of the study groups. Conclusion We documented similar improvement in insulin sensitivity in obese T2DM women after all three study operations during the 6-month postoperative follow-up. Notably, only BPD led to decreased demand on beta cells (decreased integrated insulin secretion), but without increasing the beta cell glucose sensitivity. PMID:27951535

Inhibitory G proteins and their receptors: emerging therapeutic targets for obesity and diabetes

PubMed Central

Kimple, Michelle E; Neuman, Joshua C; Linnemann, Amelia K; Casey, Patrick J

2014-01-01

The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology. PMID:24946790

Human MI-ER1 alpha and beta function as transcriptional repressors by recruitment of histone deacetylase 1 to their conserved ELM2 domain.

PubMed

Ding, Zhihu; Gillespie, Laura L; Paterno, Gary D

2003-01-01

mi-er1 (previously called er1) was first isolated from Xenopus laevis embryonic cells as a novel fibroblast growth factor-regulated immediate-early gene. Xmi-er1 was shown to encode a nuclear protein with an N-terminal acidic transcription activation domain. The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level and was shown to be upregulated in breast carcinoma cell lines and tumors. Alternative splicing at the 3' end of hmi-er1 produces two major isoforms, hMI-ER1alpha and hMI-ER1beta, which contain distinct C-terminal domains. In this study, we investigated the role of hMI-ER1alpha and hMI-ER1beta in the regulation of transcription. Using fusion proteins of hMI-ER1alpha or hMI-ER1beta tethered to the GAL4 DNA binding domain, we show that both isoforms, when recruited to the G5tkCAT minimal promoter, function to repress transcription. We demonstrate that this repressor activity is due to interaction and recruitment of a trichostatin A-sensitive histone deacetylase 1 (HDAC1). Furthermore, deletion analysis revealed that recruitment of HDAC1 to hMI-ER1alpha and hMI-ER1beta occurs through their common ELM2 domain. The ELM2 domain was first described in the Caenorhabditis elegans Egl-27 protein and is present in a number of SANT domain-containing transcription factors. This is the first report of a function for the ELM2 domain, highlighting its role in the regulation of transcription.

Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

PubMed Central

1995-01-01

Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium.

PubMed

Kellogg, Stephanie L; Little, Jaime L; Hoff, Jessica S; Kristich, Christopher J

2017-05-01

Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis , exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci. Copyright © 2017 American Society for Microbiology.

Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium

PubMed Central

Kellogg, Stephanie L.; Little, Jaime L.; Hoff, Jessica S.

2017-01-01

ABSTRACT Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis. Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis. Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis, exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium. Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci. PMID:28223383

The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

PubMed

Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

2011-12-01

We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. Copyright © 2011 Elsevier Ltd. All rights reserved.

Thermogelling chitosan and collagen composite hydrogels initiated with β-glycerophosphate for bone tissue engineering

PubMed Central

Wang, Limin; Stegemann, Jan P.

2010-01-01

Chitosan and collagen type I are naturally-derived materials used as cell carriers because of their ability to mimic the extracellular environment and direct cell function. In this study beta-glycerophosphate (beta-GP), an osteogenic medium supplement and a weak base, was used to simultaneously initiate gelation of pure chitosan, pure collagen, and chitosan-collagen composite materials at physiological pH and temperature. Adult human bone marrow-derived stem cells (hBMSC) encapsulated in such hydrogels at chitosan/collagen ratios of 100/0, 65/35, 25/75, and 0/100 wt% exhibited high viability at day 1 after encapsulation, but DNA content dropped by about half over 12 days in pure chitosan materials while it increased two-fold in materials containing collagen. Collagen-containing materials compacted more strongly and were significantly stiffer than pure chitosan gels. In monolayer culture, exposure of hBMSC to beta-GP resulted in decreased cell metabolic activity that varied with concentration and exposure time, but washing effectively removed excess beta-GP from hydrogels. The presence of chitosan in materials resulted in higher expression of osterix and bone sialoprotein genes in medium with and without osteogenic supplements. Chitosan also increased alkaline phosphatase activity and calcium deposition in osteogenic medium. Chitosan-collagen composite materials have potential as matrices for cell encapsulation and delivery, or as in situ gel-forming materials for tissue repair. PMID:20170955

Microarray identifies ADAM family members as key responders to TGF-beta1 in alveolar epithelial cells.

PubMed

Keating, Dominic T; Sadlier, Denise M; Patricelli, Andrea; Smith, Sinead M; Walls, Dermot; Egan, Jim J; Doran, Peter P

2006-09-01

The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF) remain elusive. Transforming Growth Factor beta 1(TGF-beta1) is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549) in response to TGF-beta1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-beta1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM) proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO) databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing) family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity) of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-beta1, suggesting a potential role for these molecules in ECM accumulation in IPF.

Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells.

PubMed Central

Matsuura, N.; Puzon-McLaughlin, W.; Irie, A.; Morikawa, Y.; Kakudo, K.; Takada, Y.

1996-01-01

Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression. The increase in integrin alpha 4 beta 1 expression on primary melanomas has been reported to significantly correlate with the development of metastases. alpha 4 beta 1 is a cell surface heterodimer that mediates cell-cell and cell-extracellular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region of fibronectin. To test the effects of alpha 4 beta 1 expression on tumor cell metastasis, Chinese hamster ovary cells were transfected with human alpha 4 cDNA. Whereas alpha 4-negative Chinese hamster ovary cells developed only pulmonary metastasis, alpha 4-positive Chinese hamster ovary cells developed bone and pulmonary metastasis in 3 to 4 weeks when injected intravenously into nude mice. Bone metastasis was inhibited by antibody against alpha 4 or VCAM-1. Expression of alpha 3 beta 1, alpha 6 beta 1, or alpha V beta 1 did not induce bone metastasis. Expression of alpha 4 beta 1 also induced bone metastasis in K562 human erythroleukemia cells injected into SCID mice. These results demonstrate that alpha 4 beta 1 can induce tumor cell trafficking to bone, probably via interaction with VCAM-1 that is constitutively expressed on bone marrow stromal cells. Images Figure 1 Figure 3 PMID:8546226

Age-related islet inflammation marks the proliferative decline of pancreatic beta-cells in zebrafish

PubMed Central

Tsakmaki, Anastasia; Mousavy Gharavy, S Neda; Murawala, Priyanka; Konantz, Judith; Birke, Sarah; Hodson, David J; Rutter, Guy A; Bewick, Gavin A

2018-01-01

The pancreatic islet, a cellular community harboring the insulin-producing beta-cells, is known to undergo age-related alterations. However, only a handful of signals associated with aging have been identified. By comparing beta-cells from younger and older zebrafish, here we show that the aging islets exhibit signs of chronic inflammation. These include recruitment of tnfα-expressing macrophages and the activation of NF-kB signaling in beta-cells. Using a transgenic reporter, we show that NF-kB activity is undetectable in juvenile beta-cells, whereas cells from older fish exhibit heterogeneous NF-kB activity. We link this heterogeneity to differences in gene expression and proliferation. Beta-cells with high NF-kB signaling proliferate significantly less compared to their neighbors with low activity. The NF-kB signalinghi cells also exhibit premature upregulation of socs2, an age-related gene that inhibits beta-cell proliferation. Together, our results show that NF-kB activity marks the asynchronous decline in beta-cell proliferation with advancing age. PMID:29624168

Cortisone Dissociates the Shaker Family K Channels from their Beta Subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Y.; Weng, J; Kabaleeswaran, V

2008-01-01

The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and are essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with {Beta} subunits (Kv{Beta}s), and certain Kv{Beta}s, for example Kv{Beta}1, have an N-terminal segment that closes the channel by the N-type inactivation mechanism. In principle, dissociation of Kv{Beta}1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases rat Kv1 channel activity by binding to Kv{Beta}1. A crystal structuremore » of the K{Beta}v-cortisone complex was solved to 1.82-{angstrom}resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kv{Beta}. The new mode of channel modulation may be explored by native or synthetic ligands to fine-tune cellular excitability.« less

Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

NASA Astrophysics Data System (ADS)

Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

1990-09-01

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang Pengfei; Jiang Bimei; Yang Xinghua

2008-10-15

Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, anmore » EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.« less

Structure of the E2 DNA-binding domain from human papillomavirus serotype 31 at 2.4 A.

PubMed

Bussiere, D E; Kong, X; Egan, D A; Walter, K; Holzman, T F; Lindh, F; Robins, T; Giranda, V L

1998-11-01

The papillomaviruses are a family of small double-stranded DNA viruses which exclusively infect epithelial cells and stimulate the proliferation of those cells. A key protein within the papillomavirus life-cycle is known as the E2 (Early 2) protein and is responsible for regulating viral transcription from all viral promoters as well as for replication of the papillomavirus genome in tandem with another protein known as E1. The E2 protein itself consists of three functional domains: an N-terminal trans-activation domain, a proline-rich linker, and a C-terminal DNA-binding domain. The first crystal structure of the human papillomavirus, serotype 31 (HPV-31), E2 DNA-binding domain has been determined at 2.4 A resolution. The HPV DNA-binding domain monomer consists of two beta-alpha-beta repeats of approximately equal length and is arranged as to have an anti-parallel beta-sheet flanked by the two alpha-helices. The monomers form the functional in vivo dimer by association of the beta-sheets of each monomer so as to form an eight-stranded anti-parallel beta-barrel at the center of the dimer, with the alpha-helices lining the outside of the barrel. The overall structure of HVP-31 E2 DNA-binding domain is similar to both the bovine papillomavirus E2-binding domain and the Epstein-Barr nuclear antigen-1 DNA-binding domain.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product.

PubMed

Thuwajit, Chanitra; Thuwajit, Peti; Uchida, Kazuhiko; Daorueang, Daoyot; Kaewkes, Sasithorn; Wongkham, Sopit; Miwa, Masanao

2006-06-14

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05). O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.

PubMed

Soikkeli, Johanna; Podlasz, Piotr; Yin, Miao; Nummela, Pirjo; Jahkola, Tiina; Virolainen, Susanna; Krogerus, Leena; Heikkilä, Päivi; von Smitten, Karl; Saksela, Olli; Hölttä, Erkki

2010-07-01

Although the outgrowth of micrometastases into macrometastases is the rate-limiting step in metastatic progression and the main determinant of cancer fatality, the molecular mechanisms involved have been little studied. Here, we compared the gene expression profiles of melanoma lymph node micro- and macrometastases and unexpectedly found no common up-regulation of any single growth factor/cytokine, except for the cytokine-like SPP1. Importantly, metastatic outgrowth was found to be consistently associated with activation of the transforming growth factor-beta signaling pathway (confirmed by phospho-SMAD2 staining) and concerted up-regulation of POSTN, FN1, COL-I, and VCAN genes-all inducible by transforming growth factor-beta. The encoded extracellular matrix proteins were found to together form intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases from various organs. Functional analyses suggested that these newly synthesized protein networks regulate adhesion, migration, and growth of tumor cells, fibroblasts, and endothelial cells. POSTN acted as an anti-adhesive molecule counteracting the adhesive functions of FN1 and COL-I. Further, cellular FN and POSTN were specifically overexpressed in the newly forming/formed tumor blood vessels. Transforming growth factor-beta receptors and the metastasis-related matrix proteins, POSTN and FN1, in particular, may thus provide attractive targets for development of new therapies against disseminated melanoma, breast cancer, and possibly other tumors, by affecting key processes of metastasis: tumor/stromal cell migration, growth, and angiogenesis.

The relationship between node degree and dissipation rate in networks of diffusively coupled oscillators and its significance for pancreatic beta cells.

PubMed

Gosak, Marko; Stožer, Andraž; Markovič, Rene; Dolenšek, Jurij; Marhl, Marko; Rupnik, Marjan Slak; Perc, Matjaž

2015-07-01

Self-sustained oscillatory dynamics is a motion along a stable limit cycle in the phase space, and it arises in a wide variety of mechanical, electrical, and biological systems. Typically, oscillations are due to a balance between energy dissipation and generation. Their stability depends on the properties of the attractor, in particular, its dissipative characteristics, which in turn determine the flexibility of a given dynamical system. In a network of oscillators, the coupling additionally contributes to the dissipation, and hence affects the robustness of the oscillatory solution. Here, we therefore investigate how a heterogeneous network structure affects the dissipation rate of individual oscillators. First, we show that in a network of diffusively coupled oscillators, the dissipation is a linearly decreasing function of the node degree, and we demonstrate this numerically by calculating the average divergence of coupled Hopf oscillators. Subsequently, we use recordings of intracellular calcium dynamics in pancreatic beta cells in mouse acute tissue slices and the corresponding functional connectivity networks for an experimental verification of the presented theory. We use methods of nonlinear time series analysis to reconstruct the phase space and calculate the sum of Lyapunov exponents. Our analysis reveals a clear tendency of cells with a higher degree, that is, more interconnected cells, having more negative values of divergence, thus confirming our theoretical predictions. We discuss these findings in the context of energetic aspects of signaling in beta cells and potential risks for pathological changes in the tissue.