PLAG1 deficiency impairs spermatogenesis and sperm motility in mice.

PubMed

Juma, Almas R; Grommen, Sylvia V H; O'Bryan, Moira K; O'Connor, Anne E; Merriner, D Jo; Hall, Nathan E; Doyle, Stephen R; Damdimopoulou, Pauliina E; Barriga, Daniel; Hart, Adam H; Van de Ven, Wim J M; De Groef, Bert

2017-07-13

Deficiency in pleomorphic adenoma gene 1 (PLAG1) leads to reduced fertility in male mice, but the mechanism by which PLAG1 contributes to reproduction is unknown. To investigate the involvement of PLAG1 in testicular function, we determined (i) the spatial distribution of PLAG1 in the testis using X-gal staining; (ii) transcriptomic consequences of PLAG1 deficiency in knock-out and heterozygous mice compared to wild-type mice using RNA-seq; and (iii) morphological and functional consequences of PLAG1 deficiency by determining testicular histology, daily sperm production and sperm motility in knock-out and wild-type mice. PLAG1 was sparsely expressed in germ cells and in Sertoli cells. Genes known to be involved in spermatogenesis were downregulated in the testes of knock-out mice, as well as Hsd17b3, which encodes a key enzyme in androgen biosynthesis. In the absence of Plag1, a number of genes involved in immune processes and epididymis-specific genes were upregulated in the testes. Finally, loss of PLAG1 resulted in significantly lowered daily sperm production, in reduced sperm motility, and in several animals, in sloughing of the germinal epithelium. Our results demonstrate that the subfertility seen in male PLAG1-deficient mice is, at least in part, the result of significantly reduced sperm output and sperm motility.

Conditional knockout of N-WASP in mouse fibroblast caused keratinocyte hyper proliferation and enhanced wound closure

PubMed Central

Jain, Neeraj; Kalailingam, Pazhanichamy; Tan, Kai Wei; Tan, Hui Bing; Sng, Ming Keat; Chan, Jeremy Soon Kiat; Tan, Nguan Soon; Thanabalu, Thirumaran

2016-01-01

Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASPFKO (N-WASPfl/fl; Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions. Histological sections of N-WASPFKO mice skin (13 weeks old) showed thicker epidermis with higher percentage of cells staining for proliferation marker (PCNA), suggesting that N-WASP deficient fibroblasts promote keratinocyte proliferation. N-WASPFKO mice skin had elevated collagen content, elevated expression of FGF7 (keratinocyte growth factor) and TGFβ signaling proteins. Wound healing was faster in N-WASPFKO mice compared to control mice and N-WASP deficient fibroblasts were found to have enhanced collagen gel contraction properties. These results suggest that N-WASP deficiency in fibroblasts improves wound healing by growth factor-mediated enhancement of keratinocyte proliferation and increased wound contraction in mice. PMID:27909303

FOXO1 expression in keratinocytes promotes connective tissue healing

PubMed Central

Zhang, Chenying; Lim, Jason; Liu, Jian; Ponugoti, Bhaskar; Alsadun, Sarah; Tian, Chen; Vafa, Rameen; Graves, Dana T.

2017-01-01

Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFβ1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome. PMID:28220813

Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes.

PubMed

Sayedyahossein, Samar; Nini, Lylia; Irvine, Timothy S; Dagnino, Lina

2012-10-01

Phagocytic melanosome uptake by epidermal keratinocytes is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal keratinocytes isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the keratinocyte growth factor (KGF) receptor or the protease-activated receptor-2. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient keratinocytes also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin β1-deficient keratinocytes also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in keratinocytes, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.

IGF-1 deficiency impairs cerebral myogenic autoregulation in hypertensive mice.

PubMed

Toth, Peter; Tucsek, Zsuzsanna; Tarantini, Stefano; Sosnowska, Danuta; Gautam, Tripti; Mitschelen, Matthew; Koller, Akos; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan

2014-12-01

Aging impairs autoregulatory protection in the brain, exacerbating hypertension-induced cerebromicrovascular injury, neuroinflammation, and development of vascular cognitive impairment. Despite the importance of the age-related decline in circulating insulin-like growth factor-1 (IGF-1) levels in cerebrovascular aging, the effects of IGF-1 deficiency on functional adaptation of cerebral arteries to high blood pressure remain elusive. To determine whether IGF-1 deficiency impairs autoregulatory protection, hypertension was induced in control and IGF-1-deficient mice (Igf1(f/f)+TBG-iCre-AAV8) by chronic infusion of angiotensin-II. In hypertensive control mice, cerebral blood flow (CBF) autoregulation was extended to higher pressure values and the pressure-induced tone of middle cerebral arteries (MCAs) was increased. In hypertensive IGF-1-deficient mice, autoregulation was markedly disrupted, and MCAs did not show adaptive increases in myogenic tone. In control mice, the mechanism of adaptation to hypertension involved upregulation of TRPC channels in MCAs and this mechanism was impaired in hypertensive IGF-1-deficient mice. Likely downstream consequences of cerebrovascular autoregulatory dysfunction in hypertensive IGF-1-deficient mice included exacerbated disruption of the blood-brain barrier and neuroinflammation (microglia activation and upregulation of proinflammatory cytokines and chemokines), which were associated with impaired hippocampal cognitive function. Collectively, IGF-1 deficiency impairs autoregulatory protection in the brain of hypertensive mice, potentially exacerbating cerebromicrovascular injury and neuroinflammation mimicking the aging phenotype.

Inbred Strain-Specific Effects of Exercise in Wild Type and Biglycan Deficient Mice

PubMed Central

Wallace, Joseph M.; Golcuk, Kurtulus; Morris, Michael D.; Kohn, David H.

2010-01-01

Biglycan (bgn)-deficient mice (KO) have defective osteoblasts which lead to changes in the amount and quality of bone. Altered tissue strength in C57BL6/129 (B6;129) KO mice, a property which is independent of tissue quantity, suggests that deficiencies in tissue quality are responsible. However, the response to bgn-deficiency is inbred strain-specific. Mechanical loading influences bone matrix quality in addition to any increase in bone mass or change in bone formation activity. Since many diseases influence the mechanical integrity of bone through altered tissue quality, loading may be a way to prevent and treat extracellular matrix deficiencies. C3H/He (C3H) mice consistently have a less vigorous response to mechanical loading vs. other inbred strains. It was therefore hypothesized that the bones from both wild type (WT) and KO B6;129 mice would be more responsive to exercise than the bones from C3H mice. To test these hypotheses at 11 weeks of age, following 21 consecutive days of exercise, we investigated cross-sectional geometry, mechanical properties, and tissue composition in the tibiae of male mice bred on B6;129 and C3H backgrounds. This study demonstrated inbred strain-specific compositional and mechanical changes following exercise in WT and KO mice, and showed evidence of genotype-specific changes in bone in response to loading in a gene disruption model. This study further shows that exercise can influence bone tissue composition and/or mechanical integrity without changes in bone geometry. Together, these data suggest that exercise may represent a possible means to alter tissue quality and mechanical deficiencies caused by many diseases of bone. PMID:20033775

Mice deficient in LMAN1 exhibit FV and FVIII deficiencies and liver accumulation of α1-antitrypsin

PubMed Central

Zheng, Chunlei; Zhu, Min; Tao, Jiayi; Vasievich, Matthew P.; Baines, Andrea; Kim, Jinoh; Schekman, Randy; Kaufman, Randal J.; Ginsburg, David

2011-01-01

The type 1-transmembrane protein LMAN1 (ERGIC-53) forms a complex with the soluble protein MCFD2 and cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). Mutations in either LMAN1 or MCFD2 cause the combined deficiency of factor V (FV) and factor VIII (FVIII; F5F8D), suggesting an ER-to-Golgi cargo receptor function for the LMAN1-MCFD2 complex. Here we report the analysis of LMAN1-deficient mice. Levels of plasma FV and FVIII, and platelet FV, are all reduced to ∼ 50% of wild-type in Lman1−/− mice, compared with the 5%-30% levels typically observed in human F5F8D patients. Despite previous reports identifying cathepsin C, cathepsin Z, and α1-antitrypsin as additional potential cargoes for LMAN1, no differences were observed between wild-type and Lman1−/− mice in the levels of cathepsin C and cathepsin Z in liver lysates or α1-antitrypsin levels in plasma. LMAN1 deficiency had no apparent effect on COPII-coated vesicle formation in an in vitro assay. However, the ER in Lman1−/− hepatocytes is slightly distended, with significant accumulation of α1-antitrypsin and GRP78. An unexpected, partially penetrant, perinatal lethality was observed for Lman1−/− mice, dependent on the specific inbred strain genetic background, suggesting a potential role for other, as yet unidentified LMAN1-dependent cargo proteins. PMID:21795745

Lethal Effect of CD3-Specific Antibody in Mice Deficient in TGF-β1 by Uncontrolled Flu-Like Syndrome1

PubMed Central

Perruche, Sylvain; Zhang, Pin; Maruyama, Takashi; Bluestone, Jeffrey A.; Saas, Philippe; Chen, WanJun

2010-01-01

CD3-specific Ab therapy results in a transient, self-limiting, cytokine-associated, flu-like syndrome in experimental animals and in patients, but the underlying mechanism for this spontaneous resolution remains elusive. By using an in vivo model of CD3-specific Ab-induced flu-like syndrome, we show in this paper that a single injection of sublethal dose of the Ab killed all TGF-β1−/− mice. The death of TGF-β1−/− mice was associated with occurrence of this uncontrolled flu-like syndrome, as demonstrated by a sustained storm of systemic inflammatory TNF and IFN-γ cytokines. We present evidence that deficiency of professional phagocytes to produce TGF-β1 after apoptotic T cell clearance may be responsible, together with hypersensitivity of T cells to both activation and apoptosis, for the uncontrolled inflammation. These findings indicate a key role for TGF-β1 and phagocytes in protecting the recipients from lethal inflammation and resolving the flu-like syndrome after CD3-specific Ab treatment. The study may also provide a novel molecular mechanism explaining the early death in TGF-β1−/− mice. PMID:19561097

MCPIP1 Deficiency in Mice Results in Severe Anemia Related to Autoimmune Mechanisms

PubMed Central

Zhou, Zhou; Miao, Ruidong; Huang, Shengping; Elder, Brandon; Quinn, Tim; Papasian, Christopher J.; Zhang, Jifeng; Fan, Daping; Chen, Y. Eugene; Fu, Mingui

2013-01-01

Autoimmune gastritis is an organ-specific autoimmune disease of the stomach associated with pernicious anemia. The previous work from us and other groups identified MCPIP1 as an essential factor controlling inflammation and immune homeostasis. MCPIP1-/- developed severe anemia. However, the mechanisms underlying this phenotype remain unclear. In the present study, we found that MCPIP1 deficiency in mice resulted in severe anemia related to autoimmune mechanisms. Although MCPIP1 deficiency did not affect erythropoiesis per se, the erythropoiesis in MCPIP1-/- bone marrow erythroblasts was significantly attenuated due to iron and vitamin B12 (VB12) deficiency, which was mainly resulted from autoimmunity-associated gastritis and parietal cell loss. Consistently, exogenous supplement of iron and VB12 greatly improved the anemia phenotype of MCPIP1-/- mice. Finally, we have evidence suggesting that autoimmune hemolysis may also contribute to anemia phenotype of MCPIP1-/- mice. Taken together, our study suggests that MCPIP1 deficiency in mice leads to the development of autoimmune gastritis and pernicious anemia. Thus, MCPIP1-/- mice may be a good mouse model for investigating the pathogenesis of pernicious anemia and testing the efficacy of some potential drugs for treatment of this disease. PMID:24324805

Programmed cell death 1 (PD-1) regulates the effector function of CD8 T cells via PD-L1 expressed on target keratinocytes.

PubMed

Okiyama, Naoko; Katz, Stephen I

2014-09-01

Programmed cell death 1 (PD-1) is an inhibitory molecule expressed by activated T cells. Its ligands (PD-L1 and -L2; PD-Ls) are expressed not only by a variety of leukocytes but also by stromal cells. To assess the role of PD-1 in CD8 T cell-mediated diseases, we used PD-1-knockout (KO) OVA-specific T cell-receptor transgenic (Tg) CD8 T cells (OT-I cells) in a murine model of mucocutaneous graft-versus-host disease (GVHD). We found that mice expressing OVA on epidermal keratinocytes (K14-mOVA mice) developed markedly enhanced GVHD-like disease after transfer of PD-1-KO OT-I cells as compared to those mice transferred with wild-type OT-I cells. In addition, K14-mOVA × OT-I double Tg (DTg) mice do not develop GVHD-like disease after adoptive transfer of OT-I cells, while transfer of PD-1-KO OT-I cells caused GVHD-like disease in a Fas/Fas-L independent manner. These results suggest that PD-1/PD-Ls-interactions have stronger inhibitory effects on pathogenic CD8 T cells than does Fas/Fas-L-interactions. Keratinocytes from K14-mOVA mice with GVHD-like skin lesions express PD-L1, while those from mice without the disease do not. These findings reflect the fact that primary keratinocytes express PD-L1 when stimulated by interferon-γ in vitro. When co-cultured with K14-mOVA keratinocytes for 2 days, PD-1-KO OT-I cells exhibited enhanced proliferation and activation compared to wild-type OT-I cells. In addition, knockdown of 50% PD-L1 expression on the keratinocytes with transfection of PD-L1-siRNA enhanced OT-I cell proliferation. In aggregate, our data strongly suggest that PD-L1, expressed on activated target keratinocytes presenting autoantigens, regulates autoaggressive CD8 T cells, and inhibits the development of mucocutaneous autoimmune diseases. Published by Elsevier Ltd.

Estrogen deficiency heterogeneously affects tissue specific stem cells in mice

PubMed Central

Kitajima, Yuriko; Doi, Hanako; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Kitajima, Michio; Miura, Kiyonori; Li, Tao-Sheng; Masuzaki, Hideaki

2015-01-01

Postmenopausal disorders are frequently observed in various organs, but their relationship with estrogen deficiency and mechanisms remain unclear. As tissue-specific stem cells have been found to express estrogen receptors, we examined the hypothesis that estrogen deficiency impairs stem cells, which consequently contributes to postmenopausal disorders. Six-week-old C57BL/6 female mice were ovariectomized, following which they received 17β-estradiol replacement or vehicle (control). Sham-operated mice were used as healthy controls. All mice were killed for evaluation 2 months after treatments. Compared with the healthy control, ovariectomy significantly decreased uterine weight, which was partially recovered by 17β-estradiol replacement. Ovariectomy significantly increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, but impaired their capacity to grow mixed cell-type colonies in vitro. Estrogen replacement further increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, without significantly affecting colony growth in vitro. The number of CD105-positive mesenchymal stem cells in bone marrow also significantly decreased after ovariectomy, but completely recovered following estrogen replacement. Otherwise, neither ovariectomy nor estrogen replacement changed the number of Pax7-positive satellite cells, which are a skeletal muscle-type stem cell. Estrogen deficiency heterogeneously affected tissue-specific stem cells, suggesting a likely and direct relationship with postmenopausal disorders. PMID:26245252

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

PubMed

Saher, G; Hildt, E

1999-09-24

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

Crif1 Deficiency Reduces Adipose OXPHOS Capacity and Triggers Inflammation and Insulin Resistance in Mice

PubMed Central

Ryu, Min Jeong; Kim, Soung Jung; Kim, Yong Kyung; Choi, Min Jeong; Tadi, Surendar; Lee, Min Hee; Lee, Seong Eun; Chung, Hyo Kyun; Jung, Saet Byel; Kim, Hyun-Jin; Jo, Young Suk; Kim, Koon Soon; Lee, Sang-Hee; Kim, Jin Man; Kweon, Gi Ryang; Park, Ki Cheol; Lee, Jung Uee; Kong, Young Yun; Lee, Chul-Ho; Chung, Jongkyeong; Shong, Minho

2013-01-01

Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS–deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA–encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance. PMID:23516375

MUC1-C ACTIVATES THE TAK1 INFLAMMATORY PATHWAY IN COLON CANCER

PubMed Central

Takahashi, Hidekazu; Jin, Caining; Rajabi, Hasan; Pitroda, Sean; Alam, Maroof; Ahmad, Rehan; Raina, Deepak; Hasegawa, Masanori; Suzuki, Yozo; Tagde, Ashujit; Bronson, Roderick T.; Weichselbaum, Ralph; Kufe, Donald

2015-01-01

The mucin 1 (MUC1) oncoprotein has been linked to the inflammatory response by promoting cytokine-mediated activation of the NF-κB pathway. The TGF-β-activated kinase 1 (TAK1) is an essential effector of proinflammatory NF-κB signaling that also regulates cancer cell survival. The present studies demonstrate that the MUC1-C transmembrane subunit induces TAK1 expression in colon cancer cells. MUC1 also induces TAK1 in a MUC1+/−/IL-10−/− mouse model of colitis and colon tumorigenesis. We show that MUC1-C promotes NF-κB-mediated activation of TAK1 transcription and, in a positive regulatory loop, MUC1-C contributes to TAK1-induced NF-κB signaling. In this way, MUC1-C binds directly to TAK1 and confers the association of TAK1 with TRAF6, which is necessary for TAK1-mediated activation of NF-κB. Targeting MUC1-C thus suppresses the TAK1→NF-κB pathway, downregulates BCL-XL, and in turn sensitizes colon cancer cells to MEK inhibition. Analysis of colon cancer databases further indicates that MUC1, TAK1 and TRAF6 are upregulated in tumors associated with decreased survival and that MUC1-C-induced gene expression patterns predict poor outcomes in patients. These results support a model in which MUC1-C-induced TAK1→NF-κB signaling contributes to intestinal inflammation and colon cancer progression. PMID:25659581

Absence of Wip1 partially rescues Atm deficiency phenotypes in mice

PubMed Central

Darlington, Yolanda; Nguyen, Thuy-Ai; Moon, Sung-Hwan; Herron, Alan; Rao, Pulivarthi; Zhu, Chengming; Lu, Xiongbin; Donehower, Lawrence A.

2011-01-01

Wildtype p53-Induced Phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may play a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm null mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared to Atm null mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared to their Atm null counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared to Atm null mice. Finally, doubly null mice were partially rescued from infertility defects observed in Atm null mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular. PMID:21765465

Sick sinus syndrome in HCN1-deficient mice.

PubMed

Fenske, Stefanie; Krause, Stefanie C; Hassan, Sami I H; Becirovic, Elvir; Auer, Franziska; Bernard, Rebekka; Kupatt, Christian; Lange, Philipp; Ziegler, Tilman; Wotjak, Carsten T; Zhang, Henggui; Hammelmann, Verena; Paparizos, Christos; Biel, Martin; Wahl-Schott, Christian A

2013-12-17

Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and requires surgical implantation of electric pacemaker devices. Frequently, SND occurs in heart failure and hypertension, conditions that lead to electric instability of the heart. Although the pathologies of acquired SND have been studied extensively, little is known about the molecular and cellular mechanisms that cause congenital SND. Here, we show that the HCN1 protein is highly expressed in the sinoatrial node and is colocalized with HCN4, the main sinoatrial pacemaker channel isoform. To characterize the cardiac phenotype of HCN1-deficient mice, a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparation, telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology was performed. On the basis of these experiments we demonstrate that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output. We propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.

Insulin resistance in striated muscle-specific integrin receptor beta1-deficient mice.

PubMed

Zong, Haihong; Bastie, Claire C; Xu, Jun; Fassler, Reinhard; Campbell, Kevin P; Kurland, Irwin J; Pessin, Jeffrey E

2009-02-13

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin beta1 subunit in striated muscle results in a near complete loss of integrin beta1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin beta1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR.Rictor.LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin beta1 signaling events in mediating cross-talk to that of insulin action.

Impaired social recognition memory in Recombination Activating Gene 1-deficient mice

PubMed Central

McGowan, Patrick O.; Hope, Thomas A.; Meck, Warren H.; Kelsoe, Garnett; Williams, Christina L.

2012-01-01

The Recombination Activating Genes (RAGs) encode two enzymes that play key roles in the adaptive immune system. RAG1 and RAG2 mediate VDJ recombination, a process necessary for the maturation of B- and T-cells. Interestingly, RAG1 is also expressed in the brain, particularly in areas of high neural density such as the hippocampus, although its function is unknown. We tested evidence that RAG1 plays a role in brain function using a social recognition memory task, an assessment of the acquisition and retention of conspecific identity. In a first experiment, we found that RAG1-deficient mice show impaired social recognition memory compared to mice wildtype for the RAG1 allele. In a second experiment, by breeding to homogenize background genotype we found that RAG1-deficient mice show impaired social recognition memory relative to heterozygous or RAG2-deficient littermates. Because RAG1 and RAG2 null mice are both immunodeficient, the results suggest that the memory impairment is not an indirect effect of immunological dysfunction. RAG1-deficient mice show normal habituation to non-socially derived odors and habituation to an open-field, indicating that the observed effect is not likely a result of a general deficit in habituation to novelty. These data trace the origin of the impairment in social recognition memory in RAG1-deficient mice to the RAG1 gene locus and implicate RAG1 in memory formation. PMID:21354115

Altered pupillary light reflex in PACAP receptor 1-deficient mice.

PubMed

Engelund, Anna; Fahrenkrug, Jan; Harrison, Adrian; Luuk, Hendrik; Hannibal, Jens

2012-05-09

The pupillary light reflex (PLR) is regulated by the classical photoreceptors, rods and cones, and by intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin. IpRGCs receive input from rods and cones and project to the olivary pretectal nucleus (OPN), which is the primary visual center involved in PLR. Mice lacking either the classical photoreceptors or melanopsin exhibit some changes in PLR, whereas the reflex is completely lost in mice deficient of all three photoreceptors. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is co-stored with melanopsin in ipRGCs and mediates light signaling to the brain via the specific PACAP receptor 1 (PAC1R). Here, we examined the occurrence of PACAP and PAC1R in the mouse OPN, and studied if lack of PAC1R affected the PLR. PACAP-immunoreactive nerve fibers were shown in the mouse OPN, and by in situ hybridization histochemistry, we demonstrated the presence of PAC1R mRNA. Mice lacking PAC1R exhibited a significantly attenuated PLR compared to wild type mice upon light stimulation, and the difference became more pronounced as light intensity was increased. Our findings accord well with observations of the PLR in the melanopsin-deficient mouse. We conclude that PACAP/PAC1R signaling is involved in the sustained phase of the PLR at high irradiances. Copyright © 2012 Elsevier B.V. All rights reserved.

An evolutionarily conserved motif in the TAB1 C-terminal region is necessary for interaction with and activation of TAK1 MAPKKK.

PubMed

Ono, K; Ohtomo, T; Sato, S; Sugamata, Y; Suzuki, M; Hisamoto, N; Ninomiya-Tsuji, J; Tsuchiya, M; Matsumoto, K

2001-06-29

TAK1, a member of the MAPKKK family, is involved in the intracellular signaling pathways mediated by transforming growth factor beta, interleukin 1, and Wnt. TAK1 kinase activity is specifically activated by the TAK1-binding protein TAB1. The C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation. Analysis of various truncated versions of TAB1-C68 defined a C-terminal 30-amino acid sequence (TAB1-C30) necessary for TAK1 binding and activation. NMR studies revealed that the TAB1-C30 region has a unique alpha-helical structure. We identified a conserved sequence motif, PYVDXA/TXF, in the C-terminal domain of mammalian TAB1, Xenopus TAB1, and its Caenorhabditis elegans homolog TAP-1, suggesting that this motif constitutes a specific TAK1 docking site. Alanine substitution mutagenesis showed that TAB1 Phe-484, located in the conserved motif, is crucial for TAK1 binding and activation. The C. elegans homolog of TAB1, TAP-1, was able to interact with and activate the C. elegans homolog of TAK1, MOM-4. However, the site in TAP-1 corresponding to Phe-484 of TAB1 is an alanine residue (Ala-364), and changing this residue to Phe abrogates the ability of TAP-1 to interact with and activate MOM-4. These results suggest that the Phe or Ala residue within the conserved motif of the TAB1-related proteins is important for interaction with and activation of specific TAK1 MAPKKK family members in vivo.

Sunlight Triggers Cutaneous Lupus through a Colony Stimulating Factor-1 (CSF-1) Dependent Mechanism in MRL-Faslpr mice

PubMed Central

Menke, Julia; Hsu, Mei-Yu; Byrne, Katelyn T.; Lucas, Julie A.; Rabacal, Whitney A.; Croker, Byron P.; Zong, Xiao-Hua; Stanley, E. Richard; Kelley, Vicki R.

2008-01-01

Sunlight (UVB) triggers cutaneous (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (Mø) -mediated mechanism in MRL-Faslpr mice. By constructing mutant MRL-Faslpr strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex-vivo gene transfer to deliver CSF-1 intra-dermally, we determined that CSF-1 induces CLE in lupus-susceptible, MRL-Faslpr mice, but not in lupus-resistant, BALB/c mice. Notably, UVB incites an increase in Mø, apoptosis in the skin and CLE in MRL-Faslpr, but not in CSF-1-deficient MRL-Faslpr mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of Mø that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, Mø-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Faslpr, but not lupus-resistant BALB/c mice. Taken together, we envision CSF-1 as the “match” and lupus-susceptibility as the “tinder” leading to CLE. PMID:18981160

Resistin deficiency in mice has no effect on pulmonary responses induced by acute ozone exposure

PubMed Central

Razvi, Shehla S.; Richards, Jeremy B.; Malik, Farhan; Cromar, Kevin R.; Price, Roger E.; Bell, Cynthia S.; Weng, Tingting; Atkins, Constance L.; Spencer, Chantal Y.; Cockerill, Katherine J.; Alexander, Amy L.; Blackburn, Michael R.; Alcorn, Joseph L.; Haque, Ikram U.

2015-01-01

Acute exposure to ozone (O3), an air pollutant, causes pulmonary inflammation, airway epithelial desquamation, and airway hyperresponsiveness (AHR). Pro-inflammatory cytokines—including IL-6 and ligands of chemokine (C-X-C motif) receptor 2 [keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-2], TNF receptor 1 and 2 (TNF), and type I IL-1 receptor (IL-1α and IL-1β)—promote these sequelae. Human resistin, a pleiotropic hormone and cytokine, induces expression of IL-1α, IL-1β, IL-6, IL-8 (the human ortholog of murine KC and MIP-2), and TNF. Functional differences exist between human and murine resistin; yet given the aforementioned observations, we hypothesized that murine resistin promotes O3-induced lung pathology by inducing expression of the same inflammatory cytokines as human resistin. Consequently, we examined indexes of O3-induced lung pathology in wild-type and resistin-deficient mice following acute exposure to either filtered room air or O3. In wild-type mice, O3 increased bronchoalveolar lavage fluid (BALF) resistin. Furthermore, O3 increased lung tissue or BALF IL-1α, IL-6, KC, TNF, macrophages, neutrophils, and epithelial cells in wild-type and resistin-deficient mice. With the exception of KC, which was significantly greater in resistin-deficient compared with wild-type mice, no genotype-related differences in the other indexes existed following O3 exposure. O3 caused AHR to acetyl-β-methylcholine chloride (methacholine) in wild-type and resistin-deficient mice. However, genotype-related differences in airway responsiveness to methacholine were nonexistent subsequent to O3 exposure. Taken together, these data demonstrate that murine resistin is increased in the lungs of wild-type mice following acute O3 exposure but does not promote O3-induced lung pathology. PMID:26386120

Osteoblast-Specific Krm2 Overexpression and Lrp5 Deficiency Have Different Effects on Fracture Healing in Mice

PubMed Central

Liedert, Astrid; Röntgen, Viktoria; Schinke, Thorsten; Benisch, Peggy; Ebert, Regina; Jakob, Franz; Klein-Hitpass, Ludger; Lennerz, Jochen K.; Amling, Michael; Ignatius, Anita

2014-01-01

The canonical Wnt/β-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5−/−) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5−/− mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5−/− mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active β-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis. PMID:25061805

Mice with hepatocyte-specific FXR deficiency are resistant to spontaneous but susceptible to cholic acid-induced hepatocarcinogenesis

PubMed Central

Zhu, Yan; Li, Guodong; Williams, Jessica A.; Buckley, Kyle; Tawfik, Ossama; Luyendyk, James P.

2016-01-01

Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily with its endogenous ligands bile acids. Mice with whole body FXR deficiency develop liver tumors spontaneously, but the underlying mechanism is unclear. Moreover, it is unknown whether FXR deficiency in liver alone serves as a tumor initiator or promoter during liver carcinogenesis. This study aims to evaluate the effects of hepatocyte-specific FXR deficiency (FXRhep−/−) in liver tumor formation. The results showed that FXRhep−/− mice did not show spontaneous liver tumorigenesis with aging (up to 24 mo of age). Therefore FXRhep−/− mice were fed a bile acid (cholic acid)-containing diet alone or along with a liver tumor initiator, diethylnitrosamine (DEN). Thirty weeks later, no tumors were found in wild-type or FXRhep−/− mice without any treatment or with DEN only. However, with cholic acid, while only some wild-type mice developed tumors, all FXRhep−/− mice presented with severe liver injury and tumors. Interestingly, FXRhep−/− mouse livers increased basal expression of tumor suppressor p53 protein, apoptosis, and decreased basal cyclin D1 expression, which may prevent tumor development in FXRhep−/− mice. However, cholic acid feeding reversed these effects in FXRhep−/− mice, which is associated with an increased cyclin D1 and decreased cell cycle inhibitors. More in-depth analysis indicates that the increased in cell growth might result from disturbance of the MAPK and JAK/Stat3 signaling pathways. In conclusion, this study shows that hepatic FXR deficiency may only serve as a tumor initiator, and increased bile acids is required for tumor formation likely by promoting cell proliferation. PMID:26744468

Chronic mild stress impairs latent inhibition and induces region-specific neural activation in CHL1-deficient mice, a mouse model of schizophrenia.

PubMed

Buhusi, Mona; Obray, Daniel; Guercio, Bret; Bartlett, Mitchell J; Buhusi, Catalin V

2017-08-30

Schizophrenia is a neurodevelopmental disorder characterized by abnormal processing of information and attentional deficits. Schizophrenia has a high genetic component but is precipitated by environmental factors, as proposed by the 'two-hit' theory of schizophrenia. Here we compared latent inhibition as a measure of learning and attention, in CHL1-deficient mice, an animal model of schizophrenia, and their wild-type littermates, under no-stress and chronic mild stress conditions. All unstressed mice as well as the stressed wild-type mice showed latent inhibition. In contrast, CHL1-deficient mice did not show latent inhibition after exposure to chronic stress. Differences in neuronal activation (c-Fos-positive cell counts) were noted in brain regions associated with latent inhibition: Neuronal activation in the prelimbic/infralimbic cortices and the nucleus accumbens shell was affected solely by stress. Neuronal activation in basolateral amygdala and ventral hippocampus was affected independently by stress and genotype. Most importantly, neural activation in nucleus accumbens core was affected by the interaction between stress and genotype. These results provide strong support for a 'two-hit' (genes x environment) effect on latent inhibition in CHL1-deficient mice, and identify CHL1-deficient mice as a model of schizophrenia-like learning and attention impairments. Copyright © 2017 Elsevier B.V. All rights reserved.

Melanocortin 1 Receptor Deficiency Promotes Atherosclerosis in Apolipoprotein E-/- Mice.

PubMed

Rinne, Petteri; Kadiri, James J; Velasco-Delgado, Mauricio; Nuutinen, Salla; Viitala, Miro; Hollmén, Maija; Rami, Martina; Savontaus, Eriika; Steffens, Sabine

2018-02-01

The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates anti-inflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis. Apoe -/- (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1r e/e ) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe -/- Mc1r e/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe -/- controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe -/- Mc1r e/e mice showed a defect in bile acid metabolism that aggravated high-fat diet-induced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6C high monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6C high monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1. The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation. © 2017 The Authors.

Nuclear expression of IL-33 in epidermal keratinocytes promotes wound healing in mice.

PubMed

Oshio, Tomoyuki; Komine, Mayumi; Tsuda, Hidetoshi; Tominaga, Shin-Ichi; Saito, Hirohisa; Nakae, Susumu; Ohtsuki, Mamitaro

2017-02-01

Skin is the outermost tissue of the human body, and works as a mechanical, chemical, and biological barrier. The epidermis is the uppermost layer of the skin, and keratinocytes constitute the majority of epidermal cells. Wounds are disruptions of skin integrity, and cause tremendous disadvantages to humans; accordingly, rapid wound healing is very important. Interleukin (IL)-33 is expressed in barrier tissue cells, such as epithelial and endothelial cells. Upon injury, IL-33 is released to stimulate immune cells, functioning as an "alarmin." ST2 is a receptor for IL-33; its soluble form (s)ST2 acts as a decoy receptor and competes for IL-33 binding. We aimed to clarify the role of IL-33 in wound healing. Wild-type (WT), IL-33 knockout (IL33 KO) mice, and sST2 transgenic (Tg) mice were wounded with a 4-mm punch, and the wound healing process was compared. Immunohistochemical analyses were performed to detect macrophages, neutrophils, and mast cells. Total RNA was extracted from the skin samples and real-time PCR was performed. An in vitro scratch wound assay was performed. Wound healing was delayed in IL33 KO mice compared to WT mice, while wound healing in sST2 Tg mice was comparable to that of WT mice. A histological examination showed delayed elongation of the epidermal tongue in IL-33 KO mice. An immunohistochemical study revealed prolonged neutrophilic infiltration at a later stage in IL-33 KO mice. IL-6, IL-1β, and CXCL1 transcripts were more abundant in the wounds of IL-33 KO mice than WT mice. Intraperitoneal administration of an NFκB inhibitor to IL-33 KO mice normalized the delayed wound healing and the enhanced expression of IL-6 in IL-33 KO mice. Epidermal keratinocytes from IL-33 KO mice showed delayed wound closure compared to those from WT mice. Our results indicate that nuclear IL-33, but not IL-33 as a cytokine, has beneficial effects on wound healing in mice, probably by suppressing NFκB to inhibit excessive inflammation and by maintaining

Lipid Absorption Defects in Intestine-specific Microsomal Triglyceride Transfer Protein and ATP-binding Cassette Transporter A1-deficient Mice*

PubMed Central

Iqbal, Jahangir; Parks, John S.; Hussain, M. Mahmood

2013-01-01

We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92–95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and carnitine palmitoyltransferase 1α (CPT1α). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations. PMID:24019513

Smad4 disruption accelerates keratinocyte reepithelialization in murine cutaneous wound repair.

PubMed

Yang, Leilei; Li, Wenlong; Wang, Shaoxia; Wang, Lijuan; Li, Yang; Yang, Xiao; Peng, Ruiyun

2012-10-01

Keratinocyte reepithelialization is a rate-limiting event in cutaneous wound repair, which involves the migration and proliferation of keratinocytes to cover the denuded dermal surface. Transforming growth factor-β1 (TGF-β1) has the ability to induce epithelial cell migration while inhibiting proliferation, and controversial results have been generated regarding the effect of TGF-β signaling on reepithelialization. In this study, full-thickness skin wounds were made in keratinocyte-specific Smad4 knockout and the control mice. The wound closure, reepithelialization, keratinocyte proliferation, myofibroblast numbers and collagen deposition of were assessed. The results showed that the proliferation of keratinocytes increased, which accelerated the reepithelialization, and led to faster wound repair in the epidermis of Smad4 mutant mice. Upregulation of keratin 17, 14-3-3 sigma and phosphorylated AKT in the hyperproliferative epidermis may be correlated with the accelerated reepithelialization. We conclude that Smad4 plays an inhibitory role in the keratinocyte-mediated reepithelialization of wound healing.

Diminished pheromone-induced sexual behavior in neurokinin-1 receptor deficient (TACR1(-/-)) mice.

PubMed

Berger, A; Tran, A H; Dida, J; Minkin, S; Gerard, N P; Yeomans, J; Paige, C J

2012-07-01

Studies in mice with targeted deletions of tachykinin genes suggest that tachykinins and their receptors influence emotional behaviors such as aggression, depression and anxiety. Here, we investigated whether TAC1- and TAC4-encoded peptides (substance P and hemokinin-1, respectively) and the neurokinin-1 receptor (NK-1R) are involved in the modulation of sexual behaviors. Male mice deficient for the NK-1R (TACR1 (-/-)) exhibited decreased exploration of female urine in contrast to C57BL/6 control mice and mice deficient for NK-1R ligands such as TAC1 (-/-), TAC4 (-/-) and the newly generated TAC1 (-/-) /TAC4 (-/-) mice. In comparison to C57BL/6 mice, mounting frequency and duration were decreased in male TACR1 (-/-) mice, while mounting latency was increased. Decreased preference for sexual pheromones was also seen in female TACR1 (-/-) mice. Furthermore, administration of the NK-1R-antagonist L-703,606 decreased investigation of female urine by male C57BL/6 mice, suggesting an involvement of NK-1R in urine sniffing behavior. Our results provide evidence for the NK-1R in facilitating sexual approach behavior, as male TACR1 (-/-) mice exhibited blunted approach behavior toward females following the initial interaction compared with C57BL/6 mice. NK-1R signaling may therefore play an important role in pheromone-induced sexual behavior. © 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis.

PubMed

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St Arnaud, René; Dedhar, Shoukat; D'Souza, Sudhir J A; Dagnino, Lina

2008-04-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

Impaired Hair Follicle Morphogenesis and Polarized Keratinocyte Movement upon Conditional Inactivation of Integrin-linked Kinase in the Epidermis

PubMed Central

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St. Arnaud, René; Dedhar, Shoukat

2008-01-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function. PMID:18234842

Sunlight triggers cutaneous lupus through a CSF-1-dependent mechanism in MRL-Fas(lpr) mice.

PubMed

Menke, Julia; Hsu, Mei-Yu; Byrne, Katelyn T; Lucas, Julie A; Rabacal, Whitney A; Croker, Byron P; Zong, Xiao-Hua; Stanley, E Richard; Kelley, Vicki R

2008-11-15

Sunlight (UVB) triggers cutaneous lupus erythematosus (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (Mø)-mediated mechanism in MRL-Fas(lpr) mice. By constructing mutant MRL-Fas(lpr) strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex vivo gene transfer to deliver CSF-1 intradermally, we determined that CSF-1 induces CLE in lupus-susceptible MRL-Fas(lpr) mice, but not in lupus-resistant BALB/c mice. UVB incites an increase in Møs, apoptosis in the skin, and CLE in MRL-Fas(lpr), but not in CSF-1-deficient MRL-Fas(lpr) mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of Møs that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, Mø-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Fas(lpr) but not lupus-resistant BALB/c mice. Taken together, CSF-1 is envisioned as the match and lupus susceptibility as the tinder leading to CLE.

Suppression of the hypothalamic-pituitary-gonadal axis by TAK-385 (relugolix), a novel, investigational, orally active, small molecule gonadotropin-releasing hormone (GnRH) antagonist: studies in human GnRH receptor knock-in mice.

PubMed

Nakata, Daisuke; Masaki, Tsuneo; Tanaka, Akira; Yoshimatsu, Mie; Akinaga, Yumiko; Asada, Mari; Sasada, Reiko; Takeyama, Michiyasu; Miwa, Kazuhiro; Watanabe, Tatsuya; Kusaka, Masami

2014-01-15

TAK-385 (relugolix) is a novel, non-peptide, orally active gonadotropin-releasing hormone (GnRH) antagonist, which builds on previous work with non-peptide GnRH antagonist TAK-013. TAK-385 possesses higher affinity and more potent antagonistic activity for human and monkey GnRH receptors compared with TAK-013. Both TAK-385 and TAK-013 have low affinity for the rat GnRH receptor, making them difficult to evaluate in rodent models. Here we report the human GnRH receptor knock-in mouse as a humanized model to investigate pharmacological properties of these compounds on gonadal function. Twice-daily oral administration of TAK-013 (10mg/kg) for 4 weeks decreased the weights of testes and ventral prostate in male knock-in mice but not in male wild-type mice, demonstrating the validity of this model to evaluate antagonists for the human GnRH receptor. The same dose of TAK-385 also reduced the prostate weight to castrate levels in male knock-in mice. In female knock-in mice, twice-daily oral administration of TAK-385 (100mg/kg) induced constant diestrous phases within the first week, decreased the uterus weight to ovariectomized levels and downregulated GnRH receptor mRNA in the pituitary after 4 weeks. Gonadal function of TAK-385-treated knock-in mice began to recover after 5 days and almost completely recovered within 14 days after drug withdrawal in both sexes. Our findings demonstrate that TAK-385 acts as an antagonist for human GnRH receptor in vivo and daily oral administration potently, continuously and reversibly suppresses the hypothalamic-pituitary-gonadal axis. TAK-385 may provide useful therapeutic interventions in hormone-dependent diseases including endometriosis, uterine fibroids and prostate cancer. Copyright © 2013 Elsevier B.V. All rights reserved.

[Correlation of insulin-like growth factor-1 (IGF-1) to angiogenesis of breast cancer in IGF-1-deficient mice].

PubMed

Tang, Hong-Bo; Ren, Yu-Ping; Zhang, Jun; Ma, Shi-Hui; Gao, Feng; Wu, Yi-Ping

2007-11-01

Insulin-like growth factors (IGFs) play important roles in the development and progression of tumors. But the mechanism of tumorigenesis in relation to IGF-1 is unclear yet. This study was to explore the correlation of circulating IGF-1 level to the angiogenesis of breast cancer in IGF-1-deficient mice. The liver-specific IGF-1-deficient (LID) mice and control mice were injected with 7,12-dimethybenz(a)anthracene (DMBA) to develop breast cancer. Ginsenoside Rg3 was used to intervene tumor growth. The occurrence rates of breast cancer were compared. The expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) was detected by immunohistochemistry. The occurrence rate of breast cancer was 66.67% in untreated control mice, 33.33% in untreated LID mice, 36.00% in Rg3-treated control mice, and 12.00% in Rg3-treated LID mice. The tumor size was (0.79+/-0.20) cm in untreated control mice, (0.37+/-0.08) cm in untreated LID mice, (0.32+/-0.08) cm in Rg3-treated control mice, and (0.15+/-0.05) cm in Rg3-treated LID mice. The average light density and positive rate of VEGF were the highest in untreated control mice (0.34+/-0.10 and 0.04+/-0.02, P<0.05), and the lowest in Rg3-treated LID mice (0.13+/-0.03 and 0.01+/-0.00, P<0.05). The MVD was 31.9+/-5.3 in untreated control mice, 26.8+/-4.9 in untreated LID mice, 20.1+/-4.9 in Rg3-treated control mice, and 14.4+/-4.9 in Rg3-treated LID mice. Circulating IGF-1 plays a role in the onset and development of breast cancer. Degrading serum IGF-1 level could inhibit angiogenesis and growth of breast cancer. Rg3 could promote this effect.

Circadian rhythms and food anticipatory behavior in Wfs1-deficient mice.

PubMed

Luuk, Hendrik; Fahrenkrug, Jan; Hannibal, Jens

2012-08-10

The dorsomedial hypothalamic nucleus (DMH) has been proposed as a candidate for the neural substrate of a food-entrainable oscillator. The existence of a food-entrainable oscillator in the mammalian nervous system was inferred previously from restricted feeding-induced behavioral rhythmicity in rodents with suprachiasmatic nucleus lesions. In the present study, we have characterized the circadian rhythmicity of behavior in Wfs1-deficient mice during ad libitum and restricted feeding. Based on the expression of Wfs1 protein in the DMH it was hypothesized that Wfs1-deficient mice will display reduced or otherwise altered food anticipatory activity. Wfs1 immunoreactivity in DMH was found almost exclusively in the compact part. Restricted feeding induced c-Fos immunoreactivity primarily in the ventral and lateral aspects of DMH and it was similar in both genotypes. Wfs1-deficiency resulted in significantly lower body weight and reduced wheel-running activity. Circadian rhythmicity of behavior was normal in Wfs1-deficient mice under ad libitum feeding apart from elongated free-running period in constant light. The amount of food anticipatory activity induced by restricted feeding was not significantly different between the genotypes. Present results indicate that the effects of Wfs1-deficiency on behavioral rhythmicity are subtle suggesting that Wfs1 is not a major player in the neural networks responsible for circadian rhythmicity of behavior. Copyright © 2012 Elsevier Inc. All rights reserved.

Slitrk1-deficient mice display elevated anxiety-like behavior and noradrenergic abnormalities.

PubMed

Katayama, K; Yamada, K; Ornthanalai, V G; Inoue, T; Ota, M; Murphy, N P; Aruga, J

2010-02-01

Mutations in SLITRK1 are found in patients with Tourette's syndrome and trichotillomania. SLITRK1 encodes a transmembrane protein containing leucine-rich repeats that is produced predominantly in the nervous system. However, the role of this protein is largely unknown, except that it can modulate neurite outgrowth in vitro. To clarify the role of Slitrk1 in vivo, we developed Slitrk1-knockout mice and analyzed their behavioral and neurochemical phenotypes. Slitrk1-deficient mice exhibited elevated anxiety-like behavior in the elevated plus-maze test as well as increased immobility time in forced swimming and tail suspension tests. Neurochemical analysis revealed that Slitrk1-knockout mice had increased levels of norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylglycol. Administration of clonidine, an alpha2-adrenergic agonist that is frequently used to treat patients with Tourette's syndrome, attenuated the anxiety-like behavior of Slitrk1-deficient mice in the elevated plus-maze test. These results lead us to conclude that noradrenergic mechanisms are involved in the behavioral abnormalities of Slitrk1-deficient mice. Elevated anxiety due to Slitrk1 dysfunction may contribute to the pathogenesis of neuropsychiatric diseases such as Tourette's syndrome and trichotillomania.

Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing

PubMed Central

McNamee, Kay; Przybycien, Paulina M.; Lu, Xin; Williams, Richard O.; Bou-Gharios, George; Saklatvala, Jeremy; Dean, Jonathan L. E.

2013-01-01

There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation. PMID:24143227

Gender-specific effects of endogenous testosterone: female alpha-estrogen receptor-deficient C57Bl/6J mice develop glomerulosclerosis.

PubMed

Elliot, S J; Berho, M; Korach, K; Doublier, S; Lupia, E; Striker, G E; Karl, M

2007-08-01

Young female mice on a C57Bl/6J (B6) background are considered glomerulosclerosis (GS)-resistant but aging B6 mice develop mild GS. Estrogen deficiency accelerates while estrogen replacement retards GS in young sclerosis-prone oligosyndactyly mutant mice on an ROP background. To explore the effects of sex hormones on glomerular structure and function in the context of gender and genetic background, we studied mice in which the estrogen-receptor (ER) genes alpha- or -beta were deleted (alpha- or betaER knockout (KO)) and crossed into the B6 background. We also studied ovariectomized (Ovx) B6 mice given testosterone. Male and female betaERKO and male alphaERKO mice had no glomerular dysfunction at 9 months of age; however, alphaERKO female mice displayed albuminuria and GS. Ovx prevented glomerular dysfunction in alphaERKO female mice by eliminating endogenous testosterone production while exogenous testosterone induced GS in Ovx B6 mice. Androgen receptor (AR) expression and function was found in microdissected glomeruli and cultured mesangial cells. Testosterone compared to placebo increased both AR expression and TGF-beta1 mRNA levels in glomeruli isolated from female B6 mice. Estrogen deficiency had no deleterious effects on the glomeruli in B6 mice. Our study shows that genetic traits strongly influence the GS-promoting effects of estrogen deficiency while testosterone induces GS in a gender-specific manner.

Circadian Behaviour in Neuroglobin Deficient Mice

PubMed Central

Hundahl, Christian A.; Fahrenkrug, Jan; Hay-Schmidt, Anders; Georg, Birgitte; Faltoft, Birgitte; Hannibal, Jens

2012-01-01

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night. PMID:22496809

Circadian behaviour in neuroglobin deficient mice.

PubMed

Hundahl, Christian A; Fahrenkrug, Jan; Hay-Schmidt, Anders; Georg, Birgitte; Faltoft, Birgitte; Hannibal, Jens

2012-01-01

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night.

Deficiency of Gpr1 improves steroid hormone abnormality in hyperandrogenized mice.

PubMed

Yang, Ya-Li; Sun, Li-Feng; Yu, Yan; Xiao, Tian-Xia; Wang, Bao-Bei; Ren, Pei-Gen; Tang, Hui-Ru; Zhang, Jian V

2018-05-24

Polycystic ovary syndrome (PCOS) is a complex genetic disease with multifarious phenotypes. Many researches use dehydroepiandrosterone (DHEA) to induce PCOS in pubertal mouse models. The aim of this study was to investigate the role of GPR1 in dehydroepiandrosterone (DHEA)-induced hyperandrogenized mice. Prepubertal C57BL/6 mice (25 days of age) and Gpr1-deficient mice were each divided into two groups and injected daily with sesame oil with or without DHEA (6 mg/100 g) for 21 consecutive days. Hematoxylin and eosin (H&E) staining was performed to determine the characteristics of the DHEA-treated ovaries. Real-time PCR was used to examine steroid synthesis enzymes gene expression. Granulosa cell was cultured to explore the mechanism of DHEA-induced, GPR1-mediated estradiol secretion. DHEA treatment induced some aspects of PCOS in wild-type mice, such as increased body weight, elevated serum testosterone, increased number of small, cystic, atretic follicles, and absence of corpus luteum in ovaries. However, Gpr1 deficiency significantly attenuated the DHEA-induced weight gain and ovarian phenotype, improving steroidogenesis in ovaries and estradiol synthesis in cultured granulosa cells, partially through mTOR signaling. In conclusion, Gpr1 deficiency leads to the improvement of steroid synthesis in mice hyperandrogenized with DHEA, indicating that GPR1 may be a therapeutic target for DHEA-induced hyperandrogenism.

Deficiency of PTP1B in leptin receptor-expressing neurons leads to decreased body weight and adiposity in mice.

PubMed

Tsou, Ryan C; Zimmer, Derek J; De Jonghe, Bart C; Bence, Kendra K

2012-09-01

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed tyrosine phosphatase implicated in the negative regulation of leptin and insulin receptor signaling. PTP1B(-/-) mice possess a lean metabolic phenotype attributed at least partially to improved hypothalamic leptin sensitivity. Interestingly, mice lacking both leptin and PTP1B (ob/ob:PTP1B(-/-)) have reduced body weight compared with mice lacking leptin only, suggesting that PTP1B may have important leptin-independent metabolic effects. We generated mice with PTP1B deficiency specifically in leptin receptor (LepRb)-expressing neurons (LepRb-PTP1B(-/-)) and compared them with LepRb-Cre-only wild-type (WT) controls and global PTP1B(-/-) mice. Consistent with PTP1B's role as a negative regulator of leptin signaling, our results show that LepRb-PTP1B(-/-) mice are leptin hypersensitive and have significantly reduced body weight when maintained on chow or high-fat diet (HFD) compared with WT controls. LepRb-PTP1B(-/-) mice have a significant decrease in adiposity on HFD compared with controls. Notably, the extent of attenuated body weight gain on HFD, as well as the extent of leptin hypersensitivity, is similar between LepRb-PTP1B(-/-) mice and global PTP1B(-/-) mice. Overall, these results demonstrate that PTP1B deficiency in LepRb-expressing neurons results in reduced body weight and adiposity compared with WT controls and likely underlies the improved metabolic phenotype of global and brain-specific PTP1B-deficient models. Subtle phenotypic differences between LepRb-PTP1B(-/-) and global PTP1B(-/-) mice, however, suggest that PTP1B independent of leptin signaling may also contribute to energy balance in mice.

Susceptible cytotoxicity to ultraviolet B light in fibroblasts and keratinocytes cultured from autoimmune-prone MRL/Mp-lpr/lpr mice

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

Furukawa, F.; Lyon, M.B.; Norris, D.A.

1989-09-01

The MRL/Mp-lpr/lpr (MRL/l) mouse is an autoimmune model of spontaneous lupus erythematosus (LE), in addition to lupus nephritis. In order to better understand the mechanisms of photosensitivity in LE, in vitro photocytotoxicity was examined by using fibroblasts and keratinocytes cultured from MRL/l mice, control MRL/Mp- +/+ (MRL/n) mice, and normal BALB/c mice. A colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and the acridine orange/ethidium bromide assay were used for determination of cytotoxicity. Fibroblasts cultured from newborn MRL/l mice showed higher susceptibility to single ultraviolet light B (UVB) light irradiation at a dose of 100-500 mJ than those from MRL/n, F1 hybrid ofmore » (MRL/l x MRL/n mice), and BALB/c mice. However, the susceptibility to UVB was not observed in young (1-month-old) and adult (4-month-old) MRL/l mice. UVA light irradiation was not cytotoxic. Keratinocytes cultured from MRL mice showed lower cytotoxicity to UVB irradiation than fibroblasts cultured. However, keratinocytes from newborn MRL/l mice showed higher cytotoxicity to 50 mJ UVB irradiation than cells from MRL/n mice. Syngeneic or allogeneic sera augmented UVB-induced cytotoxicity of fibroblasts cultured. UVB irradiation of spleen cells induced no significant difference of cytotoxicity between MRL/l and MRL/n mice. Based on the results of F1 hybrid of (MRL/l x MRL/n) mice, the susceptibility seemed to be associated with autoimmune traits and to be regulated by genetical background.« less

The mechanical phenotype of biglycan-deficient mice is bone- and gender-specific.

PubMed

Wallace, Joseph M; Rajachar, Rupak M; Chen, Xiao-Dong; Shi, Songtao; Allen, Matthew R; Bloomfield, Susan A; Les, Clifford M; Robey, Pamela G; Young, Marian F; Kohn, David H

2006-07-01

Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix of skeletal tissues. While bgn is known to be involved in the growth and differentiation of osteoblast precursor cells and regulation of collagen fibril formation, it is unclear how these functions impact bone's geometric and mechanical properties, properties which are integral to the structural function of bone. Because the genetic control of bone structure and function is both local- and gender-specific and because there is evidence of gender-specific effects associated with genetic deficiencies, it was hypothesized that the engineered deletion of the gene encoding bgn would result in a cortical bone mechanical phenotype that was bone- and gender-specific. In 11-week-old C57BL6/129 mice, the cortical bone in the mid-diaphyses of the femora and tibiae of both genders was examined. Phenotypic changes in bgn-deficient mice relative to wild type controls were assayed by four-point bending tests to determine mechanical properties at the whole bone (structural) and tissue levels, as well as analyses of bone geometry and bone formation using histomorphometry. Of the bones examined, bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure and suggests a new understanding of the functional role of bgn in regulating bone mineralization in vivo.

Myelin/oligodendrocyte glycoprotein–deficient (MOG-deficient) mice reveal lack of immune tolerance to MOG in wild-type mice

PubMed Central

Delarasse, Cécile; Daubas, Philippe; Mars, Lennart T.; Vizler, Csaba; Litzenburger, Tobias; Iglesias, Antonio; Bauer, Jan; Della Gaspera, Bruno; Schubart, Anna; Decker, Laurence; Dimitri, Dalia; Roussel, Guy; Dierich, Andrée; Amor, Sandra; Dautigny, André; Liblau, Roland; Pham-Dinh, Danielle

2003-01-01

We studied the immunological basis for the very potent encephalitogenicity of myelin/oligodendrocyte glycoprotein (MOG), a minor component of myelin in the CNS that is widely used to induce experimental autoimmune encephalomyelitis (EAE). For this purpose, we generated a mutant mouse lacking a functional mog gene. This MOG-deficient mouse presents no clinical or histological abnormalities, permitting us to directly assess the role of MOG as a target autoantigen in EAE. In contrast to WT mice, which developed severe EAE following immunization with whole myelin, MOG-deficient mice had a mild phenotype, demonstrating that the anti-MOG response is a major pathogenic component of the autoimmune response directed against myelin. Moreover, while MOG transcripts are expressed in lymphoid organs in minute amounts, both MOG-deficient and WT mice show similar T and B cell responses against the extracellular domain of MOG, including the immunodominant MOG 35–55 T cell epitope. Furthermore, no differences in the fine specificity of the T cell responses to overlapping peptides covering the complete mouse MOG sequence were observed between MOG+/+ and MOG–/– mice. In addition, upon adoptive transfer, MOG-specific T cells from WT mice and those from MOG-deficient mice are equally pathogenic. This total lack of immune tolerance to MOG in WT C57BL/6 mice may be responsible for the high pathogenicity of the anti-MOG immune response as well as the high susceptibility of most animal strains to MOG-induced EAE. PMID:12925695

Lipid-lowering effects of TAK-475, a squalene synthase inhibitor, in animal models of familial hypercholesterolemia.

PubMed

Amano, Yuichiro; Nishimoto, Tomoyuki; Tozawa, Ryu ichi; Ishikawa, Eiichiro; Imura, Yoshimi; Sugiyama, Yasuo

2003-04-11

The lipid-lowering effects of 1-[2-[(3R,5S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-1,2,3,5-tetrahydro-2-oxo-5-(2,3-dimethoxyphenyl)-4,1-benzoxazepine-3-yl] acetyl] piperidin-4-acetic acid (TAK-475), a novel squalene synthase inhibitor, were examined in two models of familial hypercholesterolemia, low-density lipoprotein (LDL) receptor knockout mice and Watanabe heritable hyperlipidemic (WHHL) rabbits. Two weeks of treatment with TAK-475 in a diet admixture (0.02% and 0.07%; approximately 30 and 110 mg/kg/day, respectively) significantly lowered plasma non-high-density lipoprotein (HDL) cholesterol levels by 19% and 41%, respectively, in homozygous LDL receptor knockout mice. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, simvastatin and atorvastatin (in 0.02% and 0.07% admixtures), also reduced plasma levels of non-HDL cholesterol. In homozygous WHHL rabbits, 4 weeks of treatment with TAK-475 (0.27%; approximately 100 mg/kg/day) lowered plasma total cholesterol, triglyceride and phospholipid levels by 17%, 52% and 26%, respectively. In Triton WR-1339-treated rabbits, TAK-475 inhibited to the same extent the rate of secretion from the liver of the cholesterol, triglyceride and phospholipid components of very-low-density lipoprotein (VLDL). These results suggest that the lipid-lowering effects of TAK-475 in WHHL rabbits are based partially on the inhibition of secretion of VLDL from the liver. TAK-475 had no effect on plasma aspartate aminotransferase and alanine aminotransferase activities. Thus, the squalene synthase inhibitor TAK-475 revealed lipid-lowering effects in both LDL receptor knockout mice and WHHL rabbits.

Behavioral Characteristics of Ubiquitin-Specific Peptidase 46-Deficient Mice

PubMed Central

Imai, Saki; Kano, Makoto; Nonoyama, Keiko; Ebihara, Shizufumi

2013-01-01

We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST) and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92), and mice with this mutation (MT mice), as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABAA) receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT) or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system. PMID:23472206

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling in Drosophila

PubMed Central

Stronach, Beth; Lennox, Ashley L.; Garlena, Rebecca A.

2014-01-01

A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability. PMID:24429281

Plasmalogen modulation attenuates atherosclerosis in ApoE- and ApoE/GPx1-deficient mice.

PubMed

Rasmiena, Aliki A; Barlow, Christopher K; Stefanovic, Nada; Huynh, Kevin; Tan, Ricardo; Sharma, Arpeeta; Tull, Dedreia; de Haan, Judy B; Meikle, Peter J

2015-12-01

We previously reported a negative association of circulating plasmalogens (phospholipids with proposed atheroprotective properties) with coronary artery disease. Plasmalogen modulation was previously demonstrated in animals but its effect on atherosclerosis was unknown. We assessed the effect of plasmalogen enrichment on atherosclerosis of murine models with differing levels of oxidative stress. Six-week old ApoE- and ApoE/glutathione peroxidase-1 (GPx1)-deficient mice were fed a high-fat diet with/without 2% batyl alcohol (precursor to plasmalogen synthesis) for 12 weeks. Mass spectrometry analysis of lipids showed that batyl alcohol supplementation to ApoE- and ApoE/GPx1-deficient mice increased the total plasmalogen levels in both plasma and heart. Oxidation of plasmalogen in the treated mice was evident from increased level of plasmalogen oxidative by-product, sn-2 lysophospholipids. Atherosclerotic plaque in the aorta was reduced by 70% (P = 5.69E-07) and 69% (P = 2.00E-04) in treated ApoE- and ApoE/GPx1-deficient mice, respectively. A 40% reduction in plaque (P = 7.74E-03) was also seen in the aortic sinus of only the treated ApoE/GPx1-deficient mice. Only the treated ApoE/GPx1-deficient mice showed a decrease in VCAM-1 staining (-28%, P = 2.43E-02) in the aortic sinus and nitrotyrosine staining (-78%, P = 5.11E-06) in the aorta. Plasmalogen enrichment via batyl alcohol supplementation attenuated atherosclerosis in ApoE- and ApoE/GPx1-deficient mice, with a greater effect in the latter group. Plasmalogen enrichment may represent a viable therapeutic strategy to prevent atherosclerosis and reduce cardiovascular disease risk, particularly under conditions of elevated oxidative stress and inflammation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation

PubMed Central

Cotter, David G.; Schugar, Rebecca C.; Wentz, Anna E.; André d'Avignon, D.

2013-01-01

During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1+/− mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states. PMID:23233542

Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation.

PubMed

Cotter, David G; Schugar, Rebecca C; Wentz, Anna E; d'Avignon, D André; Crawford, Peter A

2013-02-15

During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1(+/-) mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states.

Cell type-specific deficiency of c-kit gene expression in mutant mice of mi/mi genotype.

PubMed Central

Isozaki, K.; Tsujimura, T.; Nomura, S.; Morii, E.; Koshimizu, U.; Nishimune, Y.; Kitamura, Y.

1994-01-01

The mi locus of mice encodes a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called mi factor). In addition to microphthalmus, osteopetrosis, and lack of melanocytes, mice of mi/mi genotype are deficient in mast cells. Since the c-kit receptor tyrosine kinase plays an important role in the development of mast cells, and since the c-kit expression by cultured mast cells from mi/mi mice is deficient in both mRNA and protein levels, the mast cell deficiency of mi/mi mice has been attributed at least in part to the deficient expression of c-kit. However, it remained to be examined whether the c-kit expression was also deficient in tissues of mi/mi mice. In the present study, we examined the c-kit expression by mi/mi skin mast cells using in situ hybridization and immunohistochemistry. Moreover, we examined the c-kit expression by various cells other than mast cells in tissues of mi/mi mice. We found that the c-kit expression was deficient in mast cells but not in erythroid precursors, testicular germ cells, and neurons of mi/mi mice. This suggested that the regulation of the c-kit transcription by the mi factor was dependent on cell types. Mice of mi/mi genotype appeared to be a useful model to analyze the function of transcription factors in the whole-animal level. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7524330

BTB and CNC homolog 1 (Bach1) deficiency ameliorates TNBS colitis in mice: role of M2 macrophages and heme oxygenase-1.

PubMed

Harusato, Akihito; Naito, Yuji; Takagi, Tomohisa; Uchiyama, Kazuhiko; Mizushima, Katsura; Hirai, Yasuko; Higashimura, Yasuki; Katada, Kazuhiro; Handa, Osamu; Ishikawa, Takeshi; Yagi, Nobuaki; Kokura, Satoshi; Ichikawa, Hiroshi; Muto, Akihiko; Igarashi, Kazuhiko; Yoshikawa, Toshikazu

2013-01-01

BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1), which plays an important role in the protection of cells and tissues against acute and chronic inflammation. However, the role of Bach1 in the gastrointestinal mucosal defense system remains little understood. HO-1 supports the suppression of experimental colitis and localizes mainly in macrophages in colonic mucosa. This study was undertaken to elucidate the Bach1/HO-1 system's effects on the pathogenesis of experimental colitis. This study used C57BL/6 (wild-type) and homozygous Bach1-deficient C57BL/6 mice in which colonic damage was induced by the administration of an enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Subsequently, they were evaluated macroscopically, histologically, and biochemically. Peritoneal macrophages from the respective mice were isolated and analyzed. Then, wild-type mice were injected with peritoneal macrophages from the respective mice. Acute colitis was induced similarly. TNBS-induced colitis was inhibited in Bach1-deficient mice. TNBS administration increased the expression of HO-1 messenger RNA and protein in colonic mucosa in Bach1-deficient mice. The expression of HO-1 mainly localized in F4/80-immunopositive and CD11b-immunopositive macrophages. Isolated peritoneal macrophages from Bach1-deficient mice highly expressed HO-1 and also manifested M2 macrophage markers, such as Arginase-1, Fizz-1, Ym1, and MRC1. Furthermore, TNBS-induced colitis was inhibited by the transfer of Bach1-deficient macrophages into wild-type mice. Deficiency of Bach1 ameliorated TNBS-induced colitis. Bach1-deficient macrophages played a key role in protection against colitis. Targeting of this mechanism is applicable to cell therapy for human inflammatory bowel disease.

TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation.

PubMed

Morioka, Sho; Broglie, Peter; Omori, Emily; Ikeda, Yuka; Takaesu, Giichi; Matsumoto, Kunihiro; Ninomiya-Tsuji, Jun

2014-02-17

TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8-mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)-dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.

Angiopoietin-1 deficiency increases tumor metastasis in mice.

PubMed

Michael, Iacovos P; Orebrand, Martina; Lima, Marta; Pereira, Beatriz; Volpert, Olga; Quaggin, Susan E; Jeansson, Marie

2017-08-11

Angipoietin-1 activation of the tyrosine kinase receptor Tek expressed mainly on endothelial cells leads to survival and stabilization of endothelial cells. Studies have shown that Angiopoietin-1 counteracts permeability induced by a number of stimuli. Here, we test the hypothesis that loss of Angiopoietin-1/Tek signaling in the vasculature would increase metastasis. Angiopoietin-1 was deleted in mice just before birth using floxed Angiopoietin-1 and Tek mice crossed to doxycycline-inducible bitransgenic ROSA-rtTA/tetO-Cre mice. By crossing Angiopoietin-1 knockout mice to the MMTV-PyMT autochthonous mouse breast cancer model, we investigated primary tumor growth and metastasis to the lung. Furthermore, we utilized B16F10 melanoma cells subcutaneous and experimental lung metastasis models in Angiopoietin-1 and Tek knockout mice. We found that primary tumor growth in MMTV-PyMT mice was unaffected, while metastasis to the lung was significantly increased in Angiopoietin-1 knockout MMTV-PyMT mice. In addition, angiopoietin-1 deficient mice exhibited a significant increase in lung metastasis of B16F10 melanoma cells, compared to wild type mice 3 weeks after injection. Additional experiments showed that this was likely an early event due to increased attachment or extravasation of tumor cells, since seeding of tumor cells was significantly increased 4 and 24 h post tail vein injection. Finally, using inducible Tek knockout mice, we showed a significant increase in tumor cell seeding to the lung, suggesting that Angiopoietin-1/Tek signaling is important for vascular integrity to limit metastasis. This study show that loss of the Angiopoietin-1/Tek vascular growth factor system leads to increased metastasis without affecting primary tumor growth.

Aberrant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis.

PubMed Central

Nickoloff, B. J.; Mitra, R. S.; Varani, J.; Dixit, V. M.; Polverini, P. J.

1994-01-01

Psoriasis is a common inherited skin disease that is characterized by hyperproliferation of epidermal keratinocytes and excessive dermal angiogenesis. A growing body of evidence supports a key pathogenetic role for activated keratinocytes in the angiogenic response that accompanies psoriasis. We investigated the role of psoriatic epidermis in the aberrant expression of angiogenesis by examining the ability of pure populations of multipassaged keratinocytes obtained from the skin of normal individuals and psoriatic patients to induce angiogenesis in vivo in the rat corneal bioassay and endothelial cell chemotaxis in vitro. Media conditioned by keratinocytes from psoriatic patients, including both symptomless skin and psoriatic plaques, induced vigorous angiogenic responses in over 90% of corneas tested and potently stimulated directional migration of capillary endothelial cells in vitro. In contrast, conditioned medium from normal keratinocyte cultures was weakly positive in less than 10% of corneas assayed and failed to stimulate endothelial cell chemotaxis. Furthermore, keratinocytes from psoriatic skin exhibited a 10- to 20-fold increase in interleukin-8 production and a seven-fold reduction in thrombospondin-1 production. The angiogenic activity present in keratinocyte-conditioned media from psoriatic patients was suppressed by adding either highly purified thrombospondin-1 (125 ng) or following the addition of either normal keratinocyte-conditioned media or neutralizing interleukin-8 antibody. We conclude that psoriatic keratinocytes are phenotypically different from normal keratinocytes with respect to their angiogenic capacity and that this aberrant phenotype is attributable to a defect in the overproduction of interleukin-8 and a deficiency in the production of the angiogenesis inhibitor thrombospondin-1. Images Figure 1 PMID:7512793

Aged PROP1 Deficient Dwarf Mice Maintain ACTH Production

PubMed Central

Bavers, David L.; Beuschlein, Felix; Mortensen, Amanda H.; Keegan, Catherine E.; Hammer, Gary D.; Camper, Sally A.

2011-01-01

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1null (Prop1-/-) and the Ames dwarf (Prop1df/df) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism. PMID:22145038

Skin Barrier Development Depends on CGI-58 Protein Expression during Late-Stage Keratinocyte Differentiation

PubMed Central

Grond, Susanne; Radner, Franz P.W.; Eichmann, Thomas O.; Kolb, Dagmar; Grabner, Gernot F.; Wolinski, Heimo; Gruber, Robert; Hofer, Peter; Heier, Christoph; Schauer, Silvia; Rülicke, Thomas; Hoefler, Gerald; Schmuth, Matthias; Elias, Peter M.; Lass, Achim; Zechner, Rudolf; Haemmerle, Guenter

2017-01-01

Adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) are limiting in cellular triglyceride catabolism. Although ATGL deficiency is compatible with normal skin development, mice globally lacking CGI-58 die postnatally and exhibit a severe epidermal permeability barrier defect, which may originate from epidermal and/or peripheral changes in lipid and energy metabolism. Here, we show that epidermis-specific disruption of CGI-58 is sufficient to provoke a defect in the formation of a functional corneocyte lipid envelope linked to impaired ω-O-acylceramide synthesis. As a result, epidermis-specific CGI-58-deficient mice show severe skin dysfunction, arguing for a tissue autonomous cause of disease development. Defective skin permeability barrier formation in global CGI-58-deficient mice could be reversed via transgenic restoration of CGI-58 expression in differentiated but not basal keratinocytes suggesting that CGI-58 is essential for lipid metabolism in suprabasal epidermal layers. The compatibility of ATGL deficiency with normal epidermal function indicated that CGI-58 may stimulate an epidermal triglyceride lipase beyond ATGL required for the adequate provision of fatty acids as a substrate for ω-O-acylceramide synthesis. Pharmacological inhibition of ATGL enzyme activity similarly reduced triglyceride-hydrolytic activities in wild-type and CGI-58 overexpressing epidermis implicating that CGI-58 participates in ω-O-acylceramide biogenesis independent of its role as a coactivator of epidermal triglyceride catabolism. PMID:27725204

Osteoblast Specific Overexpression of Human Interleukin-7 Rescues the Bone Mass Phenotype of Interleukin-7 Deficient Female Mice

PubMed Central

Aguila, Hector L.; Mun, Se Hwan; Kalinowski, Judith; Adams, Douglas J.; Lorenzo, Joseph A.; Lee, Sun-Kyeong

2012-01-01

Interleukin-7 is a critical cytokine for lymphoid development and a direct inhibitor of in vitro osteoclastogenesis in murine bone marrow cultures. To explore the role of IL-7 in bone, we generated transgenic mouse lines bearing the 2.3 Kb rat collagen 1A1 promoter driving the expression of human IL-7 specifically in osteoblasts. In addition we crossed these mice with IL-7 deficient mice to determine if the alterations in lymphopoiesis, bone mass and osteoclast formation observed in the IL-7 KO mice could be rescued by osteoblast-specific overexpression of IL-7. Here we show that mice overexpressing human IL-7 in the osteoblast lineage demonstrated increased trabecular bone volume in vivo by µCT and decreased osteoclast formation in vitro. Furthermore, targeted overexpression of IL-7 in osteoblasts rescued the osteopenic bone phenotype and B cell development of IL-7 KO mice but did not have an effect on T lymphopoiesis, which occurs in the periphery. The bone phenotypes in IL-7 KO mice and targeted IL-7 overexpressing mouse models were observed only in females. These results likely reflect both a direct inhibitory effects of IL-7 on osteoclastogenesis in vivo and gender specific differences in responses to IL-7. PMID:22258693

Adipose tissue deficiency of hormone-sensitive lipase causes fatty liver in mice

PubMed Central

Yang, Hao; Wang, Shu Pei; Mitchell, Grant A.

2017-01-01

Fatty liver is a major health problem worldwide. People with hereditary deficiency of hormone-sensitive lipase (HSL) are reported to develop fatty liver. In this study, systemic and tissue-specific HSL-deficient mice were used as models to explore the underlying mechanism of this association. We found that systemic HSL deficient mice developed fatty liver in an age-dependent fashion between 3 and 8 months of age. To further explore the mechanism of fatty liver in HSL deficiency, liver-specific HSL knockout mice were created. Surprisingly, liver HSL deficiency did not influence liver fat content, suggesting that fatty liver in HSL deficiency is not liver autonomous. Given the importance of adipose tissue in systemic triglyceride metabolism, we created adipose-specific HSL knockout mice and found that adipose HSL deficiency, to a similar extent as systemic HSL deficiency, causes age-dependent fatty liver in mice. Mechanistic study revealed that deficiency of HSL in adipose tissue caused inflammatory macrophage infiltrates, progressive lipodystrophy, abnormal adipokine secretion and systemic insulin resistance. These changes in adipose tissue were associated with a constellation of changes in liver: low levels of fatty acid oxidation, of very low density lipoprotein secretion and of triglyceride hydrolase activity, each favoring the development of hepatic steatosis. In conclusion, HSL-deficient mice revealed a complex interorgan interaction between adipose tissue and liver: the role of HSL in the liver is minimal but adipose tissue deficiency of HSL can cause age-dependent hepatic steatosis. Adipose tissue is a potential target for treating the hepatic steatosis of HSL deficiency. PMID:29232702

Adipose tissue deficiency of hormone-sensitive lipase causes fatty liver in mice.

PubMed

Xia, Bo; Cai, Guo He; Yang, Hao; Wang, Shu Pei; Mitchell, Grant A; Wu, Jiang Wei

2017-12-01

Fatty liver is a major health problem worldwide. People with hereditary deficiency of hormone-sensitive lipase (HSL) are reported to develop fatty liver. In this study, systemic and tissue-specific HSL-deficient mice were used as models to explore the underlying mechanism of this association. We found that systemic HSL deficient mice developed fatty liver in an age-dependent fashion between 3 and 8 months of age. To further explore the mechanism of fatty liver in HSL deficiency, liver-specific HSL knockout mice were created. Surprisingly, liver HSL deficiency did not influence liver fat content, suggesting that fatty liver in HSL deficiency is not liver autonomous. Given the importance of adipose tissue in systemic triglyceride metabolism, we created adipose-specific HSL knockout mice and found that adipose HSL deficiency, to a similar extent as systemic HSL deficiency, causes age-dependent fatty liver in mice. Mechanistic study revealed that deficiency of HSL in adipose tissue caused inflammatory macrophage infiltrates, progressive lipodystrophy, abnormal adipokine secretion and systemic insulin resistance. These changes in adipose tissue were associated with a constellation of changes in liver: low levels of fatty acid oxidation, of very low density lipoprotein secretion and of triglyceride hydrolase activity, each favoring the development of hepatic steatosis. In conclusion, HSL-deficient mice revealed a complex interorgan interaction between adipose tissue and liver: the role of HSL in the liver is minimal but adipose tissue deficiency of HSL can cause age-dependent hepatic steatosis. Adipose tissue is a potential target for treating the hepatic steatosis of HSL deficiency.

Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1−/− mice

PubMed Central

Prasher, Joanna M; Lalai, Astrid S; Heijmans-Antonissen, Claudia; Ploemacher, Robert E; Hoeijmakers, Jan H J; Touw, Ivo P; Niedernhofer, Laura J

2005-01-01

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1−/− mice was compared to that in young and old wild-type mice. Ercc1−/− mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1−/− mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa−/− mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging. PMID:15692571

Type 1 diabetes in NOD mice unaffected by mast cell deficiency.

PubMed

Gutierrez, Dario A; Fu, Wenxian; Schonefeldt, Susann; Feyerabend, Thorsten B; Ortiz-Lopez, Adriana; Lampi, Yulia; Liston, Adrian; Mathis, Diane; Rodewald, Hans-Reimer

2014-11-01

Mast cells have been invoked as important players in immune responses associated with autoimmune diseases. Based on in vitro studies, or in vivo through the use of Kit mutant mice, mast cells have been suggested to play immunological roles in direct antigen presentation to both CD4(+) and CD8(+) T cells, in the regulation of T-cell and dendritic cell migration to lymph nodes, and in Th1 versus Th2 polarization, all of which could significantly impact the immune response against self-antigens in autoimmune disease, including type 1 diabetes (T1D). Until now, the role of mast cells in the onset and incidence of T1D has only been indirectly tested through the use of low-specificity mast cell inhibitors and activators, and published studies reported contrasting results. Our three laboratories have generated independently two strains of mast cell-deficient nonobese diabetic (NOD) mice, NOD.Cpa3(Cre/+) (Heidelberg) and NOD.Kit(W-sh/W-sh) (Leuven and Boston), to address the effects of mast cell deficiency on the development of T1D in the NOD strain. Our collective data demonstrate that both incidence and progression of T1D in NOD mice are independent of mast cells. Moreover, analysis of pancreatic lymph node cells indicated that lack of mast cells has no discernible effect on the autoimmune response, which involves both innate and adaptive immune components. Our results demonstrate that mast cells are not involved in T1D in the NOD strain, making their role in this process nonessential and excluding them as potential therapeutic targets. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Candidate EDA targets revealed by expression profiling of primary keratinocytes from Tabby mutant mice

PubMed Central

Esibizione, Diana; Cui, Chang-Yi; Schlessinger, David

2009-01-01

EDA, the gene mutated in anhidrotic ectodermal dysplasia, encodes ectodysplasin, a TNF superfamily member that activates NF-kB mediated transcription. To identify EDA target genes, we have earlier used expression profiling to infer genes differentially expressed at various developmental time points in Tabby (Eda-deficient) compared to wild-type mouse skin. To increase the resolution to find genes whose expression may be restricted to epidermal cells, we have now extended studies to primary keratinocyte cultures established from E19 wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 preliminary candidate genes whose expression was significantly affected by Eda loss. By comparing expression profiles to those from Eda-A1 transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. We confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in keratinocytes and in whole skin, by Q-PCR and Western blotting analyses. Thus, by the analysis of keratinocytes, novel candidate pathways downstream of EDA were detected. PMID:18848976

Loss of stearoyl-CoA desaturase 1 rescues cardiac function in obese leptin-deficient mice.

PubMed

Dobrzyn, Pawel; Dobrzyn, Agnieszka; Miyazaki, Makoto; Ntambi, James M

2010-08-01

The heart of leptin-deficient ob/ob mice is characterized by pathologic left ventricular hypertrophy along with elevated triglyceride (TG) content, increased stearoyl-CoA desaturase (SCD) activity, and increased myocyte apoptosis. In the present study, using an ob/ob;SCD1(-/-) mouse model, we tested the hypothesis that lack of SCD1 could improve steatosis and left ventricle (LV) function in leptin deficiency. We show that disruption of the SCD1 gene improves cardiac function in ob/ob mice by correcting systolic and diastolic dysfunction without affecting levels of plasma TG and FFA. The improvement is associated with reduced expression of genes involved in FA transport and lipid synthesis in the heart, as well as reduction in cardiac FFA, diacylglycerol, TG, and ceramide levels. The rate of FA beta-oxidation is also significantly lower in the heart of ob/ob;SCD1(-/-) mice compared with ob/ob controls. Moreover, SCD1 deficiency reduces cardiac apoptosis in ob/ob mice due to increased expression of antiapoptotic factor Bcl-2 and inhibition of inducible nitric oxide synthase and caspase-3 activities. Reduction in myocardial lipid accumulation and inhibition of apoptosis appear to be one of the main mechanisms responsible for improved LV function in ob/ob mice caused by SCD1 deficiency.

Plasminogen activator inhibitor-1 deficiency ameliorates insulin resistance and hyperlipidemia but not bone loss in obese female mice.

PubMed

Tamura, Yukinori; Kawao, Naoyuki; Yano, Masato; Okada, Kiyotaka; Matsuo, Osamu; Kaji, Hiroshi

2014-05-01

We previously demonstrated that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is involved in type 1 diabetic bone loss in female mice. PAI-1 is well known as an adipogenic factor induced by obesity. We therefore examined the effects of PAI-1 deficiency on bone and glucose and lipid metabolism in high-fat and high-sucrose diet (HF/HSD)-induced obese female mice. Female wild-type (WT) and PAI-1-deficient mice were fed with HF/HSD or normal diet for 20 weeks from 10 weeks of age. HF/HSD increased the levels of plasma PAI-1 in WT mice. PAI-1 deficiency suppressed the levels of blood glucose, plasma insulin, and total cholesterol elevated by obesity. Moreover, PAI-1 deficiency improved glucose intolerance and insulin resistance induced by obesity. Bone mineral density (BMD) at trabecular bone as well as the levels of osterix, alkaline phosphatase, and receptor activator of nuclear factor κB ligand mRNA in tibia were decreased by HF/HSD in WT mice, and those changes by HF/HSD were not affected by PAI-1 deficiency. HF/HSD increased the levels of plasma TNF-α in both WT and PAI-1-deficient mice, and the levels of plasma TNF-α were negatively correlated with trabecular BMD in tibia of female mice. In conclusion, we revealed that PAI-1 deficiency does not affect the trabecular bone loss induced by obesity despite the amelioration of insulin resistance and hyperlipidemia in female mice. Our data suggest that the changes of BMD and bone metabolism by obesity might be independent of PAI-1 as well as glucose and lipid metabolism.

Protective Role of Mitochondrial Peroxiredoxin III against UVB-Induced Apoptosis of Epidermal Keratinocytes.

PubMed

Baek, Jin Young; Park, Sujin; Park, Jiyoung; Jang, Ji Yong; Wang, Su Bin; Kim, Sin Ri; Woo, Hyun Ae; Lim, Kyung Min; Chang, Tong-Shin

2017-06-01

UVB light induces generation of reactive oxygen species, ultimately leading to skin cell damage. Mitochondria are a major source of reactive oxygen species in UVB-irradiated skin cells, with increased levels of mitochondrial reactive oxygen species having been implicated in keratinocyte apoptosis. Peroxiredoxin III (PrxIII) is the most abundant and potent H 2 O 2 -removing enzyme in the mitochondria of most cell types. Here, the protective role of PrxIII against UVB-induced apoptosis of epidermal keratinocytes was investigated. Mitochondrial H 2 O 2 levels were differentiated from other types of ROS using mitochondria-specific fluorescent H 2 O 2 indicators. Upon UVB irradiation, PrxIII-knockdown HaCaT human keratinocytes and PrxIII-deficient (PrxIII -/- ) mouse primary keratinocytes exhibited enhanced accumulation of mitochondrial H 2 O 2 compared with PrxIII-expressing controls. Keratinocytes lacking PrxIII were subsequently sensitized to apoptosis through mitochondrial membrane potential loss, cardiolipin oxidation, cytochrome c release, and caspase activation. Increased UVB-induced epidermal tissue damage in PrxIII -/- mice was attributable to increased caspase-dependent keratinocyte apoptosis. Our findings show that mitochondrial H 2 O 2 is a key mediator in UVB-induced apoptosis of keratinocytes and that PrxIII plays a critical role in protecting epidermal keratinocytes against UVB-induced apoptosis through eliminating mitochondrial H 2 O 2 . These findings support the concept that reinforcing mitochondrial PrxIII defenses may help prevent UVB-induced skin damage such as inflammation, sunburn, and photoaging. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Relative adrenal insufficiency in mice deficient in 5α-reductase 1

PubMed Central

Livingstone, Dawn E W; Di Rollo, Emma M; Yang, Chenjing; Codrington, Lucy E; Mathews, John A; Kara, Madina; Hughes, Katherine A; Kenyon, Christopher J; Walker, Brian R; Andrew, Ruth

2014-01-01

Patients with critical illness or hepatic failure exhibit impaired cortisol responses to ACTH, a phenomenon known as ‘relative adrenal insufficiency’. A putative mechanism is that elevated bile acids inhibit inactivation of cortisol in liver by 5α-reductases type 1 and type 2 and 5β-reductase, resulting in compensatory downregulation of the hypothalamic–pituitary–adrenal axis and adrenocortical atrophy. To test the hypothesis that impaired glucocorticoid clearance can cause relative adrenal insufficiency, we investigated the consequences of 5α-reductase type 1 deficiency in mice. In adrenalectomised male mice with targeted disruption of 5α-reductase type 1, clearance of corticosterone was lower after acute or chronic (eightfold, P<0.05) administration, compared with WT control mice. In intact 5α-reductase-deficient male mice, although resting plasma corticosterone levels were maintained, corticosterone responses were impaired after ACTH administration (26% lower, P<0.05), handling stress (2.5-fold lower, P<0.05) and restraint stress (43% lower, P<0.05) compared with WT mice. mRNA levels of Nr3c1 (glucocorticoid receptor), Crh and Avp in pituitary or hypothalamus were altered, consistent with enhanced negative feedback. These findings confirm that impaired peripheral clearance of glucocorticoids can cause ‘relative adrenal insufficiency’ in mice, an observation with important implications for patients with critical illness or hepatic failure, and for patients receiving 5α-reductase inhibitors for prostatic disease. PMID:24872577

Isoflurane anesthesia exacerbates learning and memory impairment in zinc-deficient APP/PS1 transgenic mice.

PubMed

Feng, Chunsheng; Liu, Ya; Yuan, Ye; Cui, Weiwei; Zheng, Feng; Ma, Yuan; Piao, Meihua

2016-12-01

Zinc (Zn) is known to play crucial roles in numerous brain functions including learning and memory. Zn deficiency is believed to be widespread throughout the world, particularly in patients with Alzheimer's disease (AD). A number of studies have shown that volatile anesthetics, such as isoflurane, might be potential risk factors for the development of AD. However, whether isoflurane exposure accelerates the process of AD and cognitive impairment in AD patients with Zn deficiency is yet to be documented. The aim of the present study was to explore the effects of 1.4% isoflurane exposure for 2 h on learning and memory function, and neuropathogenesis in 10-month-old Zn-adequate, Zn-deficient, and Zn-treated APP/PS1 mice with the following parameters: behavioral tests, neuronal apoptosis, Aβ, and tau pathology. The results demonstrated that isoflurane exposure showed no impact on learning and memory function, but induced transient elevation of neuroapoptosis in Zn-adequate APP/PS1 mice. Exposure of isoflurane exhibited significant neuroapoptosis, Aβ generation, tau phosphorylation, and learning and memory impairment in APP/PS1 mice in the presence of Zn deficiency. Appropriate Zn treatment improved learning and memory function, and prevented isoflurane-induced neuroapoptosis in APP/PS1 mice. Isoflurane exposure may cause potential neurotoxicity, which is tolerated to some extent in Zn-adequate APP/PS1 mice. When this tolerance is limited, like in AD with Zn deficiency, isoflurane exposure markedly exacerbated learning and memory impairment, and neuropathology, indicating that AD patients with certain conditions such as Zn deficiency may be vulnerable to volatile anesthetic isoflurane. Copyright © 2016 Elsevier Ltd. All rights reserved.

GFPT1 deficiency in muscle leads to myasthenia and myopathy in mice.

PubMed

Issop, Yasmin; Hathazi, Denisa; Khan, Muzamil Majid; Rudolf, Rüdiger; Weis, Joachim; Spendiff, Sally; Slater, Clarke R; Roos, Andreas; Lochmüller, Hanns

2018-06-14

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway which yields precursors required for protein and lipid glycosylation. Mutations in GFPT1 and other genes downstream of this pathway cause congenital myasthenic syndrome (CMS) characterised by fatigable muscle weakness due to impaired neurotransmission. The precise pathomechanisms at the neuromuscular junction (NMJ) due to a deficiency in GFPT1 is yet to be discovered. One of the challenges we face is the viability of Gfpt1 -/- knockout mice. In this study, we use Cre/LoxP technology to generate a muscle-specific GFPT1 knockout mouse model, Gfpt1tm1d/tm1d, characteristic of the human CMS phenotype. Our data suggests a critical role for muscle derived GFPT1 in the development of the NMJ, neurotransmission, skeletal muscle integrity, and highlights that a deficiency in skeletal muscle alone is sufficient to cause morphological postsynaptic NMJ changes that are accompanied by presynaptic alterations despite the conservation of neuronal GFPT1 expression. In addition to the conventional morphological NMJ changes and fatigable muscle weakness, Gfpt1tm1d/tm1d mice display a progressive myopathic phenotype with the presence of tubular aggregates in muscle, characteristic of the GFPT1-CMS phenotype. We further identify an upregulation of skeletal muscle proteins glypican-1, farnesyltransferase/geranylgeranyltransferase type-1 subunit alpha and Muscle-specific kinase which are known to be involved in the differentiation and maintenance of the NMJ. The Gfpt1tm1d/tm1d model allows for further investigation of pathophysiological consequences on genes and pathways downstream of GFPT1 likely to involve misglycosylation or hypoglycosylation of NMJs and muscle targets.

Tissue-specific oxidative stress and loss of mitochondria in CoQ-deficient Pdss2 mutant mice.

PubMed

Quinzii, Catarina M; Garone, Caterina; Emmanuele, Valentina; Tadesse, Saba; Krishna, Sindu; Dorado, Beatriz; Hirano, Michio

2013-02-01

Primary human CoQ(10) deficiencies are clinically heterogeneous diseases caused by mutations in PDSS2 and other genes required for CoQ(10) biosynthesis. Our in vitro studies of PDSS2 mutant fibroblasts, with <20% CoQ(10) of control cells, revealed reduced activity of CoQ(10)-dependent complex II+III and ATP synthesis, without amplification of reactive oxygen species (ROS), markers of oxidative damage, or antioxidant defenses. In contrast, COQ2 and ADCK3 mutant fibroblasts, with 30-50% CoQ(10) of controls, showed milder bioenergetic defects but significantly increased ROS and oxidation of lipids and proteins. We hypothesized that absence of oxidative stress markers and cell death in PDSS2 mutant fibroblasts were due to the extreme severity of CoQ(10) deficiency. Here, we have investigated in vivo effects of Pdss2 deficiency in affected and unaffected organs of CBA/Pdss2(kd/kd) mice at presymptomatic, phenotypic-onset, and end-stages of the disease. Although Pdss2 mutant mice manifest widespread CoQ(9) deficiency and mitochondrial respiratory chain abnormalities, only affected organs show increased ROS production, oxidative stress, mitochondrial DNA depletion, and reduced citrate synthase activity, an index of mitochondrial mass. Our data indicate that kidney-specific loss of mitochondria triggered by oxidative stress may be the cause of renal failure in Pdss2(kd/kd) mice.

CD22 x Siglec-G double-deficient mice have massively increased B1 cell numbers and develop systemic autoimmunity.

PubMed

Jellusova, Julia; Wellmann, Ute; Amann, Kerstin; Winkler, Thomas H; Nitschke, Lars

2010-04-01

CD22 and Siglec-G are inhibitory coreceptors for BCR-mediated signaling. Although CD22-deficient mice show increased calcium signaling in their conventional B2 cells and a quite normal B cell maturation, Siglec-G-deficient mice have increased calcium mobilization just in B1 cells and show a large expansion of the B1 cell population. Neither CD22-deficient, nor Siglec-G-deficient mice on a pure C57BL/6 or BALB/c background, respectively, develop autoimmunity. Using Siglec-G x CD22 double-deficient mice, we addressed whether Siglec-G and CD22 have redundant functions. Siglec-G x CD22 double-deficient mice show elevated calcium responses in both B1 cells and B2 cells, increased serum IgM levels and an enlarged population of B1 cells. The enlargement of B1 cell numbers is even higher than in Siglecg(-/-) mice. This expansion seems to happen at the expense of B2 cells, which are reduced in absolute cell numbers, but show an activated phenotype. Furthermore, Siglec-G x CD22 double-deficient mice show a diminished immune response to both thymus-dependent and thymus-independent type II Ags. In contrast, B cells from Siglec-G x CD22 double-deficient mice exhibit a hyperproliferative response to stimulation with several TLR ligands. Aged Siglec-G x CD22 double-deficient mice spontaneously develop anti-DNA and antinuclear autoantibodies. These resulted in a moderate form of immune complex glomerulonephritis. These results show that Siglec-G and CD22 have partly compensatory functions and together are crucial in maintaining the B cell tolerance.

Commensal Microbiota Contributes to Chronic Endocarditis in TAX1BP1 Deficient Mice

PubMed Central

Nakano, Satoko; Ikebe, Emi; Tsukamoto, Yoshiyuki; Wang, Yan; Matsumoto, Takashi; Mitsui, Takahiro; Yahiro, Takaaki; Inoue, Kunimitsu; Kawazato, Hiroaki; Yasuda, Aiko; Ito, Kanako; Yokoyama, Shigeo; Takahashi, Naohiko; Hori, Mitsuo; Shimada, Tatsuo; Moriyama, Masatsugu; Kubota, Toshiaki; Ono, Katsushige; Fujibuchi, Wataru; Jeang, Kuan-Teh; Iha, Hidekatsu; Nishizono, Akira

2013-01-01

Tax1-binding protein 1 (Tax1bp1) negatively regulates NF-κB by editing the ubiquitylation of target molecules by its catalytic partner A20. Genetically engineered TAX1BP1-deficient (KO) mice develop age-dependent inflammatory constitutions in multiple organs manifested as valvulitis or dermatitis and succumb to premature death. Laser capture dissection and gene expression microarray analysis on the mitral valves of TAX1BP1-KO mice (8 and 16 week old) revealed 588 gene transcription alterations from the wild type. SAA3 (serum amyloid A3), CHI3L1, HP, IL1B and SPP1/OPN were induced 1,180-, 361-, 187-, 122- and 101-fold respectively. WIF1 (Wnt inhibitory factor 1) exhibited 11-fold reduction. Intense Saa3 staining and significant I-κBα reduction were reconfirmed and massive infiltration of inflammatory lymphocytes and edema formation were seen in the area. Antibiotics-induced ‘germ free’ status or the additional MyD88 deficiency significantly ameliorated TAX1BP1-KO mice's inflammatory lesions. These pathological conditions, as we named ‘pseudo-infective endocarditis’ were boosted by the commensal microbiota who are usually harmless by their nature. This experimental outcome raises a novel mechanistic linkage between endothelial inflammation caused by the ubiquitin remodeling immune regulators and fatal cardiac dysfunction. PMID:24086273

Severe dermatitis with loss of epidermal Langerhans cells in human and mouse zinc deficiency

PubMed Central

Kawamura, Tatsuyoshi; Ogawa, Youichi; Nakamura, Yuumi; Nakamizo, Satoshi; Ohta, Yoshihiro; Nakano, Hajime; Kabashima, Kenji; Katayama, Ichiro; Koizumi, Schuichi; Kodama, Tatsuhiko; Nakao, Atsuhito; Shimada, Shinji

2012-01-01

Zinc deficiency can be an inherited disorder, in which case it is known as acrodermatitis enteropathica (AE), or an acquired disorder caused by low dietary intake of zinc. Even though zinc deficiency diminishes cellular and humoral immunity, patients develop immunostimulating skin inflammation. Here, we have demonstrated that despite diminished allergic contact dermatitis in mice fed a zinc-deficient (ZD) diet, irritant contact dermatitis (ICD) in these mice was more severe and prolonged than that in controls. Further, histological examination of ICD lesions in ZD mice revealed subcorneal vacuolization and epidermal pallor, histological features of AE. Consistent with the fact that ATP release from chemically injured keratinocytes serves as a causative mediator of ICD, we found that the severe ICD response in ZD mice was attenuated by local injection of soluble nucleoside triphosphate diphosphohydrolase. In addition, skin tissue from ZD mice with ICD showed increased levels of ATP, as did cultured wild-type keratinocytes treated with chemical irritants and the zinc-chelating reagent TPEN. Interestingly, numbers of epidermal Langerhans cells (LCs), which play a protective role against ATP-mediated inflammatory signals, were decreased in ZD mice as well as samples from ZD patients. These findings suggest that upon exposure to irritants, aberrant ATP release from keratinocytes and impaired LC-dependent hydrolysis of nucleotides may be important in the pathogenesis of AE. PMID:22214844

Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

PubMed

Salerno, Simona; Morelli, Sabrina; Giordano, Francesca; Gordano, Amalia; Bartolo, Loredana De

2016-10-01

In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system. Copyright © 2016 Elsevier B.V. All rights reserved.

Fucosylation Deficiency in Mice Leads to Colitis and Adenocarcinoma.

PubMed

Wang, Yiwei; Huang, Dan; Chen, Kai-Yuan; Cui, Min; Wang, Weihuan; Huang, Xiaoran; Awadellah, Amad; Li, Qing; Friedman, Ann; Xin, William W; Di Martino, Luca; Cominelli, Fabio; Miron, Alex; Chan, Ricky; Fox, James G; Xu, Yan; Shen, Xiling; Kalady, Mathew F; Markowitz, Sanford; Maillard, Ivan; Lowe, John B; Xin, Wei; Zhou, Lan

2017-01-01

De novo synthesis of guanosine diphosphate (GDP)-fucose, a substrate for fucosylglycans, requires sequential reactions mediated by GDP-mannose 4,6-dehydratase (GMDS) and GDP-4-keto-6-deoxymannose 3,5-epimerase-4-reductase (FX or tissue specific transplantation antigen P35B [TSTA3]). GMDS deletions and mutations are found in 6%-13% of colorectal cancers; these mostly affect the ascending and transverse colon. We investigated whether a lack of fucosylation consequent to loss of GDP-fucose synthesis contributes to colon carcinogenesis. FX deficiency and GMDS deletion produce the same biochemical phenotype of GDP-fucose deficiency. We studied a mouse model of fucosylation deficiency (Fx-/- mice) and mice with the full-length Fx gene (controls). Mice were placed on standard chow or fucose-containing diet (equivalent to a control fucosylglycan phenotype). Colon tissues were collected and analyzed histologically or by enzyme-linked immunosorbent assays to measure cytokine levels; T cells also were collected and analyzed. Fecal samples were analyzed by 16s ribosomal RNA sequencing. Mucosal barrier function was measured by uptake of fluorescent dextran. We transplanted bone marrow cells from Fx-/- or control mice (Ly5.2) into irradiated 8-week-old Fx-/- or control mice (Ly5.1). We performed immunohistochemical analyses for expression of Notch and the hes family bHLH transcription factor (HES1) in colon tissues from mice and a panel of 60 human colorectal cancer specimens (27 left-sided, 33 right-sided). Fx-/- mice developed colitis and serrated-like lesions. The intestinal pathology of Fx-/- mice was reversed by addition of fucose to the diet, which restored fucosylation via a salvage pathway. In the absence of fucosylation, dysplasia appeared and progressed to adenocarcinoma in up to 40% of mice, affecting mainly the right colon and cecum. Notch was not activated in Fx-/- mice fed standard chow, leading to decreased expression of its target Hes1. Fucosylation deficiency

Induction of senescence pathways in Kindler syndrome primary keratinocytes.

PubMed

Piccinni, E; Di Zenzo, G; Maurelli, R; Dellambra, E; Teson, M; Has, C; Zambruno, G; Castiglia, D

2013-05-01

Individuals with Kindler syndrome (KS) have loss-of-function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin-1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated. To investigate how kindlin-1 deficiency affects the proliferative potential of primary human keratinocytes. We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony-forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin-1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology. We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi-1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle-arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin-1 proved that its deficiency was directly responsible for p63, Bmi-1 and pRb downregulation and p16 induction. Our data directly implicate kindlin-1 in preventing premature senescence of keratinocytes. © 2013 The Authors. BJD © 2013 British Association of Dermatologists.

Marrow Adipose Tissue Expansion Coincides with Insulin Resistance in MAGP1-Deficient Mice

PubMed Central

Walji, Tezin A.; Turecamo, Sarah E.; Sanchez, Alejandro Coca; Anthony, Bryan A.; Abou-Ezzi, Grazia; Scheller, Erica L.; Link, Daniel C.; Mecham, Robert P.; Craft, Clarissa S.

2016-01-01

Marrow adipose tissue (MAT) is an endocrine organ with the potential to influence skeletal remodeling and hematopoiesis. Pathologic MAT expansion has been studied in the context of severe metabolic challenge, including caloric restriction, high fat diet feeding, and leptin deficiency. However, the rapid change in peripheral fat and glucose metabolism associated with these models impedes our ability to examine which metabolic parameters precede or coincide with MAT expansion. Microfibril-associated glycoprotein-1 (MAGP1) is a matricellular protein that influences cellular processes by tethering signaling molecules to extracellular matrix structures. MAGP1-deficient (Mfap2−/−) mice display a progressive excess adiposity phenotype, which precedes insulin resistance and occurs without changes in caloric intake or ambulation. Mfap2−/− mice were, therefore, used as a model to associate parameters of metabolic disease, bone remodeling, and hematopoiesis with MAT expansion. Marrow adiposity was normal in Mfap2−/− mice until 6 months of age; however, by 10 months, marrow fat volume had increased fivefold relative to wild-type control at the same age. Increased gonadal fat pad mass and hyperglycemia were detectable in Mfap2−/− mice by 2 months, but peaked by 6 months. The development of insulin resistance coincided with MAT expansion. Longitudinal characterization of bone mass demonstrated a disconnection in MAT volume and bone volume. Specifically, Mfap2−/− mice had reduced trabecular bone volume by 2 months, but this phenotype did not progress with age or MAT expansion. Interestingly, MAT expansion in the 10-month-old Mfap2−/− mice was associated with modest alterations in basal hematopoiesis, including a shift from granulopoiesis to B lymphopoiesis. Together, these findings indicate MAT expansion is coincident with insulin resistance, but not excess peripheral adiposity or hyperglycemia in Mfap2−/− mice; and substantial MAT

Iron Overload and Heart Fibrosis in Mice Deficient for Both β2-Microglobulin and Rag1

PubMed Central

Santos, Manuela M.; de Sousa, Maria; Rademakers, Luke H. P. M.; Clevers, Hans; Marx, J. J. M.; Schilham, Marco W.

2000-01-01

Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, a β2-microglobulin (β2m)-associated major histocompatibility complex class I-like protein. Accordingly, mutant β2m−/− mice have increased intestinal iron absorption and develop parenchymal iron overload in the liver. In humans, other genetic and environmental factors have been suggested to influence the pathology and severity of HH. Previously, an association has been reported between low numbers of lymphocytes and the severity of clinical expression of the iron overload in HH. In the present study, the effect of a total absence of lymphocytes on iron overload was investigated by crossing β2m−/− mice (which develop iron overload resembling human disease) with mice deficient in recombinase activator gene 1 (Rag1), which is required for normal B and T lymphocyte development. Iron overload was more severe in β2mRag1 double-deficient mice than in each of the single deficient mice, with iron accumulation in parenchymal cells of the liver, in acinar cells of the pancreas, and in heart myocytes. With increasing age β2mRag1−/− mice develop extensive heart fibrosis, which could be prevented by reconstitution with normal hematopoietic cells. Thus, the development of iron-mediated cellular damage is substantially enhanced when a Rag1 mutation, which causes a lack of mature lymphocytes, is introduced into β2m−/− mice. Mice deficient in β2m and Rag1 thus offer a new experimental model of iron-related cardiomyopathy. PMID:11106561

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

PubMed

deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

2003-02-01

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the

Brain endothelial TAK1 and NEMO safeguard the neurovascular unit

PubMed Central

Ridder, Dirk A.; Wenzel, Jan; Müller, Kristin; Töllner, Kathrin; Tong, Xin-Kang; Assmann, Julian C.; Stroobants, Stijn; Weber, Tobias; Niturad, Cristina; Fischer, Lisanne; Lembrich, Beate; Wolburg, Hartwig; Grand’Maison, Marilyn; Papadopoulos, Panayiota; Korpos, Eva; Truchetet, Francois; Rades, Dirk; Sorokin, Lydia M.; Schmidt-Supprian, Marc; Bedell, Barry J.; Pasparakis, Manolis; Balschun, Detlef; D’Hooge, Rudi; Löscher, Wolfgang; Hamel, Edith

2015-01-01

Inactivating mutations of the NF-κB essential modulator (NEMO), a key component of NF-κB signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood–brain barrier (BBB), and epileptic seizures. TAK1 and NEMO protected the BBB by activating the transcription factor NF-κB and stabilizing the tight junction protein occludin. They also prevented brain endothelial cell death in a NF-κB–independent manner by reducing oxidative damage. Our data identify crucial functions of inflammatory TAK1–NEMO signaling in protecting the brain endothelium and maintaining normal brain function, thus explaining the neurological symptoms associated with IP. PMID:26347470

Cardiovascular phenotype in Smad3 deficient mice with renovascular hypertension.

PubMed

Kashyap, Sonu; Warner, Gina; Hu, Zeng; Gao, Feng; Osman, Mazen; Al Saiegh, Yousif; Lien, Karen R; Nath, Karl; Grande, Joseph P

2017-01-01

Renovascular hypertension (RVH) has deleterious effects on both the kidney and the heart. TGF-β signaling through Smad3 directs tissue fibrosis in chronic injury models. In the 2-kidney 1-clip (2K1C) model of RVH, employing mice on the 129 genetic background, Smad3 deficiency (KO) protects the stenotic kidney (STK) from development of interstitial fibrosis. However, these mice have an increased incidence of sudden cardiac death following 2K1C surgery. The purpose of this study was to characterize the cardiovascular phenotype of these mice. Renal artery stenosis (RAS) was established in Wild-type (WT) and Smad3 KO mice (129 genetic background) by placement of a polytetrafluoroethylene cuff on the right renal artery. Mortality was 25.5% for KO mice with RAS, 4.1% for KO sham mice, 1.2% for WT with RAS, and 1.8% for WT sham mice. Myocardial tissue of mice studied at 3 days following surgery showed extensive myocyte necrosis in KO but not WT mice. Myocyte necrosis was associated with a rapid induction of Ccl2 expression, macrophage influx, and increased MMP-9 activity. At later time points, both KO and WT mice developed myocardial fibrosis. No aortic aneurysms or dissections were observed at any time point. Smad3 KO mice were backcrossed to the C57BL/6J strain and subjected to RAS. Sudden death was observed at 10-14 days following surgery in 62.5% of mice; necropsy revealed aortic dissections as the cause of death. As observed in the 129 mice, the STK of Smad3 KO mice on the C57BL/6J background did not develop significant chronic renal damage. We conclude that the cardiovascular manifestations of Smad3 deficient mice are strain-specific, with myocyte necrosis in 129 mice and aortic rupture in C57BL/6J mice. Future studies will define mechanisms underlying this strain-specific effect on the cardiovascular system.

Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D -driven tumor promotion.

PubMed

Kurosawa, Koreyuki; Inoue, Yui; Kakugawa, Yoichiro; Yamashita, Yoji; Kanazawa, Kosuke; Kishimoto, Kazuhiro; Nomura, Miyuki; Momoi, Yuki; Sato, Ikuro; Chiba, Natsuko; Suzuki, Mai; Ogoh, Honami; Yamada, Hidekazu; Miura, Koh; Watanabe, Toshio; Tanuma, Nobuhiro; Tachi, Masahiro; Shima, Hiroshi

2018-05-14

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-ras G12D -expressing and Ppp6c-deficient) mice in which K-ras G12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lip, nipples, external genitalia, anus and palms, and had to be sacrificed by three weeks after induction by tamoxifen, while comparably-treated K-ras G12D -expressing mice did not. HE-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinoma. Immunohistochemical analysis of lip of doubly-mutant versus K-ras G12D mice revealed that cell proliferation and cell size increased approximately two-fold relative to K-ras G12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-ras G12D only mice. Moreover, AKT phosphorylation increased in K-ras G12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6, and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-ras G12D -dependent tumor promotion. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Absence of γ-Chain in Keratinocytes Alters Chemokine Secretion, Resulting in Reduced Immune Cell Recruitment.

PubMed

Nowak, Karolin; Linzner, Daniela; Thrasher, Adrian J; Lambert, Paul F; Di, Wei-Li; Burns, Siobhan O

2017-10-01

Loss-of-function mutations in the common gamma (γc) chain cytokine receptor subunit give rise to severe combined immunodeficiency characterized by lack of T and natural killer cells and infant death from infection. Hematopoietic stem cell transplantation or gene therapy offer a cure, but despite successful replacement of lymphoid immune lineages, a long-term risk of severe cutaneous human papilloma virus infections persists, possibly related to persistent γc-deficiency in other cell types. Here we show that keratinocytes, the only cell type directly infected by human papilloma virus, express functional γc and its co-receptors. After stimulation with the γc-ligand IL-15, γc-deficient keratinocytes show significantly impaired secretion of specific chemokines including CXCL1, CXCL8, and CCL20, resulting in reduced chemotaxis of dendritic cells and CD4 + T cells. Furthermore, γc-deficient keratinocytes also exhibit defective induction of T-cell chemotaxis in a model of stable human papilloma virus-18 infection. These findings suggest that persistent γc-deficiency in keratinocytes alters immune cell recruitment to the skin, which may contribute to the development and persistence of warts in this condition and would require different treatment approaches. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Gender Affects Skin Wound Healing in Plasminogen Deficient Mice

PubMed Central

Rønø, Birgitte; Engelholm, Lars Henning; Lund, Leif Røge; Hald, Andreas

2013-01-01

The fibrinolytic activity of plasmin plays a fundamental role in resolution of blood clots and clearance of extravascular deposited fibrin in damaged tissues. These vital functions of plasmin are exploited by malignant cells to accelerate tumor growth and facilitate metastases. Mice lacking functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency or if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin accumulation in neither wildtype nor plasmin deficient mice, and the existing differences in skin composition between males and females were unaffected by plasmin deficiency. In contrast, gender had a marked effect on the ability of plasmin deficient mice to heal skin wounds, which was seen as an accelerated wound closure in female versus male plasmin deficient mice. Further studies showed that this gender effect could not be reversed by ovariectomy, suggesting that female sex-hormones did not mediate the accelerated skin wound healing in plasmin deficient female mice. Histological examination of healed wounds revealed larger amounts of fibrotic scars in the provisional matrix of plasmin deficient male mice compared to female mice. These fibrotic scars correlated to an obstruction of cell infiltration of the granulation tissue, which is a prerequisite for wound healing. In conclusion, the presented data show that the gender dependent effect of plasmin deficiency is tissue specific and may be secondary to already established differences between genders, such as skin thickness and

Abnormalities in Osteoclastogenesis and Decreased Tumorigenesis in Mice Deficient for Ovarian Cancer G Protein-Coupled Receptor 1

PubMed Central

Li, Hui; Wang, Dongmei; Singh, Lisam Shanjukumar; Berk, Michael; Tan, Haiyan; Zhao, Zhenwen; Steinmetz, Rosemary; Kirmani, Kashif; Wei, Gang; Xu, Yan

2009-01-01

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK) activation and nitric oxide (NO) production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1's role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases. PMID:19479052

Altered synaptic phospholipid signaling in PRG-1 deficient mice induces exploratory behavior and motor hyperactivity resembling psychiatric disorders.

PubMed

Schneider, Patrick; Petzold, Sandra; Sommer, Angela; Nitsch, Robert; Schwegler, Herbert; Vogt, Johannes; Roskoden, Thomas

2018-01-15

Plasticity related gene 1 (PRG-1) is a neuron specific membrane protein located at the postsynaptic density of glutamatergic synapses. PRG-1 modulates signaling pathways of phosphorylated lipid substrates such as lysophosphatidic acid (LPA). Deletion of PRG-1 increases presynaptic glutamate release probability leading to neuronal over-excitation. However, due to its cortical expression, PRG-1 deficiency leading to increased glutamatergic transmission is supposed to also affect motor pathways. We therefore analyzed the effects of PRG-1 function on exploratory and motor behavior using homozygous PRG-1 knockout (PRG-1 -/- ) mice and PRG-1/LPA 2 -receptor double knockout (PRG-1 -/- /LPA 2 -/- ) mice in two open field settings of different size and assessing motor behavior in the Rota Rod test. PRG-1 -/- mice displayed significantly longer path lengths and higher running speed in both open field conditions. In addition, PRG-1 -/- mice spent significantly longer time in the larger open field and displayed rearing and self-grooming behavior. Furthermore PRG-1 -/- mice displayed stereotypical behavior resembling phenotypes of psychiatric disorders in the smaller sized open field arena. Altogether, this behavior is similar to the stereotypical behavior observed in animal models for psychiatric disease of autistic spectrum disorders which reflects a disrupted balance between glutamatergic and GABAergic synapses. These differences indicate an altered excitation/inhibition balance in neuronal circuits in PRG-1 -/- mice as recently shown in the somatosensory cortex [38]. In contrast, PRG-1 -/- /LPA 2 -/- did not show significant changes in behavior in the open field suggesting that these specific alterations were abolished when the LPA 2 -receptor was lacking. Our findings indicate that PRG-1 deficiency led to over-excitability caused by an altered LPA/LPA 2 -R signaling inducing a behavioral phenotype typically observed in animal models for psychiatric disorders. Copyright �

Osteoblast-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Enhanced Bone Formation With Aberrant Mineralization.

PubMed

Azuma, Kotaro; Shiba, Sachiko; Hasegawa, Tomoka; Ikeda, Kazuhiro; Urano, Tomohiko; Horie-Inoue, Kuniko; Ouchi, Yasuyoshi; Amizuka, Norio; Inoue, Satoshi

2015-07-01

Vitamin K is a fat-soluble vitamin that is necessary for blood coagulation. In addition, it has bone-protective effects. Vitamin K functions as a cofactor of γ-glutamyl carboxylase (GGCX), which activates its substrates by carboxylation. These substrates are found throughout the body and examples include hepatic blood coagulation factors. Furthermore, vitamin K functions as a ligand of the nuclear receptor known as steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR). We have previously reported on the bone-protective role of SXR/PXR signaling by demonstrating that systemic Pxr-knockout mice displayed osteopenia. Because systemic Ggcx-knockout mice die shortly after birth from severe hemorrhage, the GGCX-mediated effect of vitamin K on bone metabolism has been difficult to evaluate. In this work, we utilized Ggcx-floxed mice to generate osteoblast-specific GGCX-deficient (Ggcx(Δobl/Δobl)) mice by crossing them with Col1-Cre mice. The bone mineral density (BMD) of Ggcx(Δobl/Δobl) mice was significantly higher than that of control Col1-Cre (Ggcx(+/+)) mice. Histomorphometrical analysis of trabecular bones in the proximal tibia showed increased osteoid volume and a higher rate of bone formation in Ggcx(Δobl/Δobl) mice. Histomorphometrical analysis of cortical bones revealed a thicker cortical width and a higher rate of bone formation in Ggcx(Δobl/Δobl) mice. Electron microscopic examination revealed disassembly of mineralized nodules and aberrant calcification of collagen fibers in Ggcx(Δobl/Δobl) mice. The mechanical properties of bones from Ggcx(Δobl/Δobl) mice tended to be stronger than those from control Ggcx(+/+) mice. These results suggest that GGCX in osteoblasts functions to prevent abnormal mineralization in bone formation, although this function may not be a prerequisite for the bone-protective effect of vitamin K. © 2015 American Society for Bone and Mineral Research.

Epidermal keratinocytes initiate wound healing and pro-inflammatory immune responses following percutaneous schistosome infection

PubMed Central

Bourke, Claire D.; Prendergast, Catriona T.; Sanin, David E.; Oulton, Tate E.; Hall, Rebecca J.; Mountford, Adrian P.

2015-01-01

Keratinocytes constitute the majority of cells in the skin’s epidermis, the first line of defence against percutaneous pathogens. Schistosome larvae (cercariae) actively penetrate the epidermis to establish infection, however the response of keratinocytes to invading cercariae has not been investigated. Here we address the hypothesis that cercariae activate epidermal keratinocytes to promote the development of a pro-inflammatory immune response in the skin. C57BL/6 mice were exposed to Schistosoma mansoni cercariae via each pinna and non-haematopoietic cells isolated from epidermal tissue were characterised for the presence of different keratinocyte sub-sets at 6, 24 and 96 h p.i. We identified an expansion of epidermal keratinocyte precursors (CD45−, CD326−, CD34+) within 24 h of infection relative to naïve animals. Following infection, cells within the precursor population displayed a more differentiated phenotype (α6integrin−) than in uninfected skin. Parallel immunohistochemical analysis of pinnae cryosections showed that this expansion corresponded to an increase in the intensity of CD34 staining, specifically in the basal bulge region of hair follicles of infected mice, and a higher frequency of keratinocyte Ki67+ nuclei in both the hair follicle and interfollicular epidermis. Expression of pro-inflammatory cytokine and stress-associated keratin 6b genes was also transiently upregulated in the epidermal tissue of infected mice. In vitro exposure of keratinocyte precursors isolated from neonatal mouse skin to excretory/secretory antigens released by penetrating cercariae elicited IL-1α and IL-1β production, supporting a role for keratinocyte precursors in initiating cutaneous inflammatory immune responses. Together, these observations indicate that S.mansoni cercariae and their excretory/secretory products act directly upon epidermal keratinocytes, which respond by initiating barrier repair and pro-inflammatory mechanisms similar to those

TGF-β–Activated Kinase 1 Is Crucial in Podocyte Differentiation and Glomerular Capillary Formation

PubMed Central

Lee, So-Young; Wang, Zhibo; Ding, Yan; Haque, Nadeem; Zhang, Jiwang; Zhou, Jing

2014-01-01

TGF-β–activated kinase 1 (TAK1) is a key intermediate in signal transduction induced by TGF-β or inflammatory cytokines, such as TNF-α and IL-1, which are potent inducers of podocyte injury responses that lead to proteinuria and glomerulosclerosis. Nevertheless, little is known about the physiologic and pathologic roles of TAK1 in podocytes. To examine the in vivo role of TAK1, we generated podocyte-specific Tak1 knockout mice (Nphs2-Cre+:Tak1fx/fx; Tak1∆/∆). Targeted deletion of Tak1 in podocytes resulted in perinatal lethality, with approximately 50% of animals dying soon after birth and 90% of animals dying within 1 week of birth. Tak1∆/∆ mice developed proteinuria from P1 and exhibited delayed glomerulogenesis and reduced expression of Wilms’ tumor suppressor 1 and nephrin in podocytes. Compared with Tak1fx/fx mice, Tak1∆/∆ mice exhibited impaired formation of podocyte foot processes that caused disruption of the podocyte architecture with prominent foot process effacement. Intriguingly, Tak1∆/∆ mice displayed increased expression of vascular endothelial growth factor within the glomerulus and abnormally enlarged glomerular capillaries. Furthermore, 4- and 7-week-old Tak1∆/∆ mice with proteinuria had increased collagen deposition in the mesangium and the adjacent tubulointerstitial area. Thus, loss of Tak1 in podocytes is associated with the development of proteinuria and glomerulosclerosis. Taken together, our data show that TAK1 regulates the expression of Wilms’ tumor suppressor 1, nephrin, and vascular endothelial growth factor and that TAK1 signaling has a crucial role in podocyte differentiation and attainment of normal glomerular microvasculature during kidney development and glomerular filtration barrier homeostasis. PMID:24652804

Mice deficient for ERAD machinery component Sel1L develop central diabetes insipidus.

PubMed

Bichet, Daniel G; Lussier, Yoann

2017-10-02

Deficiency of the antidiuretic hormone arginine vasopressin (AVP) underlies diabetes insipidus, which is characterized by the excretion of abnormally large volumes of dilute urine and persistent thirst. In this issue of the JCI, Shi et al. report that Sel1L-Hrd1 ER-associated degradation (ERAD) is responsible for the clearance of misfolded pro-arginine vasopressin (proAVP) in the ER. Additionally, mice with Sel1L deficiency, either globally or specifically within AVP-expressing neurons, developed central diabetes insipidus. The results of this study demonstrate a role for ERAD in neuroendocrine cells and serve as a clinical example of the effect of misfolded ER proteins retrotranslocated through the membrane into the cytosol, where they are polyubiquitinated, extracted from the ER membrane, and degraded by the proteasome. Moreover, proAVP misfolding in hereditary central diabetes insipidus likely shares common physiopathological mechanisms with proinsulin misfolding in hereditary diabetes mellitus of youth.

Partial thyrocyte-specific Gαs deficiency leads to rapid-onset hypothyroidism, hyperplasia, and papillary thyroid carcinoma-like lesions in mice.

PubMed

Patyra, Konrad; Jaeschke, Holger; Löf, Christoffer; Jännäri, Meeri; Ruohonen, Suvi T; Undeutsch, Henriette; Khalil, Moosa; Kero, Andreina; Poutanen, Matti; Toppari, Jorma; Chen, Min; Weinstein, Lee S; Paschke, Ralf; Kero, Jukka

2018-05-25

Thyroid function is controlled by thyroid-stimulating hormone (TSH), which binds to its G protein-coupled receptor [thyroid-stimulating hormone receptor (TSHR)] on thyrocytes. TSHR can potentially couple to all G protein families, but it mainly activates the G s - and G q/11 -mediated signaling cascades. To date, there is a knowledge gap concerning the role of the individual G protein cascades in thyroid pathophysiology. Here, we demonstrate that the thyrocyte-specific deletion of G s -protein α subunit (Gα s ) in adult mice [tamoxifen-inducible G s protein α subunit deficient (iTGα s KO) mice] rapidly impairs thyrocyte function and leads to hypothyroidism. Consequently, iTGα s KO mice show reduced food intake and activity. However, body weight and the amount of white adipose tissue were decreased only in male iTGα s KO mice. Unexpectedly, hyperplastic follicles and papillary thyroid cancer-like tumor lesions with increased proliferation and slightly increased phospho-ERK1/2 staining were found in iTGα s KO mice at an older age. These tumors developed from nonrecombined thyrocytes still expressing Gα s in the presence of highly elevated serum TSH. In summary, we report that partial thyrocyte-specific Gα s deletion leads to hypothyroidism but also to tumor development in thyrocytes with remaining Gα s expression. Thus, these mice are a novel model to elucidate the pathophysiological consequences of hypothyroidism and TSHR/G s /cAMP-mediated tumorigenesis.-Patyra, K., Jaeschke, H., Löf, C., Jännäri, M., Ruohonen, S. T., Undeutsch, H., Khalil, M., Kero, A., Poutanen, M., Toppari, J., Chen, M., Weinstein, L. S., Paschke, R., Kero, J. Partial thyrocyte-specific Gα s deficiency leads to rapid-onset hypothyroidism, hyperplasia, and papillary thyroid carcinoma-like lesions in mice.

Dual specificity phosphatase 6 deficiency is associated with impaired systemic glucose tolerance and reversible weight retardation in mice

PubMed Central

Schriever, Sonja C.; Müller, Timo D.; Tschöp, Matthias H.

2017-01-01

Here, we aimed to investigate the potential role of DUSP6, a dual specificity phosphatase, that specifically inactivates extracellular signal-regulated kinase (ERK), for the regulation of body weight and glucose homeostasis. We further assessed whether metabolic challenges affect Dusp6 expression in selected brain areas or white adipose tissue. Hypothalamic Dusp6 mRNA levels remained unchanged in chow-fed lean vs. high fat diet (HFD) fed obese C57Bl/6J mice, and in C57Bl/6J mice undergoing prolonged fasting or refeeding with fat free diet (FFD) or HFD. Similarly, Dusp6 expression levels were unchanged in selected brain regions of Lepob mice treated with 1 mg/kg of leptin for 6 days, compared to pair-fed or saline-treated Lepob controls. Dusp6 expression levels remained unaltered in vitro in primary adipocytes undergoing differentiation, but were increased in eWAT of HFD-fed obese C57Bl/6J mice, compared to chow-fed lean controls. Global chow-fed DUSP6 KO mice displayed reduced body weight and lean mass and slightly increased fat mass at a young age, which is indicative for early-age weight retardation. Subsequent exposure to HFD led to a significant increase in lean mass and body weight in DUSP6 deficient mice, compared to WT controls. Nevertheless, after 26 weeks of high-fat diet exposure, we observed comparable body weight, fat and lean mass in DUSP6 WT and KO mice, suggesting overall normal susceptibility to develop obesity. In line with the increased weight gain to compensate for early-age weight retardation, HFD-fed DUSP6 KO displayed increased expression levels of anabolic genes involved in lipid and cholesterol metabolism in the epididymal white adipose tissue (eWAT), compared to WT controls. Glucose tolerance was perturbed in both chow-fed lean or HFD-fed obese DUSP6 KO, compared to their respective WT controls. Overall, our data indicate that DUSP6 deficiency has limited impact on the regulation of energy metabolism, but impairs systemic glucose tolerance

Parental origin impairment of synaptic functions and behaviors in cytoplasmic FMRP interacting protein 1 (Cyfip1) deficient mice.

PubMed

Chung, Leeyup; Wang, Xiaoming; Zhu, Li; Towers, Aaron J; Cao, Xinyu; Kim, Il Hwan; Jiang, Yong-hui

2015-12-10

CYFIP1 maps to the interval between proximal breakpoint 1 (BP1) and breakpoint 2 (BP2) of chromosomal 15q11-q13 deletions that are implicated in the Angelman (AS) and Prader-Willi syndrome (PWS). There is only one breakpoint (BP3) at the distal end of deletion. CYFIP1 is deleted in AS patients with the larger class I deletion (BP1 to BP3) and the neurological presentations in these patients are more severe than that of patients with class II (BP2 to BP3) deletion. The haploinsufficiency of CYFIP1 is hypothesized to contribute to more severe clinical presentations in class I AS patients. The expression of CYFIP1 is suggested to be bi-allelic in literature but the possibility of parental origin of expression is not completely excluded. We generated and characterized Cyfip1 mutant mice. Homozygous Cyfip1 mice were early embryonic lethal. However, there was a parental origin specific effect between paternal Cyfip1 deficiency (m+/p-) and maternal deficiency (m-/p+) on both synaptic transmissions and behaviors in hippocampal CA1 synapses despite no evidence supporting the parental origin difference for the expression. Both m-/p+ and m+/p- showed the impaired input-output response and paired-pulse facilitation. While the long term-potentiation and group I mGluR mediated long term depression induced by DHPG was not different between Cyfip1 m-/p+ and m+/p- mice, the initial DHPG induced response was significantly enhanced in m-/p+ but not in m+/p- mice. m+/p- but not m-/p+ mice displayed increased freezing in cued fear conditioning and abnormal transitions in zero-maze test. The impaired synaptic transmission and behaviors in haploinsufficiency of Cyfip1 mice provide the evidence supporting the role of CYFIP1 modifying the clinical presentation of class I AS patients and in human neuropsychiatric disorders. Copyright © 2015 Elsevier B.V. All rights reserved.

Procarcinogenic effects of cyclosporine A are mediated through the activation of TAK1/TAB1 signaling pathway

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

Xu, Jianmin; Walsh, Stephanie B.; Verney, Zoe M.

Research highlights: {yields} Organ transplant recipients are highly susceptible to early skin cancer development. {yields} CsA-mediated TGFB1-dependent TAK1/TAB1 signaling augments invasive tumor growth. {yields} CsA enhances accumulation of upstream kinases, ZMP, AMPK and IRAK to activate TAK1. {yields} TAK1 mediates enhanced proliferation and reduced apoptosis via CsA-dependent NF{kappa}B. -- Abstract: Cyclosporine A (CsA) is an immunosuppressive drug commonly used for maintaining chronic immune suppression in organ transplant recipients. It is known that patients receiving CsA manifest increased growth of aggressive non-melanoma skin cancers. However, the underlying mechanism by which CsA augments tumor growth is not fully understood. Here, we showmore » that CsA augments the growth of A431 epidermoid carcinoma xenograft tumors by activating tumor growth factor {beta}-activated kinase1 (TAK1). The activation of TAK1 by CsA occurs at multiple levels by kinases ZMP, AMPK and IRAK. TAK1 forms heterodimeric complexes with TAK binding protein 1 and 2 (TAB1/TAB2) which in term activate nuclear factor {kappa}B (NF{kappa}B) and p38 MAP kinase. Transcriptional activation of NF{kappa}B is evidenced by IKK{beta}-mediated phosphorylation-dependent degradation of I{kappa}B and consequent nuclear translocation of p65. This also leads to enhancement in the expression of its transcriptional target genes cyclin D1, Bcl2 and COX-2. Similarly, activation of p38 leads to enhanced inflammation-related signaling shown by increased phosphorylation of MAPKAPK2 and which in turn phosphorylates its substrate HSP27. Activation of both NF{kappa}B and p38 MAP kinase provide mitogenic stimuli to augment the growth of SCCs.« less

High Mutation Levels are Compatible with Normal Embryonic Development in Mlh1-Deficient Mice.

PubMed

Fan, Xiaoyan; Li, Yan; Zhang, Yulong; Sang, Meixiang; Cai, Jianhui; Li, Qiaoxia; Ozaki, Toshinori; Ono, Tetsuya; He, Dongwei

2016-10-01

To elucidate the role of the mismatch repair gene Mlh1 in genome instability during the fetal stage, spontaneous mutations were studied in Mlh1-deficient lacZ-transgenic mouse fetuses. Mutation levels were high at 9.5 days post coitum (dpc) and gradually increased during the embryonic stage, after which they remained unchanged. In addition, mutations that were found in brain, liver, spleen, small intestine and thymus showed similar levels and no statistically significant difference was found. The molecular nature of mutations at 12.5 dpc in fetuses of Mlh1 +/+ and Mlh1 -/- mice showed their own unique spectra, suggesting that deletion mutations were the main causes in the deficiency of the Mlh1 gene. Of note, fetuses of irradiated mice exhibited marked differences such as post-implantation loss and Mendelian distribution. Collectively, these results strongly suggest that high mutation ofMlh1 -/- -deficient fetuses has little effect on the fetuses during their early developmental stages, whereas Mlh1 -/- -deficient fetuses from X-ray irradiated mothers are clearly effected.

Overexpressed TRPV3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2

PubMed Central

Huang, Susan M.; Lee, Hyosang; Chung, Man-Kyo; Park, Una; Yu, Yin Yin; Bradshaw, Heather B.; Coulombe, Pierre A.; Walker, J. Michael; Caterina, Michael J.

2009-01-01

The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive TRPV ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared to wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E2 (PGE2) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naïve mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-7203212, however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Co-administration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE2. PMID:19091963

Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model

PubMed Central

Takashima, K; Matsunaga, N; Yoshimatsu, M; Hazeki, K; Kaisho, T; Uekata, M; Hazeki, O; Akira, S; Iizawa, Y; Ii, M

2009-01-01

Background and purpose: TAK-242, a novel synthetic small-molecule, suppresses production of multiple cytokines by inhibiting Toll-like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK-242 and examined its therapeutic effect in a mouse sepsis model. Experimental approach: Binding assay with [3H]-TAK-242 and nuclear factor-κB reporter assay were used to identify the target molecule and binding site of TAK-242. Bacillus calmette guerin (BCG)-primed mouse sepsis model using live Escherichia coli was used to estimate the efficacy of TAK-242 in sepsis. Key results: TAK-242 strongly bound to TLR4, but binding to TLR2, 3, 5, 9, TLR-related adaptor molecules and MD-2 was either not observed or marginal. Mutational analysis using TLR4 mutants indicated that TAK-242 inhibits TLR4 signalling by binding to Cys747 in the intracellular domain of TLR4. TAK-242 inhibited MyD88-independent pathway as well as MyD88-dependent pathway and its inhibitory effect was largely unaffected by lipopolysaccharide (LPS) concentration and types of TLR4 ligands. TAK-242 had no effect on the LPS-induced conformational change of TLR4-MD-2 and TLR4 homodimerization. In mouse sepsis model, although TAK-242 alone did not affect bacterial counts in blood, if co-administered with ceftazidime it inhibited the increases in serum cytokine levels and improved survival of mice. Conclusions and implications: TAK-242 suppressed TLR4 signalling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. When co-administered with antibiotics, TAK-242 showed potent therapeutic effects in an E. coli-induced sepsis model using BCG-primed mice. Thus, TAK-242 may be a promising therapeutic agent for sepsis. PMID:19563534

IGF-1 deficiency impairs neurovascular coupling in mice: implications for cerebromicrovascular aging.

PubMed

Toth, Peter; Tarantini, Stefano; Ashpole, Nicole M; Tucsek, Zsuzsanna; Milne, Ginger L; Valcarcel-Ares, Noa M; Menyhart, Akos; Farkas, Eszter; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan

2015-12-01

Aging is associated with marked deficiency in circulating IGF-1, which has been shown to contribute to age-related cognitive decline. Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of age-related cognitive impairment. To establish the link between IGF-1 deficiency and cerebromicrovascular impairment, neurovascular coupling mechanisms were studied in a novel mouse model of IGF-1 deficiency (Igf1(f/f) -TBG-Cre-AAV8) and accelerated vascular aging. We found that IGF-1-deficient mice exhibit neurovascular uncoupling and show a deficit in hippocampal-dependent spatial memory test, mimicking the aging phenotype. IGF-1 deficiency significantly impaired cerebromicrovascular endothelial function decreasing NO mediation of neurovascular coupling. IGF-1 deficiency also impaired glutamate-mediated CBF responses, likely due to dysregulation of astrocytic expression of metabotropic glutamate receptors and impairing mediation of CBF responses by eicosanoid gliotransmitters. Collectively, we demonstrate that IGF-1 deficiency promotes cerebromicrovascular dysfunction and neurovascular uncoupling mimicking the aging phenotype, which are likely to contribute to cognitive impairment. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Glucocorticoids exacerbate obesity and insulin resistance in neuron-specific proopiomelanocortin-deficient mice

PubMed Central

Smart, James L.; Tolle, Virginie; Low, Malcolm J.

2006-01-01

Null mutations of the proopiomelanocortin gene (Pomc–/–) cause obesity in humans and rodents, but the contributions of central versus pituitary POMC deficiency are not fully established. To elucidate these roles, we introduced a POMC transgene (Tg) that selectively restored peripheral melanocortin and corticosterone secretion in Pomc–/– mice. Rather than improving energy balance, the genetic replacement of pituitary POMC in Pomc–/–Tg+ mice aggravated their metabolic syndrome with increased caloric intake and feed efficiency, reduced oxygen consumption, increased subcutaneous, visceral, and hepatic fat, and severe insulin resistance. Pair-feeding of Pomc–/–Tg+ mice to the daily intake of lean controls normalized their rate of weight gain but did not abolish obesity, indicating that hyperphagia is a major but not sole determinant of the phenotype. Replacement of corticosterone in the drinking water of Pomc–/– mice recapitulated the hyperphagia, excess weight gain and fat accumulation, and hyperleptinemia characteristic of genetically rescued Pomc–/–Tg+ mice. These data demonstrate that CNS POMC peptides play a critical role in energy homeostasis that is not substituted by peripheral POMC. Restoration of pituitary POMC expression to create a de facto neuronal POMC deficiency exacerbated the development of obesity, largely via glucocorticoid modulation of appetite, metabolism, and energy partitioning. PMID:16440060

Enhanced susceptibility to acute pneumococcal otitis media in mice deficient in complement C1qa, factor B, and factor B/C2.

PubMed

Tong, Hua Hua; Li, Yong Xing; Stahl, Gregory L; Thurman, Joshua M

2010-03-01

To define the roles of specific complement activation pathways in host defense against Streptococcus pneumoniae in acute otitis media (AOM), we investigated the susceptibility to AOM in mice deficient in complement factor B and C2 (Bf/C2(-/)(-)), C1qa (C1qa(-/)(-)), and factor B (Bf(-)(/)(-)). Bacterial titers of both S. pneumoniae serotype 6A and 14 in the middle ear lavage fluid samples from Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice were significantly higher than in samples from wild-type mice 24 h after transtympanical infection (P < 0.05) and remained persistently higher in samples from Bf/C2(-/)(-) mice than in samples from wild-type mice. Bacteremia occurred in Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice infected with both strains, but not in wild-type mice. Recruitment of inflammatory cells was paralleled by enhanced production of inflammatory mediators in the middle ear lavage samples from Bf/C2(-/)(-) mice. C3b deposition on both strains was greatest for sera obtained from wild-type mice, followed by C1qa(-)(/)(-) and Bf(-)(/)(-) mice, and least for Bf/C2(-)(/)(-) mice. Opsonophagocytosis and whole-blood killing capacity of both strains were significantly decreased in the presence of sera or whole blood from complement-deficient mice compared to wild-type mice. These findings indicate that both the classical and alternative complement pathways are critical for middle ear immune defense against S. pneumoniae. The reduced capacity of complement-mediated opsonization and phagocytosis in the complement-deficient mice appears to be responsible for the impaired clearance of S. pneumoniae from the middle ear and dissemination to the bloodstream during AOM.

TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers

PubMed Central

Singh, Anurag; Sweeney, Michael F.; Yu, Min; Burger, Alexa; Greninger, Patricia; Benes, Cyril; Haber, Daniel A.; Settleman, Jeff

2012-01-01

Summary Colon cancers frequently harbor KRAS mutations, yet only a subset of KRAS-mutant colon cancer cell lines are dependent upon KRAS signaling for survival. In a screen for kinases that promote survival of KRAS-dependent colon cancer cells, we found that the TAK1 kinase (MAP3K7) is required for tumor cell viability. The induction of apoptosis by RNAi-mediated depletion or pharmacologic inhibition of TAK1 is linked to its suppression of hyperactivated Wnt signaling, evident in both endogenous and genetically reconstituted cells. In APC-mutant/KRAS-dependent cells, KRAS stimulates BMP-7 secretion and BMP signaling, leading to TAK1 activation and enhancement of Wnt-dependent transcription. An in vitro-derived “TAK1-dependency signature” is enriched in primary human colon cancers with mutations in both APC and KRAS, suggesting potential clinical utility in stratifying patient populations. Together, these findings identify TAK1 inhibition as a potential therapeutic strategy for a treatment-refractory subset of colon cancers exhibiting aberrant KRAS and Wnt pathway activation. PMID:22341439

4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice.

PubMed

Patti, M E; Sun, X J; Bruening, J C; Araki, E; Lipes, M A; White, M F; Kahn, C R

1995-10-20

Insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate of the insulin and insulin-like growth factor (IGF)-1 receptors. Transgenic mice lacking IRS-1 are resistant to insulin and IGF-1, but exhibit significant residual insulin action which corresponds to the presence of an alternative high molecular weight substrate in liver and muscle. Recently, Sun et al. (Sun, X.-J., Wang, L.-M., Zhang, Y., Yenush, L. P., Myers, M. G., Jr., Glasheen, E., Lane, W.S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177) purified and cloned 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, which has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, we performed immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle, binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling.

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen.

PubMed

Levin, Rebecca S; Hertz, Nicholas T; Burlingame, Alma L; Shokat, Kevan M; Mukherjee, Shaeri

2016-08-16

TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPase-activating protein (GAP) enzymes, and is exclusive to membrane-localized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection.

Loss of neutrophil polarization in colon carcinoma liver metastases of mice with an inducible, liver-specific IGF-I deficiency.

PubMed

Rayes, Roni F; Milette, Simon; Fernandez, Maria Celia; Ham, Boram; Wang, Ni; Bourdeau, France; Perrino, Stephanie; Yakar, Shoshana; Brodt, Pnina

2018-03-20

The growth of cancer metastases in the liver depends on a permissive interaction with the hepatic microenvironment and neutrophils can contribute to this interaction, either positively or negatively, depending on their phenotype. Here we investigated the role of IGF-I in the control of the tumor microenvironment in the liver, using mice with a conditional, liver-specific, IGF-I deficiency (iLID) induced by a single tamoxifen injection. In mice that had a sustained (3 weeks) IGF-I deficiency prior to the intrasplenic/portal inoculation of colon carcinoma MC-38 cells, we observed an increase in neutrophil accumulation in the liver relative to controls. However, unlike controls, these neutrophils did not acquire the (anti-inflammatory) tumor-promoting phenotype, as evidenced by retention of high ICAM-1 expression and nitric oxide production and low CXCR4, CCL5, and VEGF expression and arginase production, all characteristic of the (pro-inflammatory) phenotype. This coincided with an increase in apoptotic tumor cells and reduced metastasis. Neutrophils isolated from these mice also had reduced IGF-IR expression levels. These changes were not observed in iLID mice with a short-term (2 days) IGF-I depletion, despite a 70% reduction in their circulating IGF-I levels, indicating that a sustained IGF-I deficiency was necessary to alter the neutrophil phenotype. Similar results were obtained with the highly metastatic Lewis lung carcinoma subline H-59 cells and in mice injected with an IGF-Trap that blocks IGF-IR signaling by reducing ligand bioavailability. Our results implicate the IGF axis in neutrophil polarization and the induction of a pro-metastatic microenvironment in the liver.

Barrier Function of the Repaired Skin Is Disrupted Following Arrest of Dicer in Keratinocytes

PubMed Central

Ghatak, Subhadip; Chan, Yuk Cheung; Khanna, Savita; Banerjee, Jaideep; Weist, Jessica; Roy, Sashwati; Sen, Chandan K

2015-01-01

Tissue injury transiently silences miRNA-dependent posttranscriptional gene silencing in its effort to unleash adult tissue repair. Once the wound is closed, miRNA biogenesis is induced averting neoplasia. In this work, we report that Dicer plays an important role in reestablishing the barrier function of the skin post-wounding via a miRNA-dependent mechanism. MicroRNA expression profiling of skin and wound-edge tissue revealed global upregulation of miRNAs following wound closure at day 14 post-wounding with significant induction of Dicer expression. Barrier function of the skin, as measured by trans-epidermal water loss, was compromised in keratinocyte-specific conditional (K14/Lox-Cre) Dicer-ablated mice because of malformed cornified epithelium lacking loricrin expression. Studies on human keratinocytes recognized that loricrin expression was inversely related to the expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1. Compared to healthy epidermis, wound-edge keratinocytes from Dicer-ablated skin epidermis revealed elevated p21Waf1/Cip1 expression. Adenoviral and pharmacological suppression of p21Waf1/Cip1 in keratinocyte-specific conditional Dicer-ablated mice improved wound healing indicating a role of Dicer in the suppression of p21Waf1/Cip1. This work upholds p21Waf1/Cip1 as a druggable target to restore barrier function of skin suffering from loss of Dicer function as would be expected in diabetes and other forms of oxidant insult. PMID:25896246

Peripubertal Vitamin D3 Deficiency Delays Puberty and Disrupts the Estrous Cycle in Adult Female Mice1

PubMed Central

Dicken, Cary L.; Israel, Davelene D.; Davis, Joe B.; Sun, Yan; Shu, Jun; Hardin, John; Neal-Perry, Genevieve

2012-01-01

ABSTRACT The mechanism(s) by which vitamin D3 regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D3 deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D3) null mice to study the effect of vitamin D3 deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D3-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D3 deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D3-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D3-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D3-replete diet. Estrous cycles were restored when vitamin D3-deficient Cyp27b1 null young adult females were transferred to a vitamin D3-replete diet. This study is the first to demonstrate that peripubertal vitamin D3 sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D3 is a key regulator of neuroendocrine and ovarian physiology. PMID:22572998

RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation.

PubMed

Lin, Juan; Kumari, Snehlata; Kim, Chun; Van, Trieu-My; Wachsmuth, Laurens; Polykratis, Apostolos; Pasparakis, Manolis

2016-12-01

Receptor-interacting protein kinase 1 (RIPK1) regulates cell death and inflammation through kinase-dependent and -independent functions. RIPK1 kinase activity induces caspase-8-dependent apoptosis and RIPK3 and mixed lineage kinase like (MLKL)-dependent necroptosis. In addition, RIPK1 inhibits apoptosis and necroptosis through kinase-independent functions, which are important for late embryonic development and the prevention of inflammation in epithelial barriers. The mechanism by which RIPK1 counteracts RIPK3-MLKL-mediated necroptosis has remained unknown. Here we show that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3-MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM1). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 (RIPK1 mRHIM ) caused perinatal lethality that was prevented by RIPK3, MLKL or ZBP1 deficiency. Furthermore, mice expressing only RIPK1 mRHIM in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 in cells expressing RIPK1 mRHIM , suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Collectively, these results show that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis. Furthermore, these findings identify ZBP1 as a critical mediator of inflammation beyond its previously known role in antiviral defence and suggest that ZBP1 might be implicated in the pathogenesis of necroptosis-associated inflammatory diseases.

Increased ethanol preference and serotonin 1A receptor-dependent attenuation of ethanol-induced hypothermia in PACAP-deficient mice.

PubMed

Tanaka, Kazuhiro; Kunishige-Yamamoto, Akiko; Hashimoto, Hitoshi; Shintani, Norihito; Hayata, Atsuko; Baba, Akemichi

2010-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice display remarkable behavioral changes including increased novelty-seeking behavior and reduced hypothermia induced by either serotonin (5-HT)(1A) receptor agonists or ethanol. Because 5-HT(1A) receptors have been implicated in the development of alcohol dependence, we have examined ethanol preference in PACAP-deficient mice using a two-bottle choice and a conditioned place preference test, as well as additive effects of ethanol and 5-HT(1A) receptor agents on hypothermia. PACAP-deficient mice showed an increased preference towards ethanol compared with wild-type mice. However, they showed no preference for the ethanol compartment after conditioning and neither preference nor aversion to sucrose or quinine. The 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) restored the attenuated hypothermic response to ethanol in the mutants to similar levels in wild-type mice, with no effect in wild-types. In contrast, the 5-HT(1A) receptor antagonist WAY-100635 attenuated the ethanol-induced hypothermia in wild-type mice, with no effect in the mutants. These results demonstrate increased ethanol preference in PACAP-deficient mice that may be mediated by 5-HT(1A) receptor-dependent attenuation of ethanol-induced central inhibition. Copyright 2009 Elsevier Inc. All rights reserved.

SAP deficiency mitigated atherosclerotic lesions in ApoE(-/-) mice.

PubMed

Zheng, Lingyun; Wu, Teng; Zeng, Cuiling; Li, Xiangli; Li, Xiaoqiang; Wen, Dingwen; Ji, Tianxing; Lan, Tian; Xing, Liying; Li, Jiangchao; He, Xiaodong; Wang, Lijing

2016-01-01

Serum amyloid P conpoent (SAP), a member of the pentraxin family, interact with pathogens and cell debris to promote their removal by macrophages and neutrophils and is co-localized with atherosclerotic plaques in patients. However, the exact mechanism of SAP in atherogenesis is still unclear. We investigated whether SAP influence macrophage recruitment and foam cell formation and ultimately affect atherosclerotic progression. we generated apoE(-/-); SAP(-/-) (DKO) mice and fed them western diet for 4 and 8 weeks to characterize atherosclerosis development. SAP deficiency effectively reduced plaque size both in the aorta (p = 0.0006 for 4 wks; p = 0.0001 for 8 wks) and the aortic root (p = 0.0061 for 4 wks; p = 0.0079 for 8wks) compared with apoE(-/-) mice. Meanwhile, SAP deficiency inhibited oxLDL-induced foam cell formation (p = 0.0004) compared with apoE(-/-) mice and SAP treatment increases oxLDL-induced foam cell formation (p = 0.002) in RAW cells. Besides, SAP deficiency reduced macrophages recruitment (p = 0.035) in vivo and in vitro (p = 0.026). Furthermore, SAP treatment enhanced CD36 (p = 0.007) and FcγRI (p = 0.031) expression induced by oxLDL through upregulating JNK and p38 MAPK phosphorylation whereas specific JNK1/2 inhibitor reduced CD36 (p = 0.0005) and FcγRI (P = 0.0007) expression in RAW cell. SAP deficiency also significantly decreased the expression of M1 and M2 macrophage markers and inflammatory cytokines in oxLDL-induced macrophages. SAP deficiency mitigated foam cell formation and atherosclerotic development in apoE(-/-) mice, due to reduction in macrophages recruitment, polarization and pro-inflammatory cytokines and inhibition the CD36/FcγR-dependent signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

TAB2 Is Essential for Prevention of Apoptosis in Fetal Liver but Not for Interleukin-1 Signaling

PubMed Central

Sanjo, Hideki; Takeda, Kiyoshi; Tsujimura, Tohru; Ninomiya-Tsuji, Jun; Matsumoto, Kunihiro; Akira, Shizuo

2003-01-01

The proinflammatory cytokine interleukin-1 (IL-1) transmits a signal via several critical cytoplasmic proteins such as MyD88, IRAKs and TRAF6. Recently, serine/threonine kinase TAK1 and TAK1 binding protein 1 and 2 (TAB1/2) have been identified as molecules involved in IL-1-induced TRAF6-mediated activation of AP-1 and NF-κB via mitogen-activated protein (MAP) kinases and IκB kinases, respectively. However, their physiological functions remain to be clarified. To elucidate their roles in vivo, we generated TAB2-deficient mice. The TAB2 deficiency was embryonic lethal due to liver degeneration and apoptosis. This phenotype was similar to that of NF-κB p65-, IKKβ-, and NEMO/IKKγ-deficient mice. However, the IL-1-induced activation of NF-κB and MAP kinases was not impaired in TAB2-deficient embryonic fibroblasts. These findings demonstrate that TAB2 is essential for embryonic development through prevention of liver apoptosis but not for the IL-1 receptor-mediated signaling pathway. PMID:12556483

Epidermal Expression of Intercellular Adhesion Molecule 1 is Not a Primary Inducer of Cutaneous Inflammation in Transgenic Mice

NASA Astrophysics Data System (ADS)

Williams, Ifor R.; Kupper, Thomas S.

1994-10-01

Keratinocytes at sites of cutaneous inflammation have increased expression of intercellular adhesion molecule 1 (ICAM-1), a cytokine-inducible adhesion molecule which binds the leukocyte integrins LFA-1 and Mac-1. Transgenic mice were prepared in which the expression of mouse ICAM-1 was targeted to basal keratinocytes by using the human K14 keratin promoter. The level of constitutive expression attained in the transgenic mice exceeded the peak level of ICAM-1 expression induced on nontransgenic mouse keratinocytes in vitro by optimal combinations of interferon γ and tumor necrosis factor α or in vivo by proinflammatory stimuli such as phorbol 12-myristate 13-acetate. In vitro adhesion assays demonstrated that cultured transgenic keratinocytes were superior to normal keratinocytes as a substrate for the LFA-1-dependent binding of mouse T cells, confirming that the transgene-encoded ICAM-1 was expressed in a functional form. However, the high level of constitutive ICAM-1 expression achieved on keratinocytes in vivo in these transgenic mice did not result in additional recruitment of CD45^+ leukocytes into transgenic epidermis, nor did it elicit dermal inflammation. Keratinocyte ICAM-1 expression also did not potentiate contact-hypersensitivity reactions to epicutaneous application of haptens. The absence of a spontaneous phenotype in these transgenic mice was not the result of increased levels of soluble ICAM-1, since serum levels of soluble ICAM-1 were equal in transgenic mice and controls. We conclude that elevated ICAM-1 expression on keratinocytes cannot act independently to influence leukocyte trafficking and elicit cutaneous inflammation.

Overexpression of TGF-ß1 in Macrophages Reduces and Stabilizes Atherosclerotic Plaques in ApoE-Deficient Mice

PubMed Central

Orning, Carolin; Crain, Jeanine; Küpper, Ines; Wiese, Elena; Protschka, Martina; Blessing, Manfred; Lackner, Karl J.; Torzewski, Michael

2012-01-01

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE−/− mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice. PMID:22829904

Chemical peeling by SA-PEG remodels photo-damaged skin: suppressing p53 expression and normalizing keratinocyte differentiation.

PubMed

Dainichi, Teruki; Amano, Satoshi; Matsunaga, Yukiko; Iriyama, Shunsuke; Hirao, Tetsuji; Hariya, Takeshi; Hibino, Toshihiko; Katagiri, Chika; Takahashi, Motoji; Ueda, Setsuko; Furue, Masutaka

2006-02-01

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG), which specifically acts on the stratum corneum, suppresses the development of skin tumors in UVB-irradiated hairless mice. To elucidate the mechanism through which chemical peeling with SA-PEG suppresses skin tumor development, the effects of chemical peeling on photodamaged keratinocytes and cornified envelopes (CEs) were evaluated in vivo. Among UVB-irradiated hairless mice, the structural atypia and expression of p53 protein in keratinocytes induced by UVB irradiation were intensely suppressed in the SA-PEG-treated mice 28 days after the start of weekly SA-PEG treatments when compared to that in the control UVB-irradiated mice. Incomplete expression of filaggrin and loricrin in keratinocytes from the control mice was also improved in keratinocytes from the SA-PEG-treated mice. In photo-exposed human facial skin, immature CEs were replaced with mature CEs 4 weeks after treatment with SA-PEG. Restoration of photodamaged stratum corneum by treatment with SA-PEG, which may affect remodeling of the structural environment of the keratinocytes, involved the normalization of keratinocyte differentiation and suppression of skin tumor development. These results suggest that the stratum corneum plays a protective role against carcinogenesis, and provide a novel strategy for the prevention of photo-induced skin tumors.

Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1)

PubMed Central

Udasin, Ronald G.; Wen, Xia; Bircsak, Kristin M.; Aleksunes, Lauren M.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2016-01-01

Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants developed as chemical warfare agents. These electrophilic, bifunctional alkylating agents cause skin injury, including inflammation, edema, and blistering. HN2 covalently modifies macromolecules such as DNA, RNA, and proteins or is scavenged by glutathione, forming adducts that can contribute to toxicity. Multidrug resistance-associated protein 1 (Mrp1/MRP1) is a transmembrane ATPase known to efflux glutathione-conjugated electrophiles. In the present studies, we examined the effects of modulating Mrp1-mediated transport activity on the sensitivity of primary and PAM212 mouse keratinocytes to HN2. Primary keratinocytes, and to a lesser extent, PAM212 cells, express Mrp1 mRNA and protein and possess Mrp1 functional activity, as measured by calcein efflux. Sulforaphane, an activator of Nrf2, increased Mrp1 mRNA, protein, and functional activity in primary keratinocytes and PAM212 cells and decreased their sensitivity to HN2-induced growth inhibition (IC50 = 1.4 and 4.8 µM in primary keratinocytes and 1 and 13 µM in PAM212 cells, in the absence and presence of sulforaphane, respectively). The Mrp1 inhibitor, MK-571, reversed the effects of sulforaphane on HN2-induced growth inhibition in both primary keratinocytes and PAM212 cells. In primary keratinocytes from Nrf2−/− mice, sulforaphane had no impact on Mrp1 expression or activity, or on sensitivity to HN2, demonstrating that its effects depend on Nrf2. These data suggest that Mrp1-mediated efflux is important in regulating HN2-induced keratinocyte growth inhibition. Enhancing HN2 efflux from keratinocytes may represent a novel strategy for mitigating vesicant-induced cytotoxicity. PMID:26454883

TAK1 regulates NF-{Kappa}B and AP-1 activation in airway epithelial cells following RSV infection

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

Dey, Nilay; Liu Tianshuang; Garofalo, Roberto P.

2011-09-30

Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKK{beta} plays a key role in viral-induced NF-{kappa}B activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases.more » Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-{kappa}B and AP-1 nuclear translocation and DNA-binding activity, as well as NF-{kappa}B-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-{kappa}B and AP-1 activation. - Highlights: > IKK{beta} is a major kinase involved in RSV-induced NF-{kappa}B activation. > JNK regulates AP-1-dependent gene transcription in RSV infection. > TAK1 is a critical upstream signaling molecule for both pathways in infected cells.« less

Deficiency of cyclin-dependent kinase inhibitors p21{sup Cip1} and p27{sup Kip1} accelerates atherogenesis in apolipoprotein E-deficient mice

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

Akyuerek, Levent M.; Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Goeteborg, SE-405 30; Boehm, Manfred

2010-05-28

Cyclin-dependent kinase inhibitors, p21{sup Cip1} and p27{sup Kip1}, are upregulated during vascular cell proliferation and negatively regulate growth of vascular cells. We hypothesized that absence of either p21{sup Cip1} or p27{sup Kip1} in apolipoprotein E (apoE)-deficiency may increase atherosclerotic plaque formation. Compared to apoE{sup -/-} aortae, both apoE{sup -/-}/p21{sup -/-} and apoE{sup -/-}/p27{sup -/-} aortae exhibited significantly more atherosclerotic plaque following a high-cholesterol regimen. This increase was particularly observed in the abdominal aortic regions. Deficiency of p27{sup Kip1} accelerated plaque formation significantly more than p21{sup -/-} in apoE{sup -/-} mice. This increased plaque formation was in parallel with increased intima/mediamore » area ratios. Deficiency of p21{sup Cip1} and p27{sup Kip1} accelerates atherogenesis in apoE{sup -/-} mice. These findings have significant implications for our understanding of the molecular basis of atherosclerosis associated with excessive proliferation of vascular cells.« less

Wound Healing in Mac-1 Deficient Mice

DTIC Science & Technology

2017-05-01

36. Rosenkranz AR, Coxon A, Maurer M, Gurish MF, Austen KF, Friend DS, Galli SJ, Mayadas TN. Impaired mast cell development and innate immunity in Mac...genetically deficient mice. 3 INTRODUCTION Wound healing is a complex yet well-regulated process in which multiple resident cells ...recruited inflammatory cells , and stem cells interact to create an environment that supports the healing process. An optimal inflammatory response is a

Deficiency of circadian clock protein BMAL1 in mice results in a low bone mass phenotype.

PubMed

Samsa, William E; Vasanji, Amit; Midura, Ronald J; Kondratov, Roman V

2016-03-01

The circadian clock is an endogenous time keeping system that controls the physiology and behavior of many organisms. The transcription factor Brain and Muscle ARNT-like Protein 1 (BMAL1) is a component of the circadian clock and necessary for clock function. Bmal1(-/-) mice display accelerated aging and many accompanying age associated pathologies. Here, we report that mice deficient for BMAL1 have a low bone mass phenotype that is absent at birth and progressively worsens over their lifespan. Accelerated aging of these mice is associated with the formation of bony bridges occurring across the metaphysis to the epiphysis, resulting in shorter long bones. Using micro-computed tomography we show that Bmal1(-/-) mice have reductions in cortical and trabecular bone volume and other micro-structural parameters and a lower bone mineral density. Histology shows a deficiency of BMAL1 results in a reduced number of active osteoblasts and osteocytes in vivo. Isolation of bone marrow derived mesenchymal stem cells from Bmal1(-/-) mice demonstrate a reduced ability to differentiate into osteoblasts in vitro, which likely explains the observed reductions in osteoblasts and osteocytes, and may contribute to the observed osteopenia. Our data support the role of the circadian clock in the regulation of bone homeostasis and shows that BMAL1 deficiency results in a low bone mass phenotype. Copyright © 2016 Elsevier Inc. All rights reserved.

Deficiency of Circadian Clock Protein BMAL1 in Mice Results in a Low Bone Mass Phenotype

PubMed Central

Samsa, William E.; Vasanji, Amit; Midura, Ronald J.; Kondratov, Roman V.

2016-01-01

The circadian clock is an endogenous time keeping system that controls the physiology and behavior of many organisms. The transcription factor Brain and Muscle ARNT-like Protein 1 (BMAL1) is a component of the circadian clock and necessary for clock function. Bmal1−/− mice display accelerated aging and many accompanying age associated pathologies. Here, we report that mice deficient for BMAL1 have a low bone mass phenotype that is absent at birth and progressively worsens over their lifespan. Accelerated aging of these mice is associated with the formation of bony bridges occurring across the metaphysis to the epiphysis, resulting in shorter long bones. Using micro-computed tomography we show that Bmal1−/− mice have reductions in cortical and trabecular bone volume and other micro-structural parameters and a lower bone mineral density. Histology shows a deficiency of BMAL1 results in a reduced number of active osteoblasts and osteocytes in vivo. Isolation of bone marrow derived mesenchymal stem cells from Bmal1−/− mice demonstrate a reduced ability to differentiate into osteoblasts in vitro, which likely explains the observed reductions in osteoblasts and osteocytes, and may contribute to the observed osteopenia. Our data support the role of the circadian clock in the regulation of bone homeostasis and shows that BMAL1 deficiency results in a low bone mass phenotype. PMID:26789548

Calpain-2 Compensation Promotes Angiotensin II-Induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice

PubMed Central

Subramanian, Venkateswaran; Moorleghen, Jessica J.; Balakrishnan, Anju; Howatt, Deborah A.; Chishti, Athar H.; Uchida, Haruhito A.

2013-01-01

Background and Objective Recently, we demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. Methodology/Results To investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr −/− mice that were either calpain-1 +/+ or −/− were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and −/− mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an

Heightened aggressive behavior in mice deficient in aldo-keto reductase 1a (Akr1a).

PubMed

Homma, Takujiro; Akihara, Ryusuke; Okano, Satoshi; Shichiri, Mototada; Yoshida, Yasukazu; Yamada, Ken-Ichi; Miyata, Satoshi; Nakajima, Osamu; Fujii, Junichi

2017-02-15

Aldehyde reductase (Akr1a) is involved in the synthesis of ascorbic acid (AsA) which may play a role in social behavior. In the current study, we performed analyses on Akr1a-deficient (Akr1a -/- ) mice that synthesize about 10% as much AsA as wild-type mice from the viewpoint of intermale aggression. The use of the resident-intruder test revealed that the Akr1a -/- mice exhibited more aggressive phenotypes than wild-type control mice. Unexpectedly, however, the oral administration of additional AsA failed to reduce the aggressive behavior of Akr1a -/- mice, suggesting that the heightened aggression was independent of AsA biosynthesis. The findings also show that the plasma levels of corticosterone, but not serotonin and testosterone, were increased in the absence of Akr1a in mice, suggesting that the mice were highly stressed. These results suggest that Akr1a might be involved in the metabolism of steroids and other carbonyl-containing compounds and, hence, the absence of Akr1a results in heightened aggression via a malfunction in a metabolic pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Onset and organ specificity of Tk2 deficiency depends on Tk1 down-regulation and transcriptional compensation.

PubMed

Dorado, Beatriz; Area, Estela; Akman, Hasan O; Hirano, Michio

2011-01-01

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2-/-). Although normal until postnatal day 8, Tk2-/- mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2-/- mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2-/- heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2-/- heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency.

MEK and TAK1 Regulate Apoptosis in Colon Cancer Cells with KRAS-Dependent Activation of Proinflammatory Signaling.

PubMed

McNew, Kelsey L; Whipple, William J; Mehta, Anita K; Grant, Trevor J; Ray, Leah; Kenny, Connor; Singh, Anurag

2016-12-01

MEK inhibitors have limited efficacy in treating RAS-RAF-MEK pathway-dependent cancers due to feedback pathway compensation and dose-limiting toxicities. Combining MEK inhibitors with other targeted agents may enhance efficacy. Here, codependencies of MEK, TAK1, and KRAS in colon cancer were investigated. Combined inhibition of MEK and TAK1 potentiates apoptosis in KRAS-dependent cells. Pharmacologic studies and cell-cycle analyses on a large panel of colon cancer cell lines demonstrate that MEK/TAK1 inhibition induces cell death, as assessed by sub-G 1 accumulation, in a distinct subset of cell lines. Furthermore, TAK1 inhibition causes G 2 -M cell-cycle blockade and polyploidy in many of the cell lines. MEK plus TAK1 inhibition causes reduced G 2 -M/polyploid cell numbers and additive cytotoxic effects in KRAS/TAK1-dependent cell lines as well as a subset of BRAF-mutant cells. Mechanistically, sensitivity to MEK/TAK1 inhibition can be conferred by KRAS and BMP receptor activation, which promote expression of NF-κB-dependent proinflammatory cytokines, driving tumor cell survival and proliferation. MEK/TAK1 inhibition causes reduced mTOR, Wnt, and NF-κB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NF-κB transcriptional signature was derived that stratifies primary tumors into three major subtypes: Wnt-high/NF-κB-low, Wnt-low/NF-κB-high and Wnt-high/NF-κB-high, designated W, N, and WN, respectively. These subtypes have distinct characteristics, including enrichment for BRAF mutations with serrated carcinoma histology in the N subtype. Both N and WN subtypes bear molecular hallmarks of MEK and TAK1 dependency seen in cell lines. Therefore, N and WN subtype signatures could be utilized to identify tumors that are most sensitive to anti-MEK/TAK1 therapeutics. This study describes a potential therapeutic strategy for a subset of colon cancers that are dependent on oncogenic KRAS signaling pathways, which are currently difficult to

Myostatin induces interstitial fibrosis in the heart via TAK1 and p38.

PubMed

Biesemann, Nadine; Mendler, Luca; Kostin, Sawa; Wietelmann, Astrid; Borchardt, Thilo; Braun, Thomas

2015-09-01

Myostatin, a member of the TGF-β superfamily of secreted growth factors, is a negative regulator of skeletal muscle growth. In the heart, it is expressed at lower levels compared to skeletal muscle but up-regulated under disease conditions. Cre recombinase-mediated inactivation of myostatin in adult cardiomyocytes leads to heart failure and increased mortality but cardiac function of surviving mice is restored after several weeks probably due to compensatory expression in non-cardiomyocytes. To study long-term effects of increased myostatin expression in the heart and to analyze the putative crosstalk between cardiomyocytes and fibroblasts, we overexpressed myostatin in cardiomyocytes. Increased expression of myostatin in heart muscle cells caused interstitial fibrosis via activation of the TAK-1-MKK3/6-p38 signaling pathway, compromising cardiac function in older mice. Our results uncover a novel role of myostatin in the heart and highlight the necessity for tight regulation of myostatin to maintain normal heart function.

Interleukin 1α-Deficient Mice Have an Altered Gut Microbiota Leading to Protection from Dextran Sodium Sulfate-Induced Colitis.

PubMed

Nunberg, Moran; Werbner, Nir; Neuman, Hadar; Bersudsky, Marina; Braiman, Alex; Ben-Shoshan, Moshe; Ben Izhak, Meirav; Louzoun, Yoram; Apte, Ron N; Voronov, Elena; Koren, Omry

2018-01-01

Inflammatory bowel diseases (IBD) are a group of chronic inflammatory disorders of the intestine, with as-yet-unclear etiologies, affecting over a million people in the United States alone. With the emergence of microbiome research, numerous studies have shown a connection between shifts in the gut microbiota composition (dysbiosis) and patterns of IBD development. In a previous study, we showed that interleukin 1α (IL-1α) deficiency in IL-1α knockout (KO) mice results in moderate dextran sodium sulfate (DSS)-induced colitis compared to that of wild-type (WT) mice, characterized by reduced inflammation and complete healing, as shown by parameters of weight loss, disease activity index (DAI) score, histology, and cytokine expression. In this study, we tested whether the protective effects of IL-1α deficiency on DSS-induced colitis correlate with changes in the gut microbiota and whether manipulation of the microbiota by cohousing can alter patterns of colon inflammation. We analyzed the gut microbiota composition in both control (WT) and IL-1α KO mice under steady-state homeostasis, during acute DSS-induced colitis, and after recovery using 16S rRNA next-generation sequencing. Additionally, we performed cohousing of both mouse groups and tested the effects on the microbiota and clinical outcomes. We demonstrate that host-derived IL-1α has a clear influence on gut microbiota composition, as well as on severity of DSS-induced acute colon inflammation. Cohousing both successfully changed the gut microbiota composition and increased the disease severity of IL-1α-deficient mice to levels similar to those of WT mice. This study shows a strong and novel correlation between IL-1α expression, microbiota composition, and clinical outcomes of DSS-induced colitis. IMPORTANCE Here, we show a connection between IL-1α expression, microbiota composition, and clinical outcomes of DSS-induced colitis. Specifically, we show that the mild colitis symptoms seen in IL-1α-deficient

Interleukin 1α-Deficient Mice Have an Altered Gut Microbiota Leading to Protection from Dextran Sodium Sulfate-Induced Colitis

PubMed Central

2018-01-01

ABSTRACT Inflammatory bowel diseases (IBD) are a group of chronic inflammatory disorders of the intestine, with as-yet-unclear etiologies, affecting over a million people in the United States alone. With the emergence of microbiome research, numerous studies have shown a connection between shifts in the gut microbiota composition (dysbiosis) and patterns of IBD development. In a previous study, we showed that interleukin 1α (IL-1α) deficiency in IL-1α knockout (KO) mice results in moderate dextran sodium sulfate (DSS)-induced colitis compared to that of wild-type (WT) mice, characterized by reduced inflammation and complete healing, as shown by parameters of weight loss, disease activity index (DAI) score, histology, and cytokine expression. In this study, we tested whether the protective effects of IL-1α deficiency on DSS-induced colitis correlate with changes in the gut microbiota and whether manipulation of the microbiota by cohousing can alter patterns of colon inflammation. We analyzed the gut microbiota composition in both control (WT) and IL-1α KO mice under steady-state homeostasis, during acute DSS-induced colitis, and after recovery using 16S rRNA next-generation sequencing. Additionally, we performed cohousing of both mouse groups and tested the effects on the microbiota and clinical outcomes. We demonstrate that host-derived IL-1α has a clear influence on gut microbiota composition, as well as on severity of DSS-induced acute colon inflammation. Cohousing both successfully changed the gut microbiota composition and increased the disease severity of IL-1α-deficient mice to levels similar to those of WT mice. This study shows a strong and novel correlation between IL-1α expression, microbiota composition, and clinical outcomes of DSS-induced colitis. IMPORTANCE Here, we show a connection between IL-1α expression, microbiota composition, and clinical outcomes of DSS-induced colitis. Specifically, we show that the mild colitis symptoms seen in IL-1

Medical Castration Using the Investigational Oral GnRH Antagonist TAK-385 (Relugolix): Phase 1 Study in Healthy Males

PubMed Central

Shi, Hongliang; Faessel, Hélène M.; Saad, Fred

2015-01-01

Context: TAK-385 is a highly selective, oral, nonpeptide GnRH antagonist being investigated as a possible prostate cancer treatment. Objective: The objectives were to evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics of TAK-385 on LH and testosterone. Design, Setting, and Participants: This was a three-part, randomized, double-blind, placebo-controlled, phase 1 dose-escalation study in 176 healthy male UK volunteers. Interventions: Part 1, single doses of TAK-385 (0 [placebo], 80, 120, 180, or 360 mg). Part 2, 14-day TAK-385 (0, 20, 40, 80, or 180 mg) daily. Part 3, 28-day TAK-385 (40 [with loading dose], 60, 80, or 160 mg) or placebo daily. Parts 2 and 3 included men aged 40–75 years. Main Outcome Measures: Main outcome measures included plasma concentrations of TAK-385, LH, and testosterone. Results: Oral TAK-385 was readily absorbed, and steady state was reached in ≤14 days. Food reduced TAK-385 systemic exposure by 47–52%. Mean serum testosterone levels declined ≤6 hours after TAK-385 administration. Loading doses up to 360 mg on day 1 or 360 mg on day 1 followed by 240 mg on day 2 reduced the time to achieve castrate testosterone levels from ≥7 to <3 days. TAK-385 doses ≥80 mg/d achieved sustained medical castration and trough TAK-385 concentrations >4 ng/mL. After discontinuation of TAK-385 on day 28, testosterone levels normalized in most subjects in ≤ 28 days. Common adverse events included bradycardia, headache, and hot flush (all grade ≤2). Conclusions: Oral TAK-385 (40–180 mg/d) was well tolerated and effectively lowered testosterone in healthy men. Planned phase 2 doses in men with hormone-sensitive prostate cancer are 80 and 120 mg/d. PMID:26502357

Toxoplasma gondii infection inhibits Th17-mediated spontaneous development of arthritis in interleukin-1 receptor antagonist-deficient mice.

PubMed

Washino, Takuya; Moroda, Masataka; Iwakura, Yoichiro; Aosai, Fumie

2012-04-01

Interleukin 1 receptor antagonist (IL-1Ra)-deficient BALB/c mice develop spontaneous arthritis resembling human rheumatoid arthritis. We herein report that infection with Toxoplasma gondii, an intracellular protozoan, is capable of ameliorating the spontaneous development of arthritis in IL-1Ra-deficient mice. The onset of arthritis development was delayed and the severity score of arthritis was significantly suppressed in T. gondii-infected mice. Expression of IL-12p40 mRNA from CD11c(+) cells of mesenteric lymph nodes (mLN) and spleen markedly increased at 1 week after peroral infection. While CD11c(+) cells also produced IL-10, IL-1β, and IL-6, CD4(+) T cells from T. gondii-infected mice expressed significantly high levels of T-bet and gamma interferon (IFN-γ) mRNA in both mLN and spleen. Levels of GATA-3/IL-4 mRNA or RORγt/IL-17 mRNA decreased in the infected mice, indicating Th1 cell polarization and the reduction of Th2 and Th17 cell polarization. The severity of arthritis was related to Th1 cell polarization accompanied by Th17 cell reduction, demonstrating the protective role of the T. gondii-derived Th1 response against Th17 cell-mediated arthritis in IL-1Ra-deficient mice.

Shrimp TAB1 interacts with TAK1 and p38 and activates the host innate immune response to bacterial infection.

PubMed

Wang, Sheng; Li, Mengqiao; Yin, Bin; Li, Haoyang; Xiao, Bang; Lǚ, Kai; Huang, Zhijian; Li, Sedong; He, Jianguo; Li, Chaozheng

2017-08-01

Mammalian TAB1 has been previously identified as transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) binding protein, which functions as the activator of TAK1 and p38. This report, for the first time, identified and characterized the homolog of TAB1 in shrimp, to be specific, the homolog gene from Litopenaeus vannamei, containing a 1560-bp open reading frame (ORF) that encoded a putative protein of 519 amino acids with the conserved PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain in N-terminal and a TAK1 binding motif in C-terminus, has been cloned and named LvTAB1. LvTAB1 was most abundant in gills and its expression could respond significantly to a series of stimuli, including LPS, Vibrio parahemolyticus and Staphylococcus aureus. Moreover, Co-immunoprecipitation (Co-IP) experiments showed that LvTAB1 could combine with LvTAK1 as well as Lvp38, two members of IMD-NF-κB/MAPK pathway, which meant LvTAB1 could have a role in regulating the activities of these kinases. Over-expression of LvTAB1 in drosophila S2 cells could improve the transcriptional levels of antimicrobial peptide genes (AMPs) such as Diptericin (Dpt), the hallmark of drosophila NF-κB activated genes, indicating its activation effect on NF-κB pathway. Furthermore, suppression of LvTAB1 expression in vivo by RNA-interference increased the sensibility of shrimps to V. parahaemolyticus infection, implying its protective role against bacterial infection. In conclusion, these results provide some insight into the function of LvTAB1 during bacterial infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hematopoietic Sphingosine 1-Phosphate Lyase Deficiency Decreases Atherosclerotic Lesion Development in LDL-Receptor Deficient Mice

PubMed Central

Bot, Martine; Van Veldhoven, Paul P.; de Jager, Saskia C. A.; Johnson, Jason; Nijstad, Niels; Van Santbrink, Peter J.; Westra, Marijke M.; Van Der Hoeven, Gerd; Gijbels, Marion J.; Müller-Tidow, Carsten; Varga, Georg; Tietge, Uwe J. F.; Kuiper, Johan; Van Berkel, Theo J. C.; Nofer, Jerzy-Roch

2013-01-01

Aims Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1−/−) deficiency on leukocyte subsets relevant to atherosclerosis. Methods and Results LDL receptor deficient mice that were transplanted with Sgpl1−/− bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1−/− chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. Conclusions Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution. PMID:23700419

2'-5' Oligoadenylate synthetase-like 1 (OASL1) deficiency in mice promotes an effective anti-tumor immune response by enhancing the production of type I interferons.

PubMed

Sim, Chan Kyu; Cho, Yeon Sook; Kim, Byung Soo; Baek, In-Jeoung; Kim, Young-Joon; Lee, Myeong Sup

2016-06-01

Type I interferon (IFN-I) plays a critical role in antiviral and antitumor defense. In our previous studies, we showed that IFN-I-inducible 2'-5' oligoadenylate synthetase-like 1 (OASL1) negatively regulates IFN-I production upon viral infection by specifically inhibiting translation of the IFN-I-regulating master transcription factor, interferon regulatory factor 7 (IRF7). In this study, we investigated whether OASL1 plays a negative role in the anti-tumor immune response by using OASL1-deficient (Oasl1 (-/-)) mice and transplantable syngeneic tumor cell models. We found that Oasl1 (-/-) mice demonstrate enhanced resistance to lung metastatic tumors and subcutaneously implanted tumors compared to wild-type (WT) mice. Additionally, we found that cytotoxic effector cells such as CD8(+) T cells (including tumor antigen-specific CD8(+) T cells) and NK cells as well as CD8α(+) DCs (the major antigen cross-presenting cells) were much more frequent (>fivefold) in the Oasl1 (-/-) mouse tumors. Furthermore, the cytotoxic effector cells in Oasl1 (-/-) mouse tumors seemed to be more functionally active. However, the proportion of immunosuppressive myeloid-derived suppressor cells within hematopoietic cells and of regulatory T cells within CD4(+) T cells in Oasl1 (-/-) mouse tumors did not differ significantly from that of WT mice. Tumor-challenged Oasl1 (-/-) mice expressed increased levels of IFN-I and IRF7 protein in the growing tumor, indicating that the enhanced antitumor immune response observed in Oasl1 (-/-) mice was caused by higher IFN-I production in Oasl1 (-/-) mice. Collectively, these results show that OASL1 deficiency promotes the antitumor immune response, and thus, OASL1 could be a good therapeutic target for treating tumors.

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

PubMed Central

Tron, Victor A.; Trotter, Martin J.; Tang, Liren; Krajewska, Maryla; Reed, John C.; Ho, Vincent C.; Li, Gang

1998-01-01

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and

Phospholipase Cε deficiency delays the early stage of cutaneous wound healing and attenuates scar formation in mice.

PubMed

Zhu, Xiaolong; Sun, Yue; Mu, Xin; Guo, Pan; Gao, Fei; Zhang, Jing; Zhu, Yunjuan; Zhang, Xianzhi; Chen, Lingling; Ning, Zhiwei; Bai, Yunfeng; Ren, Jiling; Man, Maoqiang; Liu, Peimei; Hu, Lizhi

2017-02-26

This study aimed to investigate the role of phospholipase Cε (PLCε) in the skin wound healing process. PLCε, an effect factor of Ras/Rap small G protein, plays a crucial role in skin inflammation by regulating inflammatory cytokines. Inflammatory responses are closely associated with wound healing. Full-thickness skin wounds were made in the PLCε knockout (KO) and wild-type (WT) mice, and the healing process was analyzed. The macroscopic wound closure rate declined in the PLCε KO mice on days 3, 4, and 5 after wounding, following the decreased expression of interleukin (IL)-6, chemokine (C-X-C motif) ligand (Cxcl)-1, Cxcl-2, and chemokine (C-C motif) ligand (Ccl) 20. The proliferation rate of epidermal keratinocytes was not affected by PLCε, but silencing of PLCε resulted in the delayed migration of keratinocytes. Moreover, the scars were found to be much smaller in the PLCε KO mice than in the WT mice. The mRNA expression of Ccl20, collagen (Col) 6a1, and Col17a1 decreased in the PLCε KO mice. These results were in agreement with a previous hypothesis that PLCε might delay the early stage of cutaneous wound healing by inhibiting the migration of keratinocytes, and decrease the expression of Col6a1, Col17a1, and Ccl20 by inhibiting the inflammatory response to reduce scar formation. This study shed light on a novel role of PLCε in wound healing and provided new therapeutic approaches to target PLCε for diminishing scar formation after injury. Copyright © 2017 Elsevier Inc. All rights reserved.

MicroRNA Let-7b inhibits keratinocyte migration in cutaneous wound healing by targeting IGF2BP2.

PubMed

Wu, Yan; Zhong, Julia Li; Hou, Ning; Sun, Yaolan; Ma, Benting; Nisar, Muhammad Farrukh; Teng, Yan; Tan, Zhaoli; Chen, Keping; Wang, Youliang; Yang, Xiao

2017-02-01

Wound healing is a complex process which involves proliferation and migration of keratinocyte for closure of epidermal injuries. A member of microRNA family, let-7b, has been expressed in mammalian skin, but its exact role in keratinocyte migration is still not in knowledge. Here, we showed that let-7b regulates keratinocyte migration by targeting the insulin-like growth factor IGF2BP2. Overexpression of let-7b led to reduced HaCaT cell migration, while knockdown of let-7b resulted in enhanced migration. Furthermore, let-7b was decreased during wound healing in wild-type mice, which led us to construct the transgenic mice with overexpression of let-7b in skin. The re-epithelialization of epidermis of let-7b transgenic mice was reduced during wound healing. Using bioinformatics prediction software and a reporter gene assay, we found that IGF2BP2 was a target of let-7b, which contributes to keratinocyte migration. Introduction of an expression vector of IGF2BP2 also rescued let-7b-induced migration deficiency, which confirms that IGF2BP2 is an important target for let-7b regulation. Our findings suggest that let-7b significantly delayed the re-epithelialization possibly due to reduction of keratinocyte migration and restraints IGF2BP2 during skin wound healing. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Gab3-deficient mice exhibit normal development and hematopoiesis and are immunocompetent.

PubMed

Seiffert, Martina; Custodio, Joseph M; Wolf, Ingrid; Harkey, Michael; Liu, Yan; Blattman, Joseph N; Greenberg, Philip D; Rohrschneider, Larry R

2003-04-01

Gab proteins are intracellular scaffolding and docking molecules involved in signaling pathways mediated by various growth factor, cytokine, or antigen receptors. Gab3 has been shown to act downstream of the macrophage colony-stimulating factor receptor, c-Fms, and to be important for macrophage differentiation. To analyze the physiological role of Gab3, we used homologous recombination to generate mice deficient in Gab3. Gab3(-/-) mice develop normally, are visually indistinguishable from their wild-type littermates, and are healthy and fertile. To obtain a detailed expression pattern of Gab3, we generated Gab3-specific monoclonal antibodies. Immunoblotting revealed a predominant expression of Gab3 in lymphocytes and bone marrow-derived macrophages. However, detailed analysis demonstrated that hematopoiesis in mice lacking Gab3 is not impaired and that macrophages develop in normal numbers and exhibit normal function. The lack of Gab3 expression during macrophage differentiation is not compensated for by increased levels of Gab1 or Gab2 mRNA. Furthermore, Gab3-deficient mice have no major immune deficiency in T- and B-lymphocyte responses to protein antigens or during viral infection. In addition, allergic responses in Gab3-deficient mice appeared to be normal. Together, these data demonstrate that loss of Gab3 does not result in detectable defects in normal mouse development, hematopoiesis, or immune system function.

Onset and organ specificity of Tk2 deficiency depends on Tk1 down-regulation and transcriptional compensation

PubMed Central

Dorado, Beatriz; Area, Estela; Akman, Hasan O.; Hirano, Michio

2011-01-01

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2−/−). Although normal until postnatal day 8, Tk2−/− mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2−/− mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2−/− heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2−/− heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency. PMID:20940150

Loss of Par-1a/MARK3/C-TAK1 kinase leads to reduced adiposity, resistance to hepatic steatosis, and defective gluconeogenesis.

PubMed

Lennerz, Jochen K; Hurov, Jonathan B; White, Lynn S; Lewandowski, Katherine T; Prior, Julie L; Planer, G James; Gereau, Robert W; Piwnica-Worms, David; Schmidt, Robert E; Piwnica-Worms, Helen

2010-11-01

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a(-/-) but not in control or Par-1b(-/-) mice. The intercrossing of Par-1a(-/-) with Par-1b(-/-) mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a(-/-) mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b(-/-) mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.

Monocyte chemotactic protein-1 deficiency attenuates and high-fat diet exacerbates bone loss in mice with Lewis lung carcinoma.

PubMed

Yan, Lin; Nielsen, Forrest H; Sundaram, Sneha; Cao, Jay

2017-04-04

Bone loss occurs in obesity and cancer-associated complications including wasting. This study determined whether a high-fat diet and a deficiency in monocyte chemotactic protein-1 (MCP-1) altered bone structural defects in male C57BL/6 mice with Lewis lung carcinoma (LLC) metastases in lungs. Compared to non-tumor-bearing mice, LLC reduced bone volume fraction, connectivity density, trabecular number, trabecular thickness and bone mineral density and increased trabecular separation in femurs. Similar changes occurred in vertebrae. The high-fat diet compared to the AIN93G diet exacerbated LLC-induced detrimental structural changes; the exacerbation was greater in femurs than in vertebrae. Mice deficient in MCP-1 compared to wild-type mice exhibited increases in bone volume fraction, connectivity density, trabecular number and decreases in trabecular separation in both femurs and vertebrae, and increases in trabecular thickness and bone mineral density and a decrease in structure model index in vertebrae. Lewis lung carcinoma significantly decreased osteocalcin but increased tartrate-resistant acid phosphatase 5b (TRAP 5b) in plasma. In LLC-bearing mice, the high-fat diet increased and MCP-1 deficiency decreased plasma TRAP 5b; neither the high-fat diet nor MCP-1 deficiency resulted in significant changes in plasma concentration of osteocalcin. In conclusion, pulmonary metastasis of LLC is accompanied by detrimental bone structural changes; MCP-1 deficiency attenuates and high-fat diet exacerbates the metastasis-associated bone wasting.

Microhemorrhage is an Early Event in the Pulmonary Fibrotic Disease of PECAM-1 Deficient FVB/n Mice

PubMed Central

Young, Lena C.; Woods, Steven J.; Groshong, Steven D.; Basaraba, Randall J.; Gilchrist, John M.; Higgins, David M.; Gonzalez-Juarrero, Mercedes; Bass, Todd A.; Muller, William A.; Schenkel, Alan R.

2014-01-01

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment. PMID:24972347

NADPH Oxidase-1 Plays a Key Role in Keratinocyte Responses to UV Radiation and UVB-Induced Skin Carcinogenesis.

PubMed

Raad, Houssam; Serrano-Sanchez, Martin; Harfouche, Ghida; Mahfouf, Walid; Bortolotto, Doriane; Bergeron, Vanessa; Kasraian, Zeinab; Dousset, Lea; Hosseini, Mohsen; Taieb, Alain; Rezvani, Hamid Reza

2017-06-01

The nicotinamide adenine dinucleotide phosphate oxidase (NOX) family enzymes are involved in several physiological functions. However, their roles in keratinocyte responses to UV radiation have not been clearly elucidated. This study shows that, among other NOX family members, UVB irradiation results in a biphasic activation of NOX1 that plays a critical role in defining keratinocyte fate through the modulation of the DNA damage response network. Indeed, suppression of both bursts of UVB-induced NOX1 activation by using a specific peptide inhibitor of NOX1 (InhNOX1) is associated with increased nucleotide excision repair efficiency and reduction of apoptosis, which is finally translated into decreased photocarcinogenesis. On the contrary, when only the second peak of UVB-induced NOX1 activation is blocked, both nucleotide excision repair efficiency and apoptosis are decreased. Our results show that inhibition of NOX1 activation could be a promising target for the prevention and treatment of UVB-induced skin cancer in nucleotide excision repair-proficient and -deficient patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Models of tibial fracture healing in normal and Nf1-deficient mice.

PubMed

Schindeler, Aaron; Morse, Alyson; Harry, Lorraine; Godfrey, Craig; Mikulec, Kathy; McDonald, Michelle; Gasser, Jürg A; Little, David G

2008-08-01

Delayed union and nonunion are common complications associated with tibial fractures, particularly in the distal tibia. Existing mouse tibial fracture models are typically closed and middiaphyseal, and thus poorly recapitulate the prevailing conditions following surgery on a human open distal tibial fracture. This report describes our development of two open tibial fracture models in the mouse, where the bone is broken either in the tibial midshaft (mid-diaphysis) or in the distal tibia. Fractures in the distal tibial model showed delayed repair compared to fractures in the tibial midshaft. These tibial fracture models were applied to both wild-type and Nf1-deficient (Nf1+/-) mice. Bone repair has been reported to be exceptionally problematic in human NF1 patients, and these patients can also spontaneously develop tibial nonunions (known as congenital pseudarthrosis of the tibia), which are recalcitrant to even vigorous intervention. pQCT analysis confirmed no fundamental differences in cortical or cancellous bone in Nf1-deficient mouse tibiae compared to wild-type mice. Although no difference in bone healing was seen in the tibial midshaft fracture model, the healing of distal tibial fractures was found to be impaired in Nf1+/- mice. The histological features associated with nonunited Nf1+/- fractures were variable, but included delayed cartilage removal, disproportionate fibrous invasion, insufficient new bone anabolism, and excessive catabolism. These findings imply that the pathology of tibial pseudarthrosis in human NF1 is complex and likely to be multifactorial.

Haploinsufficiency of E-selectin ligand-1 is Associated with Reduced Atherosclerotic Plaque Macrophage Content while Complete Deficiency Leads to Early Embryonic Lethality in Mice

PubMed Central

Luo, Wei; Wang, Hui; Guo, Chiao; Wang, Jintao; Kwak, Jeffrey; Bahrou, Kristina L; Eitzman, Daniel T.

2012-01-01

E-selectin-1 (ESL-1), also known as golgi complex-localized glycoprotein-1 (GLG1), homocysteine-rich fibroblast growth factor receptor (CGR-1), and latent transforming growth factor-β complex protein 1 (LTCP-1), is a multifunctional protein with widespread tissue distribution. To determine the functional consequences of ESL-1 deficiency, mice were generated carrying an ESL-1 gene trap. After backcrossing to C57BL6/J for 6 generations, mice heterozygous for the gene trap (ESL-1+/-) were intercrossed to produce ESL-1-/- mice, however ESL-1-/- mice were not viable, even at embryonic day E10.5. To determine the effect of heterozygous ESL-1 deficiency on atherosclerosis, apolipoprotein E deficient (ApoE-/-), ESL-1+/- mice were generated and fed western diet. Compared to ApoE-/-, ESL-1++ mice, atherosclerotic lesions from ApoE-/-, ESL-1+/- contained more collagen and fewer macrophages, suggesting increased plaque stability. In conclusion, heterozygous deficiency of ESL-1 is associated with features of increased atherosclerotic plaque stability while complete deficiency of ESL-1 leads to embryonic lethality. PMID:22939356

Deficiency of ABCA1 and ABCG1 in Macrophages Increases Inflammation and Accelerates Atherosclerosis in Mice

PubMed Central

Westerterp, Marit; Murphy, Andrew J.; Wang, Mi; Pagler, Tamara A.; Vengrenyuk, Yuliya; Kappus, Mojdeh S.; Gorman, Darren J.; Nagareddy, Prabhakara R.; Zhu, Xuewei; Abramowicz, Sandra; Parks, John S.; Welch, Carrie; Fisher, Edward A.; Wang, Nan; Yvan-Charvet, Laurent; Tall, Alan R.

2013-01-01

Rationale Plasma HDL levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is due to the ability of HDL to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. Objective To assess the role of macrophage cholesterol efflux pathways in atherogenesis. Methods and Results We developed MAC-ABCDKO mice with efficient deletion of the ATP Binding Cassette Transporters A1 and G1 (ABCA1 and ABCG1) in macrophages but not in hematopoietic stem or progenitor populations. MAC-ABCDKO bone marrow (BM) was transplanted into Ldlr-/- recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared to controls. On the Western type diet (WTD), MAC-ABCDKO BM transplanted Ldlr-/- mice had disproportionate atherosclerosis, considering they also had lower VLDL/LDL cholesterol levels than controls. ABCA1/G1 deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, WTD-fed MAC-ABCDKO BM transplanted Ldlr-/- mice displayed monocytosis and neutrophilia in the absence of HSPC proliferation. Mechanistic studies revealed increased expression of M-CSF and G-CSF in splenic macrophage foam cells, driving BM monocyte and neutrophil production. Conclusion These studies 1) show that macrophage deficiency of ABCA1/G1 is pro-atherogenic likely by promoting plaque inflammation and 2) uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways. PMID:23572498

Environmental factors regulate Paneth cell phenotype and host susceptibility to intestinal inflammation in Irgm1-deficient mice.

PubMed

Rogala, Allison R; Schoenborn, Alexi A; Fee, Brian E; Cantillana, Viviana A; Joyce, Maria J; Gharaibeh, Raad Z; Roy, Sayanty; Fodor, Anthony A; Sartor, R Balfour; Taylor, Gregory A; Gulati, Ajay S

2018-02-07

Crohn's disease (CD) represents a chronic inflammatory disorder of the intestinal tract. Several susceptibility genes have been linked to CD, though their precise role in the pathogenesis of this disorder remains unclear. Immunity-related GTPase M ( IRGM ) is an established risk allele in CD. We have shown previously that conventionally raised (CV) mice lacking the IRGM ortholog, Irgm1 exhibit abnormal Paneth cells (PCs) and increased susceptibility to intestinal injury. In the present study, we sought to utilize this model system to determine if environmental conditions impact these phenotypes, as is thought to be the case in human CD. To accomplish this, wild-type and Irgm1 -/- mice were rederived into specific pathogen-free (SPF) and germ-free (GF) conditions. We next assessed how these differential housing environments influenced intestinal injury patterns, and epithelial cell morphology and function in wild-type and Irgm1 -/- mice. Remarkably, in contrast to CV mice, SPF Irgm1 -/- mice showed only a slight increase in susceptibility to dextran sodium sulfate-induced inflammation. SPF Irgm1 -/- mice also displayed minimal abnormalities in PC number and morphology, and in antimicrobial peptide expression. Goblet cell numbers and epithelial proliferation were also unaffected by Irgm1 in SPF conditions. No microbial differences were observed between wild-type and Irgm1 -/- mice, but gut bacterial communities differed profoundly between CV and SPF mice. Specifically, Helicobacter sequences were significantly increased in CV mice; however, inoculating SPF Irgm1 -/- mice with Helicobacter hepaticus was not sufficient to transmit a pro-inflammatory phenotype. In summary, our findings suggest the impact of Irgm1-deficiency on susceptibility to intestinal inflammation and epithelial function is critically dependent on environmental influences. This work establishes the importance of Irgm1 -/- mice as a model to elucidate host-environment interactions that regulate

Specific TRPC6 Channel Activation, a Novel Approach to Stimulate Keratinocyte Differentiation*S⃞

PubMed Central

Müller, Margarethe; Essin, Kirill; Hill, Kerstin; Beschmann, Heike; Rubant, Simone; Schempp, Christoph M.; Gollasch, Maik; Boehncke, W. Henning; Harteneck, Christian; Müller, Walter E.; Leuner, Kristina

2008-01-01

The protective epithelial barrier in our skin undergoes constant regulation, whereby the balance between differentiation and proliferation of keratinocytes plays a major role. Impaired keratinocyte differentiation and proliferation are key elements in the pathophysiology of several important dermatological diseases, including atopic dermatitis and psoriasis. Ca2+ influx plays an essential role in this process presumably mediated by different transient receptor potential (TRP) channels. However, investigating their individual role was hampered by the lack of specific stimulators or inhibitors. Because we have recently identified hyperforin as a specific TRPC6 activator, we investigated the contribution of TRPC6 to keratinocyte differentiation and proliferation. Like the endogenous differentiation stimulus high extracellular Ca2+ concentration ([Ca2+]o), hyperforin triggers differentiation in HaCaT cells and in primary cultures of human keratinocytes by inducing Ca2+ influx via TRPC6 channels and additional inhibition of proliferation. Knocking down TRPC6 channels prevents the induction of Ca2+- and hyperforin-induced differentiation. Importantly, TRPC6 activation is sufficient to induce keratinocyte differentiation similar to the physiological stimulus [Ca2+]o. Therefore, TRPC6 activation by hyperforin may represent a new innovative therapeutic strategy in skin disorders characterized by altered keratinocyte differentiation. PMID:18818211

Allelic Variation of Ets1 Does Not Contribute to NK and NKT Cell Deficiencies in Type 1 Diabetes Susceptible NOD Mice

PubMed Central

Jordan, Margaret A.; Poulton, Lynn D.; Fletcher, Julie M.; Baxter, Alan G.

2009-01-01

The NOD mouse is a well characterized model of type 1 diabetes that shares several of the characteristics of Ets1-deficient targeted mutant mice, viz: defects in TCR allelic exclusion, susceptibility to a lupus like disease characterized by IgM and IgG autoantibodies and immune complex-mediated glomerulonephritis, and deficiencies of NK and NKT cells. Here, we sought evidence for allelic variation of Ets1 in mice contributing to the NK and NKT cell phenotypes of the NOD strain. ETS1 expression in NK and NKT cells was reduced in NOD mice, compared to C57BL/6 mice. Although NKT cells numbers were significantly correlated with ETS1 expression in both strains, NKT cell numbers were not linked to the Ets1 gene in a first backcross from NOD to C57BL/6 mice. These results indicate that allelic variation of Ets1 did not contribute to variation in NKT cell numbers in these mice. It remains possible that a third factor not linked to the Ets1 locus controls both ETS1 expression and subsequently NK and NKT cell phenotypes. PMID:19806240

Methamphetamine- and 1-methyl-4-phenyl- 1,2,3, 6-tetrahydropyridine-induced dopaminergic neurotoxicity in inducible nitric oxide synthase-deficient mice.

PubMed

Itzhak, Y; Martin, J L; Ali, S F

1999-12-15

Previous studies have suggested a role for the retrograde messenger, nitric oxide (NO), in methamphetamine (METH)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced dopaminergic neurotoxicity. Since evidence supported the involvement of the neuronal nitric oxide synthase (nNOS) isoform in the dopaminergic neurotoxicity, the present study was undertaken to investigate whether the inducible nitric oxide synthase (iNOS) isoform is also associated with METH- and MPTP-induced neurotoxicity. The administration of METH (5mg/kg x 3) to iNOS deficient mice [homozygote iNOS(-/-)] and wild type mice (C57BL/6) resulted in significantly smaller depletion of striatal dopaminergic markers in the iNOS(-/-) mice compared with the wild-type mice. METH-induced hyperthermia was also significantly lower in the iNOS(-/-) mice than in wild-type mice. In contrast to the outcome of METH administration, MPTP injections (20 mg/kg x 3) resulted in a similar decrease in striatal dopaminergic markers in iNOS(-/-) and wild-type mice. In the set of behavioral experiments, METH-induced locomotor sensitization was investigated. The acute administration of METH (1.0 mg/kg) resulted in the same intensity of locomotor activity in iNOS(-/-) and wild-type mice. Moreover, 68 to 72 h after the exposure to the high-dose METH regimen (5 mg/kg x 3), a marked sensitized response to a challenge injection of METH (1.0 mg/kg) was observed in both the iNOS(-/-) and wild-type mice. The finding that iNOS(-/-) mice were unprotected from MPTP-induced neurotoxicity suggests that the partial protection against METH-induced neurotoxicity observed was primarily associated with the diminished hyperthermic effect of METH seen in the iNOS(-/-) mice. Moreover, in contrast to nNOS deficiency, iNOS deficiency did not affect METH-induced behavioral sensitization. Copyright 1999 Wiley-Liss, Inc.

Glucagon Receptor Knockout Prevents Insulin-Deficient Type 1 Diabetes in Mice

PubMed Central

Lee, Young; Wang, May-Yun; Du, Xiu Quan; Charron, Maureen J.; Unger, Roger H.

2011-01-01

OBJECTIVE To determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency. RESEARCH DESIGN AND METHODS We compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr−/−) mice and wild-type (Gcgr+/+) controls after equivalent destruction of β-cells. We used a double dose of streptozotocin to maximize β-cell destruction. RESULTS Gcgr+/+ mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable β-cell destruction in Gcgr−/− mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (∼1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr+/+ mice with diabetes—evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting β-hydroxy butyrate levels were lower. CONCLUSIONS We conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice. PMID:21270251

Photic Resetting and Entrainment in CLOCK-Deficient Mice

PubMed Central

Dallmann, Robert; DeBruyne, Jason P.; Weaver, David R.

2012-01-01

Mice lacking CLOCK protein have a relatively subtle circadian phenotype, including a slightly shorter period in constant darkness, differences in phase resetting after 4-hr light pulses in the early and late night, and a variably advanced phase angle of entrainment in a light-dark (LD) cycle (DeBruyne et al., Neuron 50:465–477, 2006). The present series of experiments was conducted to more fully characterize the circadian phenotype of Clock−/− mice under various lighting conditions. A phase-response curve (PRC) to 4-hour light pulses in free-running mice was conducted; the results confirm that Clock−/− mice exhibit very large phase advances after 4 hrs light pulses in the late subjective night, but have relatively normal responses to light at other phases. The abnormal shape of the PRC to light may explain the tendency of CLOCK-deficient mice to begin activity before lights-out when housed in a 12 hrs light: 12 hrs dark lighting schedule. To assess this relationship further, Clock−/− and wild-type control mice were entrained to skeleton lighting cycles (1L:23D, and 1L:10D:1L:12D). Comparing entrainment under the two types of skeleton photoperiods revealed that exposure to 1 hr light in the morning leads to a phase advance of activity onset (expressed the following afternoon) in Clock−/− mice, but not in the controls. Constant light typically causes an intensity-dependent increase in circadian period in mice, but this did not occur in CLOCK-deficient mice. The failure of Clock−/− mice to respond to the period-lengthening effect of constant light likely results from the increased functional impact of light falling in the phase advance zone of the PRC. Collectively, these experiments reveal that alterations in the response of CLOCK-deficient mice to light in several paradigms are likely due to an imbalance in the shape of the PRC to light. PMID:21921293

Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis in Mice via a Toll-Like Receptor 4/p21-Activated Kinase 1 Cascade.

PubMed

Wu, Yaxin; Wu, Jiao; Chen, Ting; Li, Qing; Peng, Wei; Li, Huan; Tang, Xiaowei; Fu, Xiangsheng

2018-05-01

The underlying pathogenic mechanism of Fusobacterium nucleatum in the carcinogenesis of colorectal cancer has been poorly understood. Using C57BL/6-Apc Min/+ mice, we investigated gut microbial structures with F. nucleatum, antibiotics, and Toll-like receptor 4 (TLR4) antagonist TAK-242 treatment. In addition, we measured intestinal tumor formation and the expression of TLR4, p21-activated kinase 1 (PAK1), phosphorylated-PAK1 (p-PAK1), phosphorylated-β-catenin S675 (p-β-catenin S675), and cyclin D1 in mice with different treatments. Fusobacterium nucleatum and antibiotics treatment altered gut microbial structures in mice. In addition, F. nucleatum invaded into the intestinal mucosa in large amounts but were less abundant in the feces of F. nucleatum-fed mice. The average number and size of intestinal tumors in F. nucleatum groups was significantly increased compared to control groups in Apc Min/+ mice (P < 0.05). The expression of TLR4, PAK1, p-PAK1, p-β-catenin S675, and cyclin D1 was significantly increased in F. nucleatum groups compared to the control groups (P < 0.05). Moreover, TAK-242 significantly decreased the average number and size of intestinal tumors compared to F. nucleatum groups (P < 0.05). The expression of p-PAK1, p-β-catenin S675, and cyclin D1 was also significantly decreased in the TAK-242-treated group compared to F. nucleatum groups (P < 0.05). Fusobacterium nucleatum potentiates intestinal tumorigenesis in Apc Min/+ mice via a TLR4/p-PAK1/p-β-catenin S675 cascade. Fusobacterium nucleatum-induced intestinal tumorigenesis can be inhibited by TAK-242, implicating TLR4 as a potential target for the prevention and therapy of F. nucleatum-related colorectal cancer.

Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6.

PubMed

Hegan, Denise Campisi; Narayanan, Latha; Jirik, Frank R; Edelmann, Winfried; Liskay, R Michael; Glazer, Peter M

2006-12-01

Defects in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer. Because the MMR pathway includes multiple factors with both overlapping and divergent functions, we sought to compare the impact of deficiencies in each of several MMR genes on genetic instability using a collection of knock-out mouse models. We investigated mutation frequencies and patterns in MMR-deficient mice using two transgenic reporter genes, supFG1 and cII, in the context of mice deficient for Pms2, Mlh1, Msh2, Msh3 or Msh6 or both Msh2 and Msh3 or both Msh3 and Msh6. We found that the mean mutation frequencies of all of the MMR-deficient mice were significantly higher than the mean mutation frequencies of wild-type mice. Mlh1-deficient mice and Msh2-deficient mice had the highest mutation frequencies in a comparison of the single nullizygous mice. Of all the mice studied, mice nullizygous for both Msh2 and Msh3 and those nullizygous for both Msh3 and Msh6 displayed the greatest overall increases in mutation frequencies compared with wild-type mice. Sequence analysis of the mutated reporter genes revealed significant differences between the individual groups of MMR-deficient mice. Taken together, our results further characterize the functions of the MMR factors in mutation avoidance and provide in vivo correlation to biochemical models of the MMR pathway.

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

PubMed

Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

2008-05-02

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Indomethacin induced gastropathy in CD18, intercellular adhesion molecule 1, or P-selectin deficient mice

PubMed Central

Morise, Z; Granger, D; Fuseler, J; Anderson, D; Grisham, M

1999-01-01

BACKGROUND—Neutrophil-endothelial cell interactions are thought to play a critical role in the pathophysiology of non-steroidal anti-inflammatory drug (NSAID) induced gastropathy.
AIMS—To optimise a mouse model of NSAID induced gastropathy and to evaluate the importance of adhesion molecules using adhesion molecule deficient mice.
METHODS—Gastropathy was induced in C57BL/6 mice or their adhesion molecule deficient counterparts via oral administration of indomethacin (20 mg/kg). Lesion scores, mucosal permeability, and histopathology were used to assess gastric mucosal injury.
RESULTS—Intragastric administration of indomethacin induced linear haemorrhagic mucosal lesions, primarily in the corpus of the stomach that were first observed at six hours. These lesions continued to develop over the next six hours with maximal lesion scores and mucosal permeabilities at 12 hours. When indomethacin was administered to mice deficient in CD18, intercellular adhesion molecule 1 (ICAM-1), or P-selectin, there were significant decreases in lesion scores compared with their C57BL/6 controls. In addition, mucosal permeabilities were found to be significantly lower in CD18 or ICAM-1 deficient mice observed at 12 hours.
CONCLUSION—Certain leucocyte and endothelial cell adhesion molecules are important determinants for full expression of indomethacin induced gastropathy. It is proposed that this modification of the mouse model may be useful for the investigation of other pathophysiological mechanisms of NSAID induced gastropathy.


Keywords: indomethacin; gastropathy; cyclooxygenase; intercellular adhesion molecule; VCAM; vascular cell adhesion molecule; P-selectin PMID:10486359

TGF-β Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival

PubMed Central

Gingery, Anne; Bradley, Elizabeth W.; Pederson, Larry; Ruan, Ming; Horwood, Nikki J.; Oursler, Merry Jo

2008-01-01

To better understand the roles of TGF-β in bone metabolism, we investigated osteoclast survival in response TGF-β and found that TGF-β inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-β receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-β treatment. Since osteoclast survival involves MEK, AKT, and NFκB activation, we examined TGF-β effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IκB, and NFκB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFκB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFκB repressed TGF-β-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-β-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclXL expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-β-induced NFκB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-β to support of osteoclast survival. PMID:18586026

HIV Glycoprotein Gp120 Impairs Fast Axonal Transport by Activating Tak1 Signaling Pathways

PubMed Central

Berth, Sarah H.; Mesnard-Hoaglin, Nichole; Wang, Bin; Kim, Hajwa; Song, Yuyu; Sapar, Maria; Morfini, Gerardo

2016-01-01

Sensory neuropathies are the most common neurological complication of HIV. Of these, distal sensory polyneuropathy (DSP) is directly caused by HIV infection and characterized by length-dependent axonal degeneration of dorsal root ganglion (DRG) neurons. Mechanisms for axonal degeneration in DSP remain unclear, but recent experiments revealed that the HIV glycoprotein gp120 is internalized and localized within axons of DRG neurons. Based on these findings, we investigated whether intra-axonal gp120 might impair fast axonal transport (FAT), a cellular process critical for appropriate maintenance of the axonal compartment. Significantly, we found that gp120 severely impaired both anterograde and retrograde FAT. Providing a mechanistic basis for these effects, pharmacological experiments revealed an involvement of various phosphotransferases in this toxic effect, including members of mitogen-activated protein kinase pathways (Tak-1, p38, and c-Jun N-terminal Kinase (JNK)), inhibitor of kappa-B-kinase 2 (IKK2), and PP1. Biochemical experiments and axonal outgrowth assays in cell lines and primary cultures extended these findings. Impairments in neurite outgrowth in DRG neurons by gp120 were rescued using a Tak-1 inhibitor, implicating a Tak-1 mitogen-activated protein kinase pathway in gp120 neurotoxicity. Taken together, these observations indicate that kinase-based impairments in FAT represent a novel mechanism underlying gp120 neurotoxicity consistent with the dying-back degeneration seen in DSP. Targeting gp120-based impairments in FAT with specific kinase inhibitors might provide a novel therapeutic strategy to prevent axonal degeneration in DSP. PMID:27872270

Ionizing radiation, inflammation, and their interactions in colon carcinogenesis in Mlh1-deficient mice

PubMed Central

Morioka, Takamitsu; Miyoshi-Imamura, Tomoko; Blyth, Benjamin J; Kaminishi, Mutsumi; Kokubo, Toshiaki; Nishimura, Mayumi; Kito, Seiji; Tokairin, Yutaka; Tani, Shusuke; Murakami-Murofushi, Kimiko; Yoshimi, Naoki; Shimada, Yoshiya; Kakinuma, Shizuko

2015-01-01

Genetic, physiological and environmental factors are implicated in colorectal carcinogenesis. Mutations in the mutL homolog 1 (MLH1) gene, one of the DNA mismatch repair genes, are a main cause of hereditary colon cancer syndromes such as Lynch syndrome. Long-term chronic inflammation is also a key risk factor, responsible for colitis-associated colorectal cancer; radiation exposure is also known to increase colorectal cancer risk. Here, we studied the effects of radiation exposure on inflammation-induced colon carcinogenesis in DNA mismatch repair-proficient and repair-deficient mice. Male and female Mlh1−/− and Mlh1+/+ mice were irradiated with 2 Gy X-rays when aged 2 weeks or 7 weeks and/or were treated with 1% dextran sodium sulfate (DSS) in drinking water for 7 days at 10 weeks old to induce mild inflammatory colitis. No colon tumors developed after X-rays and/or DSS treatment in Mlh1+/+ mice. Colon tumors developed after DSS treatment alone in Mlh1−/− mice, and exposure to radiation prior to DSS treatment increased the number of tumors. Histologically, colon tumors in the mice resembled the subtype of well-to-moderately differentiated adenocarcinomas with tumor-infiltrating lymphocytes of human Lynch syndrome. Immunohistochemistry revealed that expression of both p53 and β-catenin and loss of p21 and adenomatosis polyposis coli proteins were observed at the later stages of carcinogenesis, suggesting a course of molecular pathogenesis distinct from typical sporadic or colitis-associated colon cancer in humans. In conclusion, radiation exposure could further increase the risk of colorectal carcinogenesis induced by inflammation under the conditions of Mlh1 deficiency. PMID:25529563

Colitis and Colon Cancer in WASP-Deficient Mice Require Helicobacter Spp.

PubMed Central

Nguyen, Deanna D.; Muthupalani, Suresh; Goettel, Jeremy A.; Eston, Michelle A.; Mobley, Melissa; Taylor, Nancy S.; McCabe, Amanda; Marin, Romela; Snapper, Scott B.; Fox, James G.

2014-01-01

Background Wiskott-Aldrich Syndrome protein (WASP)-deficient patients and mice are immunodeficient and can develop inflammatory bowel disease. The intestinal microbiome is critical to the development of colitis in most animal models, in which, Helicobacter spp. have been implicated in disease pathogenesis. We sought to determine the role of Helicobacter spp. in colitis development in WASP-deficient (WKO) mice. Methods Feces from WKO mice raised under specific pathogen free conditions were evaluated for the presence of Helicobacter spp., after which, a subset of mice were rederived in Helicobacter spp.-free conditions. Helicobacter spp.-free WKO animals were subsequently infected with Helicobacter bilis. Results Helicobacter spp. were detected in feces from WKO mice. After re-derivation in Helicobacter spp.-free conditions, WKO mice did not develop spontaneous colitis but were susceptible to radiation-induced colitis. Moreover, a T-cell transfer model of colitis dependent on WASP-deficient innate immune cells also required Helicobacter spp. colonization. Helicobacter bilis infection of rederived WKO mice led to typhlitis and colitis. Most notably, several H. bilis-infected animals developed dysplasia with 10% demonstrating colon carcinoma, which was not observed in uninfected controls. Conclusions Spontaneous and T-cell transfer, but not radiation-induced, colitis in WKO mice is dependent on the presence of Helicobacter spp. Furthermore, H. bilis infection is sufficient to induce typhlocolitis and colon cancer in Helicobacter spp.-free WKO mice. This animal model of a human immunodeficiency with chronic colitis and increased risk of colon cancer parallels what is seen in human colitis and implicates specific microbial constituents in promoting immune dysregulation in the intestinal mucosa. PMID:23820270

Mice with Sort1 deficiency display normal cognition but elevated anxiety-like behavior.

PubMed

Ruan, Chun-Sheng; Yang, Chun-Rui; Li, Jia-Yi; Luo, Hai-Yun; Bobrovskaya, Larisa; Zhou, Xin-Fu

2016-07-01

Exposure to stressful life events plays a central role in the development of mood disorders in vulnerable individuals. However, the mechanisms that link mood disorders to stress are poorly understood. Brain-derived neurotrophic factor (BDNF) has long been implicated in positive regulation of depression and anxiety, while its precursor (proBDNF) recently showed an opposing effect on such mental illnesses. P75(NTR) and sortilin are co-receptors of proBDNF, however, the role of these receptors in mood regulation is not established. Here, we aimed to investigate the role of sortilin in regulating mood-related behaviors and its role in the proBDNF-mediated mood abnormality in mice. We found that sortilin was up-regulated in neocortex (by 78.3%) and hippocampus (by 111%) of chronically stressed mice as assessed by western blot analysis. These changes were associated with decreased mobility in the open field test and increased depression-like behavior in the forced swimming test. We also found that sortilin deficiency in mice resulted in hyperlocomotion in the open field test and increased anxiety-like behavior in both the open field and elevated plus maze tests. No depression-like behavior in the forced swimming test and no deficit in spatial cognition in the Morris water maze test were found in the Sort1-deficient mice. Moreover, the intracellular and extracellular levels of mature BDNF and proBDNF were not changed when sortilin was absent in vivo and in vitro. Finally, we found that both WT and Sort1-deficient mice injected with proBDNF in lateral ventricle displayed increased depression-like behavior in the forced swimming test but not anxiety-like behaviors in the open field and elevated plus maze tests. The present study suggests that sortilin functions as a negative regulator of mood performance and can be a therapeutic target for the treatment of mental illness. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Lipoprotein lipase gene-deficient mice with hypertriglyceridaemia associated with acute pancreatitis.

PubMed

Tang, Maochun; Zong, Pengfei; Zhang, Ting; Wang, Dongyan; Wang, Yuhui; Zhao, Yan

2016-10-01

To investigate the severity of pancreatitis in lipoprotein lipase (LPL)-deficient hypertriglyceridaemic (HTG) heterozygous mice and to establish an experimental animal model for HTG pancreatitis study. LPL-deficient HTG heterozygous mice were rescued by somatic gene transfer and mated with wild-type mice. The plasma amylase, triglyceride, and pathologic changes in the pancreas of the LPL-deficient HTG heterozygous mice were compared with those of wild-type mice to assess the severity of pancreatitis. In addition, acute pancreatitis (AP) was induced by caerulein (50 µg/kg) for further assessment. The levels of plasma amylase and triglyceride were significantly higher in the LPL-deficient HTG heterozygous mice. According to the pancreatic histopathologic scores, the LPL-deficient HTG heterozygous mice showed more severe pathologic damage than the wild-type mice. Lipoprotein lipase deficient heterozygous mice developed severe caerulein-induced pancreatitis. In addition, their high triglyceride levels were stable. Therefore, LPL-deficient HTG heterozygous mice are a useful experimental model for studying HTG pancreatitis.

Loss of Par-1a/MARK3/C-TAK1 Kinase Leads to Reduced Adiposity, Resistance to Hepatic Steatosis, and Defective Gluconeogenesis ▿

PubMed Central

Lennerz, Jochen K.; Hurov, Jonathan B.; White, Lynn S.; Lewandowski, Katherine T.; Prior, Julie L.; Planer, G. James; Gereau, Robert W.; Piwnica-Worms, David; Schmidt, Robert E.; Piwnica-Worms, Helen

2010-01-01

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice. PMID:20733003

Aquaporin-1 Deficiency Protects Against Myocardial Infarction by Reducing Both Edema and Apoptosis in Mice

PubMed Central

Li, Lihua; Weng, Zhiyong; Yao, Chenjuan; Song, Yuanlin; Ma, Tonghui

2015-01-01

Many studies have determined that AQP1 plays an important role in edema formation and resolution in various tissues via water transport across the cell membrane. The aim of this research was to determine both if and how AQP1 is associated with cardiac ischemic injury, particularly the development of edema following myocardial infarction (MI). AQP1+/+ and AQP1−/− mice were used to create the MI model. Under physiological conditions, AQP1−/− mice develop normally; however, in the setting of MI, they exhibit cardioprotective properties, as shown by reduced cardiac infarct size determined via NBT staining, improved cardiac function determined via left ventricular catheter measurements, decreased AQP1-dependent myocardial edema determined via water content assays, and decreased apoptosis determined via TUNEL analysis. Cardiac ischemia caused by hypoxia secondary to AQP1 deficiency stabilized the expression of HIF-1α in endothelial cells and subsequently decreased microvascular permeability, resulting in the development of edema. The AQP1-dependent myocardial edema and apoptosis contributed to the development of MI. AQP1 deficiency protected cardiac function from ischemic injury following MI. Furthermore, AQP1 deficiency reduced microvascular permeability via the stabilization of HIF-1α levels in endothelial cells and decreased cellular apoptosis following MI. PMID:26348407

Differential effects of silver nanoparticles on DNA damage and DNA repair gene expression in Ogg1-deficient and wild type mice.

PubMed

Nallanthighal, Sameera; Chan, Cadia; Murray, Thomas M; Mosier, Aaron P; Cady, Nathaniel C; Reliene, Ramune

2017-10-01

Due to extensive use in consumer goods, it is important to understand the genotoxicity of silver nanoparticles (AgNPs) and identify susceptible populations. 8-Oxoguanine DNA glycosylase 1 (OGG1) excises 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), a pro-mutagenic lesion induced by oxidative stress. To understand whether defects in OGG1 is a possible genetic factor increasing an individual's susceptibly to AgNPs, we determined DNA damage, genome rearrangements, and expression of DNA repair genes in Ogg1-deficient and wild type mice exposed orally to 4 mg/kg of citrate-coated AgNPs over a period of 7 d. DNA damage was examined at 3 and 7 d of exposure and 7 and 14 d post-exposure. AgNPs induced 8-oxoG, double strand breaks (DSBs), chromosomal damage, and DNA deletions in both genotypes. However, 8-oxoG was induced earlier in Ogg1-deficient mice and 8-oxoG levels were higher after 7-d treatment and persisted longer after exposure termination. AgNPs downregulated DNA glycosylases Ogg1, Neil1, and Neil2 in wild type mice, but upregulated Myh, Neil1, and Neil2 glycosylases in Ogg1-deficient mice. Neil1 and Neil2 can repair 8-oxoG. Thus, AgNP-mediated downregulation of DNA glycosylases in wild type mice may contribute to genotoxicity, while upregulation thereof in Ogg1-deficient mice could serve as an adaptive response to AgNP-induced DNA damage. However, our data show that Ogg1 is indispensable for the efficient repair of AgNP-induced damage. In summary, citrate-coated AgNPs are genotoxic in both genotypes and Ogg1 deficiency exacerbates the effect. These data suggest that humans with genetic polymorphisms and mutations in OGG1 may have increased susceptibility to AgNP-mediated DNA damage.

Group 1B phospholipase A₂ inactivation suppresses atherosclerosis and metabolic diseases in LDL receptor-deficient mice.

PubMed

Hollie, Norris I; Konaniah, Eddy S; Goodin, Colleen; Hui, David Y

2014-06-01

Previous studies have shown that inactivation of the group 1B phospholipase A2 (Pla2g1b) suppresses diet-induced obesity, hyperglycemia, insulin resistance, and hyperlipidemia in C57BL/6 mice. A possible influence of Pla2g1b inactivation on atherosclerosis has not been addressed previously. The current study utilized LDL receptor-deficient (Ldlr(-/-)) mice with plasma lipid levels and distribution similar to hyperlipidemic human subjects as a preclinical animal model to test the effectiveness of Pla2g1b inactivation on atherosclerosis. The Pla2g1b(+/+)Ldlr(-/-) and Pla2g1b(-/-)Ldlr(-/-) mice were fed a low fat chow diet or a hypercaloric diet with 58.5 kcal% fat and 25 kcal% sucrose for 10 weeks. Minimal differences were observed between Pla2g1b(+/+)Ldlr(-/-) and Pla2g1b(-/-)Ldlr(-/-) mice when the animals were maintained on the low fat chow diet. However, when the animals were maintained on the hypercaloric diet, the Pla2g1(+/+)Ldlr(-/-) mice showed the expected body weight gain but the Pla2g1b(-/-)Ldlr(-/-) mice were resistant to diet-induced body weight gain. The Pla2g1b(-/-)Ldlr(-/-) mice also displayed lower fasting glucose, insulin, and plasma lipid levels compared to the Pla2g1b(+/+)Ldlr(-/-) mice, which displayed robust hyperglycemia, hyperinsulinemia, and hyperlipidemia in response to the hypercaloric diet. Importantly, atherosclerotic lesions in the aortic roots were also reduced 7-fold in the Pla2g1b(-/-)Ldlr(-/-) mice. The effectiveness of Pla2g1b inactivation to suppress diet-induced body weight gain and reduce diabetes and atherosclerosis in LDL receptor-deficient mice suggests that pharmacological inhibition of Pla2g1b may be a viable strategy to decrease diet-induced obesity and the risk of diabetes and atherosclerosis in humans. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Bioluminescent Imaging Reveals Divergent Viral Pathogenesis in Two Strains of Stat1-Deficient Mice, and in αßγ Interferon Receptor-Deficient Mice

PubMed Central

Pasieka, Tracy Jo; Collins, Lynne; O'Connor, Megan A.; Chen, Yufei; Parker, Zachary M.; Berwin, Brent L.; Piwnica-Worms, David R.; Leib, David A.

2011-01-01

Pivotal components of the IFN response to virus infection include the IFN receptors (IFNR), and the downstream factor signal transducer and activator of transcription 1 (Stat1). Mice deficient for Stat1 and IFNR (Stat1−/− and IFNαßγR−/− mice) lack responsiveness to IFN and exhibit high sensitivity to various pathogens. Here we examined herpes simplex virus type 1 (HSV-1) pathogenesis in Stat1−/− mice and in IFNαßγR−/− mice following corneal infection and bioluminescent imaging. Two divergent and paradoxical patterns of infection were observed. Mice with an N-terminal deletion in Stat1 (129Stat1−/− (N-term)) had transient infection of the liver and spleen, but succumbed to encephalitis by day 10 post-infection. In stark contrast, infection of IFNαßγR−/− mice was rapidly fatal, with associated viremia and fulminant infection of the liver and spleen, with infected infiltrating cells being primarily of the monocyte/macrophage lineage. To resolve the surprising difference between Stat1−/− and IFNαßγR−/− mice, we infected an additional Stat1−/− strain deleted in the DNA-binding domain (129Stat1−/− (DBD)). These 129Stat1−/− (DBD) mice recapitulated the lethal pattern of liver and spleen infection seen following infection of IFNαßγR−/− mice. This lethal pattern was also observed when 129Stat1−/− (N-term) mice were infected and treated with a Type I IFN-blocking antibody, and immune cells derived from 129Stat1−/− (N-term) mice were shown to be responsive to Type I IFN. These data therefore show significant differences in viral pathogenesis between two commonly-used Stat1−/− mouse strains. The data are consistent with the hypothesis that Stat1−/− (N-term) mice have residual Type I IFN receptor-dependent IFN responses. Complete loss of IFN signaling pathways allows viremia and rapid viral spread with a fatal infection of the liver. This study underscores the importance of careful comparisons

Ionizing radiation, inflammation, and their interactions in colon carcinogenesis in Mlh1-deficient mice.

PubMed

Morioka, Takamitsu; Miyoshi-Imamura, Tomoko; Blyth, Benjamin J; Kaminishi, Mutsumi; Kokubo, Toshiaki; Nishimura, Mayumi; Kito, Seiji; Tokairin, Yutaka; Tani, Shusuke; Murakami-Murofushi, Kimiko; Yoshimi, Naoki; Shimada, Yoshiya; Kakinuma, Shizuko

2015-03-01

Genetic, physiological and environmental factors are implicated in colorectal carcinogenesis. Mutations in the mutL homolog 1 (MLH1) gene, one of the DNA mismatch repair genes, are a main cause of hereditary colon cancer syndromes such as Lynch syndrome. Long-term chronic inflammation is also a key risk factor, responsible for colitis-associated colorectal cancer; radiation exposure is also known to increase colorectal cancer risk. Here, we studied the effects of radiation exposure on inflammation-induced colon carcinogenesis in DNA mismatch repair-proficient and repair-deficient mice. Male and female Mlh1(-/-) and Mlh1(+/+) mice were irradiated with 2 Gy X-rays when aged 2 weeks or 7 weeks and/or were treated with 1% dextran sodium sulfate (DSS) in drinking water for 7 days at 10 weeks old to induce mild inflammatory colitis. No colon tumors developed after X-rays and/or DSS treatment in Mlh1(+/+) mice. Colon tumors developed after DSS treatment alone in Mlh1(-/-) mice, and exposure to radiation prior to DSS treatment increased the number of tumors. Histologically, colon tumors in the mice resembled the subtype of well-to-moderately differentiated adenocarcinomas with tumor-infiltrating lymphocytes of human Lynch syndrome. Immunohistochemistry revealed that expression of both p53 and β-catenin and loss of p21 and adenomatosis polyposis coli proteins were observed at the later stages of carcinogenesis, suggesting a course of molecular pathogenesis distinct from typical sporadic or colitis-associated colon cancer in humans. In conclusion, radiation exposure could further increase the risk of colorectal carcinogenesis induced by inflammation under the conditions of Mlh1 deficiency. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Cyclocreatine treatment improves cognition in mice with creatine transporter deficiency.

PubMed

Kurosawa, Yuko; Degrauw, Ton J; Lindquist, Diana M; Blanco, Victor M; Pyne-Geithman, Gail J; Daikoku, Takiko; Chambers, James B; Benoit, Stephen C; Clark, Joseph F

2012-08-01

The second-largest cause of X-linked mental retardation is a deficiency in creatine transporter (CRT; encoded by SLC6A8), which leads to speech and language disorders with severe cognitive impairment. This syndrome, caused by the absence of creatine in the brain, is currently untreatable because CRT is required for creatine entry into brain cells. Here, we developed a brain-specific Slc6a8 knockout mouse (Slc6a8-/y) as an animal model of human CRT deficiency in order to explore potential therapies for this syndrome. The phenotype of the Slc6a8-/y mouse was comparable to that of human patients. We successfully treated the Slc6a8-/y mice with the creatine analog cyclocreatine. Brain cyclocreatine and cyclocreatine phosphate were detected after 9 weeks of cyclocreatine treatment in Slc6a8-/y mice, in contrast to the same mice treated with creatine or placebo. Cyclocreatine-treated Slc6a8-/y mice also exhibited a profound improvement in cognitive abilities, as seen with novel object recognition as well as spatial learning and memory tests. Thus, cyclocreatine appears promising as a potential therapy for CRT deficiency.

Antibody-based inhibition of circulating DLK1 protects from estrogen deficiency-induced bone loss in mice.

PubMed

Figeac, Florence; Andersen, Ditte C; Nipper Nielsen, Casper A; Ditzel, Nicholas; Sheikh, Søren P; Skjødt, Karsten; Kassem, Moustapha; Jensen, Charlotte H; Abdallah, Basem M

2018-05-01

Soluble delta-like 1 homolog (DLK1) is a circulating protein that belongs to the Notch/Serrate/delta family, which regulates many differentiation processes including osteogenesis and adipogenesis. We have previously demonstrated an inhibitory effect of DLK1 on bone mass via stimulation of bone resorption and inhibition of bone formation. Further, serum DLK1 levels are elevated and positively correlated to bone turnover markers in estrogen (E)-deficient rodents and women. In this report, we examined whether inhibition of serum DLK1 activity using a neutralizing monoclonal antibody protects from E deficiency-associated bone loss in mice. Thus, we generated mouse monoclonal anti-mouse DLK1 antibodies (MAb DLK1) that enabled us to reduce and also quantitate the levels of bioavailable serum DLK1 in vivo. Ovariectomized (ovx) mice were injected intraperitoneally twice weekly with MAb DLK1 over a period of one month. DEXA-, microCT scanning, and bone histomorphometric analyses were performed. Compared to controls, MAb DLK1 treated ovx mice were protected against ovx-induced bone loss, as revealed by significantly increased total bone mass (BMD) due to increased trabecular bone volume fraction (BV/TV) and inhibition of bone resorption. No significant changes were observed in total fat mass or in the number of bone marrow adipocytes. These results support the potential use of anti-DLK1 antibody therapy as a novel intervention to protect from E deficiency associated bone loss. Copyright © 2018 Elsevier Inc. All rights reserved.

Combined vitamin C and vitamin E deficiency worsens early atherosclerosis in apolipoprotein E-deficient mice.

PubMed

Babaev, Vladimir R; Li, Liying; Shah, Sanket; Fazio, Sergio; Linton, MacRae F; May, James M

2010-09-01

To assess the role of combined deficiencies of vitamins C and E on the earliest stages of atherosclerosis (an inflammatory condition associated with oxidative stress), 4 combinations of vitamin supplementation (low C/low E, low C/high E, high C/low E, and high C/high E) were studied in atherosclerosis-prone apolipoprotein E-deficient mice also unable to synthesize their own vitamin C (gulonolactone oxidase(-/-)); and to evaluate the effect of a more severe depletion of vitamin C alone in a second experiment using gulonolactone oxidase(-/-) mice carrying the hemizygous deletion of SVCT2 (the vitamin C transporter). After 8 weeks of a high-fat diet (16% lard and 0.2% cholesterol), atherosclerosis developed in the aortic sinus areas of mice in all diet groups. Each vitamin-deficient diet significantly decreased liver and brain contents of the corresponding vitamin. Combined deficiency of both vitamins increased lipid peroxidation, doubled plaque size, and increased plaque macrophage content by 2- to 3-fold in male mice, although only plaque macrophage content was increased in female mice. A more severe deficiency of vitamin C in gulonolactone oxidase(-/-) mice with defective cellular uptake of vitamin C increased both oxidative stress and atherosclerosis in apolipoprotein E(-/-) mice compared with littermates receiving a diet replete in vitamin C, again most clearly in males. Combined deficiencies of vitamins E and C are required to worsen early atherosclerosis in an apolipoprotein E-deficient mouse model. However, a more severe cellular deficiency of vitamin C alone promotes atherosclerosis when vitamin E is replete.

Podocyte-Specific GLUT4-Deficient Mice Have Fewer and Larger Podocytes and Are Protected From Diabetic Nephropathy

PubMed Central

Guzman, Johanna; Jauregui, Alexandra N.; Merscher-Gomez, Sandra; Maiguel, Dony; Muresan, Cristina; Mitrofanova, Alla; Diez-Sampedro, Ana; Szust, Joel; Yoo, Tae-Hyun; Villarreal, Rodrigo; Pedigo, Christopher; Molano, R. Damaris; Johnson, Kevin; Kahn, Barbara; Hartleben, Bjoern; Huber, Tobias B.; Saha, Jharna; Burke, George W.; Abel, E. Dale; Brosius, Frank C.; Fornoni, Alessia

2014-01-01

Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria. PMID:24101677

IL-1 receptor antagonist-deficient mice develop autoimmune arthritis due to intrinsic activation of IL-17-producing CCR2+Vγ6+γδ T cells

PubMed Central

Akitsu, Aoi; Ishigame, Harumichi; Kakuta, Shigeru; Chung, Soo-hyun; Ikeda, Satoshi; Shimizu, Kenji; Kubo, Sachiko; Liu, Yang; Umemura, Masayuki; Matsuzaki, Goro; Yoshikai, Yasunobu; Saijo, Shinobu; Iwakura, Yoichiro

2015-01-01

Interleukin-17 (IL-17)-producing γδ T (γδ17) cells have been implicated in inflammatory diseases, but the underlying pathogenic mechanisms remain unclear. Here, we show that both CD4+ and γδ17 cells are required for the development of autoimmune arthritis in IL-1 receptor antagonist (IL-1Ra)-deficient mice. Specifically, activated CD4+ T cells direct γδ T-cell infiltration by inducing CCL2 expression in joints. Furthermore, IL-17 reporter mice reveal that the Vγ6+ subset of CCR2+ γδ T cells preferentially produces IL-17 in inflamed joints. Importantly, because IL-1Ra normally suppresses IL-1R expression on γδ T cells, IL-1Ra-deficient mice exhibit elevated IL-1R expression on Vγ6+ cells, which play a critical role in inducing them to produce IL-17. Our findings demonstrate a pathogenic mechanism in which adaptive and innate immunity induce an autoimmune disease in a coordinated manner. PMID:26108163

Attenuated progression of diet-induced steatohepatitis in glutathione-deficient mice

PubMed Central

Haque, Jamil A; McMahan, Ryan S; Campbell, Jean S; Shimizu-Albergine, Masami; Wilson, Angela M; Botta, Dianne; Bammler, Theo K; Beyer, Richard P; Montine, Thomas J; Yeh, Matthew M; Kavanagh, Terrance J; Fausto, Nelson

2011-01-01

In nonalcoholic fatty liver disease (NAFLD), depletion of hepatic antioxidants may contribute to the progression of steatosis to nonalcoholic steatohepatitis (NASH) by increasing oxidative stress that produces lipid peroxidation, inflammation, and fibrosis. We investigated whether depletion of glutathione (GSH) increases NASH-associated hepatic pathology in mice fed a diet deficient in methionine and choline (MCD diet). Wild-type (wt) mice and genetically GSH-deficient mice lacking the modifier subunit of glutamate cysteine ligase (Gclm null mice), the rate-limiting enzyme for de novo synthesis of GSH, were fed the MCD diet, a methionine/choline-sufficient diet, or standard chow for 21 days. We assessed NASH-associated hepatic pathology, including steatosis, fibrosis, inflammation, and hepatocyte ballooning, and used the NAFLD Scoring System to evaluate the extent of changes. We measured triglyceride levels, determined the level of lipid peroxidation products, and measured by qPCR the expression of mRNAs for several proteins associated with lipid metabolism, oxidative stress, and fibrosis. MCD-fed GSH-deficient Gclm null mice were to a large extent protected from MCD diet-induced excessive fat accumulation, hepatocyte injury, inflammation, and fibrosis. Compared with wt animals, MCD-fed Gclm null mice had much lower levels of F2-isoprostanes, lower expression of acyl-CoA oxidase, carnitine palmitoyltransferase 1a, uncoupling protein-2, stearoyl-coenzyme A desaturase-1, transforming growth factor-β, and plas-minogen activator inhibitor-1 mRNAs, and higher activity of catalase, indicative of low oxidative stress, inhibition of triglyceride synthesis, and lower expression of profibrotic proteins. Global gene analysis of hepatic RNA showed that compared with wt mice, the livers of Gclm null mice have a high capacity to metabolize endogenous and exogenous compounds, have lower levels of lipogenic proteins, and increased antioxidant activity. Thus, metabolic adaptations

Behavioral and regulatory abnormalities in mice deficient in the NPAS1 and NPAS3 transcription factors.

PubMed

Erbel-Sieler, Claudia; Dudley, Carol; Zhou, Yudong; Wu, Xinle; Estill, Sandi Jo; Han, Tina; Diaz-Arrastia, Ramon; Brunskill, Eric W; Potter, S Steven; McKnight, Steven L

2004-09-14

Laboratory mice bearing inactivating mutations in the genes encoding the NPAS1 and NPAS3 transcription factors have been shown to exhibit a spectrum of behavioral and neurochemical abnormalities. Behavioral abnormalities included diminished startle response, as measured by prepulse inhibition, and impaired social recognition. NPAS1/NPAS3-deficient mice also exhibited stereotypic darting behavior at weaning and increased locomotor activity. Immunohistochemical staining assays showed that the NPAS1 and NPAS3 proteins are expressed in inhibitory interneurons and that the viability and anatomical distribution of these neurons are unaffected by the absence of either transcription factor. Adult brain tissues from NPAS3- and NPAS1/NPAS3-deficient mice exhibited a distinct reduction in reelin, a large, secreted protein whose expression has been reported to be attenuated in the postmortem brain tissue of patients with schizophrenia. These observations raise the possibility that a regulatory program controlled in inhibitory interneurons by the NPAS1 and NPAS3 transcription factors may be either substantively or tangentially relevant to psychosis.

Behavioral and regulatory abnormalities in mice deficient in the NPAS1 and NPAS3 transcription factors

PubMed Central

Erbel-Sieler, Claudia; Dudley, Carol; Zhou, Yudong; Wu, Xinle; Estill, Sandi Jo; Han, Tina; Diaz-Arrastia, Ramon; Brunskill, Eric W.; Potter, S. Steven; McKnight, Steven L.

2004-01-01

Laboratory mice bearing inactivating mutations in the genes encoding the NPAS1 and NPAS3 transcription factors have been shown to exhibit a spectrum of behavioral and neurochemical abnormalities. Behavioral abnormalities included diminished startle response, as measured by prepulse inhibition, and impaired social recognition. NPAS1/NPAS3-deficient mice also exhibited stereotypic darting behavior at weaning and increased locomotor activity. Immunohistochemical staining assays showed that the NPAS1 and NPAS3 proteins are expressed in inhibitory interneurons and that the viability and anatomical distribution of these neurons are unaffected by the absence of either transcription factor. Adult brain tissues from NPAS3- and NPAS1/NPAS3-deficient mice exhibited a distinct reduction in reelin, a large, secreted protein whose expression has been reported to be attenuated in the postmortem brain tissue of patients with schizophrenia. These observations raise the possibility that a regulatory program controlled in inhibitory interneurons by the NPAS1 and NPAS3 transcription factors may be either substantively or tangentially relevant to psychosis. PMID:15347806

Energy homeostasis in leptin deficient Lepob/ob mice.

PubMed

Skowronski, Alicja A; Ravussin, Yann; Leibel, Rudolph L; LeDuc, Charles A

2017-01-01

Maintenance of reduced body weight is associated both with reduced energy expenditure per unit metabolic mass and increased hunger in mice and humans. Lowered circulating leptin concentration, due to decreased fat mass, provides a primary signal for this response. However, leptin deficient (Lepob/ob) mice (and leptin receptor deficient Zucker rats) reduce energy expenditure following weight reduction by a necessarily non-leptin dependent mechanisms. To identify these mechanisms, Lepob/ob mice were fed ad libitum (AL group; n = 21) or restricted to 3 kilocalories of chow per day (CR group, n = 21). After losing 20% of initial weight (in approximately 2 weeks), the CR mice were stabilized at 80% of initial body weight for two weeks by titrated refeeding, and then released from food restriction. CR mice conserved energy (-17% below predicted based on body mass and composition during the day; -52% at night); and, when released to ad libitum feeding, CR mice regained fat and lean mass (to AL levels) within 5 weeks. CR mice did so while their ad libitum caloric intake was equal to that of the AL animals. While calorically restricted, the CR mice had a significantly lower respiratory exchange ratio (RER = 0.89) compared to AL (0.94); after release to ad libitum feeding, RER was significantly higher (1.03) than in the AL group (0.93), consistent with their anabolic state. These results confirm that, in congenitally leptin deficient animals, leptin is not required for compensatory reduction in energy expenditure accompanying weight loss, but suggest that the hyperphagia of the weight-reduced state is leptin-dependent.

Impaired liver regeneration is associated with reduced cyclin B1 in natural killer T cell-deficient mice.

PubMed

Ben Ya'acov, Ami; Meir, Hadar; Zolotaryova, Lydia; Ilan, Yaron; Shteyer, Eyal

2017-03-23

It has been shown that the proportion of natural killer T cells is markedly elevated during liver regeneration and their activation under different conditions can modulate this process. As natural killer T cells and liver injury are central in liver regeneration, elucidating their role is important. The aim of the current study is to explore the role of natural killer T cells in impaired liver regeneration. Concanvalin A was injected 4 days before partial hepatectomy to natural killer T cells- deficient mice or to anti CD1d1-treated mice. Ki-67 and proliferating cell nuclear antigen were used to measure hepatocytes proliferation. Expression of hepatic cyclin B1 and proliferating cell nuclear antigen were evaluated by Western Blot and liver injury was assessed by ALT and histology. Natural killer T cells- deficient or mice injected with anti CD1d antibodies exhibited reduced liver regeneration. These mice were considerably resistant to ConA-induced liver injury. In the absence of NKT cells hepatic proliferating cell nuclear antigen and cyclin B1 decreased in mice injected with Concanvalin A before partial hepatectomy. This was accompanied with reduced serum interleukin-6 levels. Natural killer T cells play an important role in liver regeneration, which is associated with cyclin B1 and interleukin-6.

Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease

PubMed Central

Haase, Volker H.; King, Rhonda; Polyak, Erzsebet; Selak, Mary; Yudkoff, Marc; Hancock, Wayne W.; Meade, Ray; Saiki, Ryoichi; Lunceford, Adam L.; Clarke, Catherine F.; Gasser, David L.

2008-01-01

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment. PMID:18437205

Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

PubMed Central

Sonnemann, Kevin J.; Heun-Johnson, Hanke; Turner, Amy J.; Baltgalvis, Kristen A.; Lowe, Dawn A.; Ervasti, James M.

2009-01-01

Background The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or ΔR4-21 “micro” utrophin (μUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice. Methods and Findings Recombinant TAT-Utr and TAT-μUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-μUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290±920 U versus 5,950±1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%±5% versus 37%±4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%±5% versus 40%±8% drop; PBS versus TAT), and increased specific force production (9.7±1.1 N/cm2 versus 12.8±0.9 N/cm2; PBS versus TAT). Conclusions These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin. PMID:19478831

Experimental approach to IGF-1 therapy in CCl4-induced acute liver damage in healthy controls and mice with partial IGF-1 deficiency.

PubMed

Morales-Garza, Luis A; Puche, Juan E; Aguirre, Gabriel A; Muñoz, Úrsula; García-Magariño, Mariano; De la Garza, Rocío G; Castilla-Cortazar, Inma

2017-05-04

Cell necrosis, oxidative damage, and fibrogenesis are involved in cirrhosis development, a condition in which insulin-like growth factor 1 (IGF-1) levels are diminished. This study evaluates whether the exogenous administration of low doses of IGF-1 can induce hepatoprotection in acute carbon tetrachloride (CCl 4 )-induced liver damage compared to healthy controls (Wt Igf +/+ ). Additionally, the impact of IGF-1 deficiency on a damaged liver was investigated in mice with a partial deficit of this hormone (Hz Igf1 +/- ). Three groups of 25 ± 5-week-old healthy male mice (Wt Igf +/+ ) were included in the protocol: untreated controls (Wt). Controls that received CCl 4 (Wt + CCl 4 ) and Wt + CCl 4 were treated subcutaneously with IGF-1 (2 µg/100 g body weight/day) for 10 days (Wt + CCl 4  + IGF1). In parallel, three IGF-1-deficient mice (Hz Igf1 +/- ) groups were studied: untreated Hz, Hz + CCl 4 , and Hz + CCl 4  + IGF-1. Microarray and real-time quantitative polymerase chain reaction (RT-qPCR) analyses, serum aminotransferases levels, liver histology, and malondialdehyde (MDA) levels were assessed at the end of the treatment in all groups. All data represent mean ± SEM. An altered gene coding expression pattern for proteins of the extracellular matrix, fibrosis, and cellular protection were found, as compared to healthy controls, in which IGF-1 therapy normalized in the series including healthy mice. Liver histology showed that Wt + CCl 4  + IGF1 mice had less oxidative damage, fibrosis, lymphocytic infiltrate, and cellular changes when compared to the Wt + CCl 4 . Moreover, there was a correlation between MDA levels and the histological damage score (Pearson's r = 0.858). In the IGF-1-deficient mice series, similar findings were identified, denoting a much more vulnerable hepatic parenchyma. IGF1 treatment improved the biochemistry, histology, and genetic expression of pro-regenerative and cytoprotective factors in both series

Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout.

PubMed

Ehlken, H; Krishna-Subramanian, S; Ochoa-Callejero, L; Kondylis, V; Nadi, N E; Straub, B K; Schirmacher, P; Walczak, H; Kollias, G; Pasparakis, M

2014-11-01

Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO(LPC-KO) mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO(LPC-KO) mice. To address potential functional redundancies between death receptors we generated and analysed NEMO(LPC-KO) mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO(LPC-KO) mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO(LPC-KO) mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO(LPC-KO) mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving

The Neurofibromatosis Type 1 (Nf1) Tumor Suppressor is a Modifier of Carcinogen-Induced Pigmentation and Papilloma Formation in C57BL/6 Mice

PubMed Central

Atit, Radhika P.; Mitchell, Kent; Nguyen, Lam; Warshawsky, David; Ratner, Nancy

2010-01-01

There is increasing evidence implicating the human NF1 gene in epithelial carcinogenesis. To test if NF1 can play a part in skin tumor formation, we analyzed effects of the skin cancer initiator dimethylbenzanthracene and/or the tumor promoter 12-O-tetradecanoyl-13-acetylphorbol on mice heterozygous for null mutations in Nf1 (Nf1+/−). Mice were on the C57BL/6 background, noted for resistance to chemical carcinogens. Nf1+/− mice (18 of 24) developed papillomas after treatment with dimethylbenzanthracene and 12-O-tetradecanoyl-13-acetylphorbol; papillomas did not develop in wild-type C57BL/6 mice nor Nf1+/− mice treated with 12-O-tetradecanoyl-13-acetylphorbol alone. All papillomas analyzed (six of six) had mutations in codon 61 of H-ras, demonstrating strong cooperation between the Nf1 GTPase activating protein for Ras, neurofibromin, and Ras-GTP. After exposure to 12-O-tetradecanoyl-13-acetylphorbol, Nf1+/− keratinocytes showed significant, sustained, increases in proliferation, implicating Nf1 in phorbol ester responsive pathways. Thus, Nf1 levels regulate the response of keratinocytes to 12-O-tetradecanoyl-13-acetylphorbol. Nf1+/− mice also showed a 2-fold increase in the development of pigmented skin patches stimulated by dimethylbenzanthracene; patches were characterized by hair follicles in anagen phase, implicating keratinocytes in the aberrant hyperpigmentation. Our results show that mutation in the Nf1 gene causes abnormal keratinocyte proliferation that can be revealed by environmental assaults such as carcinogen exposure. The data support a plausible role for NF1 mutation in human epithelial carcinogenesis. PMID:10844550

Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β–Slug signaling

PubMed Central

Cheng, Fang; Shen, Yue; Mohanasundaram, Ponnuswamy; Lindström, Michelle; Ivaska, Johanna; Ny, Tor; Eriksson, John E.

2016-01-01

Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial–mesenchymal transition (EMT), TGF-β1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM−/−) wounds. Correspondingly, VIM−/− wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM−/− wounds. Vimentin reconstitution in VIM−/− fibroblasts restored both their proliferation and TGF-β1 production. Similarly, restoring paracrine TGF-β–Slug–EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-β1–Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation. PMID:27466403

Protease-Activated Receptor-2 Deficiency Attenuates Atherosclerotic Lesion Progression and Instability in Apolipoprotein E-Deficient Mice

PubMed Central

Zuo, Pengfei; Zuo, Zhi; Zheng, Yueyue; Wang, Xin; Zhou, Qianxing; Chen, Long; Ma, Genshan

2017-01-01

Inflammatory mechanisms are involved in the process of atherosclerotic plaque formation and rupture. Accumulating evidence suggests that protease-activated receptor (PAR)-2 contributes to the pathophysiology of chronic inflammation on the vasculature. To directly examine the role of PAR-2 in atherosclerosis, we generated apolipoprotein E/PAR-2 double-deficient mice. Mice were fed with high-fat diet for 12 weeks starting at ages of 6 weeks. PAR-2 deficiency attenuated atherosclerotic lesion progression with reduced total lesion area, reduced percentage of stenosis and reduced total necrotic core area. PAR-2 deficiency increased fibrous cap thickness and collagen content of plaque. Moreover, PAR-2 deficiency decreased smooth muscle cell content, macrophage accumulation, matrix metallopeptidase-9 expression and neovascularization in plaque. Relative quantitative PCR assay using thoracic aorta revealed that PAR-2 deficiency reduced mRNA expression of inflammatory molecules, such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. In vitro experiment, we found that PAR-2 deficiency reduced mRNA expression of interferon-γ, interleukin-6, TNF-α and MCP-1 in macrophage under unstimulated and lipopolysaccharide-stimulated conditions. These results suggest that PAR-2 deficiency attenuates the progression and instability of atherosclerotic plaque. PMID:28959204

Live imaging of transforming growth factor-β activated kinase 1 activation in Lewis lung carcinoma 3LL cells implanted into syngeneic mice and treated with polyinosinic:polycytidylic acid.

PubMed

Takaoka, Saori; Kamioka, Yuji; Takakura, Kanako; Baba, Ai; Shime, Hiroaki; Seya, Tsukasa; Matsuda, Michiyuki

2016-05-01

Transforming growth factor-β activated kinase 1 (TAK1) has been shown to play a crucial role in cell death, differentiation, and inflammation. Here, we live-imaged robust TAK1 activation in Lewis lung carcinoma 3LL cells implanted into the s.c. tissue of syngeneic C57BL/6 mice and treated with polyinosinic:polycytidylic acid (PolyI:C). First, we developed and characterized a Förster resonance energy transfer-based biosensor for TAK1 activity. The TAK1 biosensor, named Eevee-TAK1, responded to stress-inducing reagents such as anisomycin, tumor necrosis factor-α, and interleukin1-β. The anisomycin-induced increase in Förster resonance energy transfer was abolished by the TAK1 inhibitor (5z)-7-oxozeaenol. Activity of TAK1 in 3LL cells was markedly increased by PolyI:C in the presence of macrophages. 3LL cells expressing Eevee-TAK1 were implanted into mice and observed through imaging window by two-photon excitation microscopy. During the growth of tumor, the 3LL cells at the periphery of the tumor showed higher TAK1 activity than the 3LL cells located at the center of the tumor, suggesting that cells at the periphery of the tumor mass were under stronger stress. Injection of PolyI:C, which is known to induce regression of the implanted tumors, induced marked and homogenous TAK1 activation within the tumor tissues. The effect of PolyI:C faded within 4 days. These observations suggest that Eevee-TAK1 is a versatile tool to monitor cellular stress in cancer tissues. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Characterization and functional analysis of cellular immunity in mice with biotinidase deficiency.

PubMed

Pindolia, Kirit; Li, Hong; Cardwell, Cisley; Wolf, Barry

2014-05-01

Biotinidase deficiency is an autosomal recessively inherited metabolic disorder that can be easily and effectively treated with pharmacological doses of the vitamin, biotin. Untreated children with profound biotinidase deficiency may exhibit neurological, cutaneous and cellular immunological abnormalities, specifically candida infections. To better understand the immunological dysfunction in some symptomatic individuals with biotinidase deficiency, we studied various aspects of immunological function in a genetically engineered knock-out mouse with biotinidase deficiency. The mouse has no detectable biotinidase activity and develops neurological and cutaneous symptoms similar to those seen in symptomatic children with the disorder. Mice with profound biotinidase deficiency on a biotin-restricted diet had smaller thymuses and spleens than identical mice fed a biotin-replete diet or wildtype mice on either diet; however, the organ to body weight ratios were not significantly different. Thymus histology was normal. Splenocyte subpopulation study showed a significant increase in CD4 positive cells. In addition, in vitro lymphocyte proliferation assays consistently showed diminished proliferation in response to various immunological stimuli. Not all symptomatic individuals with profound biotinidase deficiency develop immunological dysfunction; however, our results do show significant alterations in cellular immunological function that may contribute and/or provide a mechanism(s) for the cellular immunity abnormalities in individuals with biotinidase deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes.

PubMed

Dahlhoff, Maik; Gaborit, Nadège; Bultmann, Sebastian; Leonhardt, Heinrich; Yarden, Yosef; Schneider, Marlon R

2017-10-01

While the epidermal growth factor receptor (EGFR) is an established regulator of skin development and homeostasis, the functions of the related tyrosine kinase receptors ERBB2 and ERBB3 in this tissue have only recently been examined. Previously reported, skin-specific deletion of each of these receptors in mice resulted in similar defects in keratinocyte proliferation and migration, resulting in impaired wound healing and tumorigenesis. Because both ERBB2 and ERBB3 are targets for treating an array of cancer types, it is important to examine the consequences of receptor inhibition in human keratinocytes. Here, we employed the CRISPR/Cas9 technology to generate HaCaT cells (an established human keratinocyte cell line) lacking ERBB2 or ERBB3. HaCaT clones lacking ERBB2 or ERBB3 showed comparable reductions in cell proliferation as assessed by BrdU staining. Apoptosis, in contrast, was reduced in ERBB3-deficient HaCaT cells only. Assessment of cell migration using a wound healing (scratch) assay showed that the closure of the wound gaps was completed by 48 h in mock and in ERBB3 knockout clones. In contrast, this process was considerably delayed in ERBB2 knockout clones, and a complete closure of the gap in the latter cells did not occur before 72 h. In conclusion, both ERBB2 and ERBB3 are essential for normal proliferation of skin keratinocytes, but in contrast to ERBB3, ERBB2 is essential for migration of human keratinocytes. These observations might bear significance to patient adverse effects of therapeutic agents targeting ERBB2 and ERBB3. © 2017 Federation of European Biochemical Societies.

Losartan and captopril treatment rescue normal thrombus formation in microfibril associated glycoprotein-1 (MAGP1) deficient mice.

PubMed

Vassequi-Silva, Tallita; Pereira, Danielle Sousa; Nery Diez, Ana Cláudia C; Braga, Guilherme G; Godoy, Juliana A; Mendes, Camila B; Dos Santos, Leonardo; Krieger, José E; Antunes, Edson; Costa, Fábio T M; Vicente, Cristina P; Werneck, Claudio C

2016-02-01

MAGP1 is a glycoprotein present in the elastic fibers and is a part of the microfibrils components. MAGP1 interacts with von Willebrand factor and the active form of TGF-β and BMP. In mice lacking MAGP1, thrombus formation is delayed, increasing the occlusion time of carotid artery despite presenting normal blood coagulation in vitro. MAGP1-containing microfibrils may play a role in hemostasis and thrombosis. In this work, we evaluated the function of MAGP1 and its relation to TGF-β in the arterial thrombosis process. We analyzed thrombus formation time in wild type and MAGP1-deficient mice comparing Rose Bengal and Ferric Chloride induced arterial lesion. The potential participation of TGF-β in this process was accessed when we treated both wild type and MAGP1-deficient mice with losartan (an antihypertensive drug that decreases TGF-β activity) or captopril (an angiotensin converting enzyme inhibitor that was used as a control antihypertensive drug). Besides, we evaluated thrombus embolization and the gelatinolytic activity in the arterial walls in vitro and ex vivo. Losartan and captopril were able to recover the thrombus formation time without changing blood pressure, activated partial thromboplastin time (aPTT), PT (prothrombin time), platelet aggregation and adhesion, but decreased gelatinase activity. Our results suggest that both treatments are effective in the prevention of the sub-endothelial ECM degradation, allowing the recovery of normal thrombus formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes.

PubMed

Calapre, Leslie; Gray, Elin S; Kurdykowski, Sandrine; David, Anthony; Descargues, Pascal; Ziman, Mel

2017-06-10

Exposure to heat stress after UVB irradiation induces a reduction of apoptosis, resulting in survival of DNA damaged human keratinocytes. This heat-mediated evasion of apoptosis appears to be mediated by activation of SIRT1 and inactivation of p53 signalling. In this study, we assessed the role of SIRT1 in the inactivation of p53 signalling and impairment of DNA damage response in UVB plus heat exposed keratinocytes. Activation of SIRT1 after multiple UVB plus heat exposures resulted in increased p53 deacetylation at K382, which is known to affect its binding to specific target genes. Accordingly, we noted decreased apoptosis and down regulation of the p53 targeted pro-apoptotic gene BAX and the DNA repair genes ERCC1 and XPC after UVB plus heat treatments. In addition, UVB plus heat induced increased expression of the cell survival gene Survivin and the proliferation marker Ki67. Notably, keratinocytes exposed to UVB plus heat in the presence of the SIRT1 inhibitor, Ex-527, showed a similar phenotype to those exposed to UV alone; i.e. an increase in p53 acetylation, increased apoptosis and low levels of Survivin. This study demonstrate that heat-induced SIRT1 activation mediates survival of DNA damaged keratinocytes through deacetylation of p53 after exposure to UVB plus heat.

γδ T-cell-deficient mice show alterations in mucin expression, glycosylation, and goblet cells but maintain an intact mucus layer.

PubMed

Kober, Olivia I; Ahl, David; Pin, Carmen; Holm, Lena; Carding, Simon R; Juge, Nathalie

2014-04-01

Intestinal homeostasis is maintained by a hierarchy of immune defenses acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion. In this study, we used γδ T-cell-deficient (TCRδ(-/-)) mice to examine whether and how γδ T-cells modulate the properties of the intestinal mucus layer. Increased susceptibility of TCRδ(-/-) mice to dextran sodium sulfate (DSS)-induced colitis is associated with a reduced number of goblet cells. Alterations in the number of goblet cells and crypt lengths were observed in the small intestine and colon of TCRδ(-/-) mice compared with C57BL/6 wild-type (WT) mice. Addition of keratinocyte growth factor to small intestinal organoid cultures from TCRδ(-/-) mice showed a marked increase in crypt growth and in both goblet cell number and redistribution along the crypts. There was no apparent difference in the thickness or organization of the mucus layer between TCRδ(-/-) and WT mice, as measured in vivo. However, γδ T-cell deficiency led to reduced sialylated mucins in association with increased gene expression of gel-secreting Muc2 and membrane-bound mucins, including Muc13 and Muc17. Collectively, these data provide evidence that γδ T cells play an important role in the maintenance of mucosal homeostasis by regulating mucin expression and promoting goblet cell function in the small intestine.

C/EBPα regulates CRL4Cdt2-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint

PubMed Central

Hall, Jonathan R; Bereman, Michael S; Nepomuceno, Angelito I; Thompson, Elizabeth A; Muddiman, David C; Smart, Robert C

2014-01-01

The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4Cdt2. Cdt2 is the substrate recognition subunit of CRL4Cdt2 and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21. PMID:25483090

Cited1 Deficiency Suppresses Intestinal Tumorigenesis

PubMed Central

Young, Madeleine; Poetz, Oliver; Parry, Lee; Jenkins, John R.; Williams, Geraint T.; Dunwoodie, Sally L.; Watson, Alastair; Clarke, Alan R.

2013-01-01

Conditional deletion of Apc in the murine intestine alters crypt-villus architecture and function. This process is accompanied by multiple changes in gene expression, including upregulation of Cited1, whose role in colorectal carcinogenesis is unknown. Here we explore the relevance of Cited1 to intestinal tumorigenesis. We crossed Cited1 null mice with ApcMin/+ and AhCre+Apcfl/fl mice and determined the impact of Cited1 deficiency on tumour growth/initiation including tumour multiplicity, cell proliferation, apoptosis and the transcriptome. We show that Cited1 is up-regulated in both human and murine tumours, and that constitutive deficiency of Cited1 increases survival in ApcMin/+ mice from 230.5 to 515 days. However, paradoxically, Cited1 deficiency accentuated nearly all aspects of the immediate phenotype 4 days after conditional deletion of Apc, including an increase in cell death and enhanced perturbation of differentiation, including of the stem cell compartment. Transcriptome analysis revealed multiple pathway changes, including p53, PI3K and Wnt. The activation of Wnt through Cited1 deficiency correlated with increased transcription of β-catenin and increased levels of dephosphorylated β-catenin. Hence, immediately following deletion of Apc, Cited1 normally restrains the Wnt pathway at the level of β-catenin. Thus deficiency of Cited1 leads to hyper-activation of Wnt signaling and an exaggerated Wnt phenotype including elevated cell death. Cited1 deficiency decreases intestinal tumourigenesis in ApcMin/+ mice and impacts upon a number of oncogenic signaling pathways, including Wnt. This restraint imposed by Cited1 is consistent with a requirement for Cited1 to constrain Wnt activity to a level commensurate with optimal adenoma formation and maintenance, and provides one mechanism for tumour repression in the absence of Cited1. PMID:23935526

Leptin deficiency suppresses MMTV-Wnt-1 mammary tumor growth in obese mice and abrogates tumor initiating cell survival.

PubMed

Zheng, Qiao; Dunlap, Sarah M; Zhu, Jinling; Downs-Kelly, Erinn; Rich, Jeremy; Hursting, Stephen D; Berger, Nathan A; Reizes, Ofer

2011-08-01

Obesity increases both the risk and mortality associated with many types of cancer including that of the breast. In mice, obesity increases both incidence of spontaneous tumors and burden of transplanted tumors. Our findings identify leptin, an adipose secreted cytokine, in promoting increased mammary tumor burden in obese mice and provide a link between this adipokine and cancer. Using a transplantable tumor that develops spontaneously in the murine mammary tumor virus-Wnt-1 transgenic mice, we show that tumors transplanted into obese leptin receptor (LepRb)-deficient (db/db) mice grow to eight times the volume of tumors transplanted into lean wild-type (WT) mice. However, tumor outgrowth and overall tumor burden is reduced in obese, leptin-deficient (ob/ob) mice. The residual tumors in ob/ob mice contain fewer undifferentiated tumor cells (keratin 6 immunopositive) compared with WT or db/db mice. Furthermore, tumors in ob/ob mice contain fewer cells expressing phosphorylated Akt, a growth promoting kinase activated by the LepRb, compared with WT and db/db mice. In vivo limiting dilution analysis of residual tumors from ob/ob mice indicated reduced tumor initiating activity suggesting fewer cancer stem cells (CSCs). The tumor cell populations reduced by leptin deficiency were identified by fluorescence-activated cell sorting and found to express LepRb. Finally, LepRb expressing tumor cells exhibit stem cell characteristics based on the ability to form tumorspheres in vitro and leptin promotes their survival. These studies provide critical new insight on the role of leptin in tumor growth and implicate LepRb as a CSC target.

MicroRNA-146a alleviates chronic skin inflammation in atopic dermatitis through suppression of innate immune responses in keratinocytes.

PubMed

Rebane, Ana; Runnel, Toomas; Aab, Alar; Maslovskaja, Julia; Rückert, Beate; Zimmermann, Maya; Plaas, Mario; Kärner, Jaanika; Treis, Angela; Pihlap, Maire; Haljasorg, Uku; Hermann, Helen; Nagy, Nikoletta; Kemeny, Lajos; Erm, Triin; Kingo, Külli; Li, Mei; Boldin, Mark P; Akdis, Cezmi A

2014-10-01

Chronic skin inflammation in atopic dermatitis (AD) is associated with elevated expression of proinflammatory genes and activation of innate immune responses in keratinocytes. microRNAs (miRNAs) are short, single-stranded RNA molecules that silence genes via the degradation of target mRNAs or inhibition of translation. The aim of this study was to investigate the role of miR-146a in skin inflammation in AD. RNA and protein expression was analyzed using miRNA and mRNA arrays, RT-quantitative PCR, Western blotting, and immunonohistochemistry. Transfection of miR-146a precursors and inhibitors into human primary keratinocytes, luciferase assays, and MC903-dependent mouse model of AD were used to study miR-146a function. We show that miR-146a expression is increased in keratinocytes and chronic lesional skin of patients with AD. miR-146a inhibited the expression of numerous proinflammatory factors, including IFN-γ-inducible and AD-associated genes CCL5, CCL8, and ubiquitin D (UBD) in human primary keratinocytes stimulated with IFN-γ, TNF-α, or IL-1β. In a mouse model of AD, miR-146a-deficient mice developed stronger inflammation characterized by increased accumulation of infiltrating cells in the dermis, elevated expression of IFN-γ, CCL5, CCL8, and UBD in the skin, and IFN-γ, IL-1β, and UBD in draining lymph nodes. Both tissue culture and in vivo experiments in mice demonstrated that miR-146a-mediated suppression in allergic skin inflammation partially occurs through direct targeting of upstream nuclear factor kappa B signal transducers caspase recruitment domain-containing protein 10 and IL-1 receptor-associated kinase 1. In addition, human CCL5 was determined as a novel, direct target of miR-146a. Our data demonstrate that miR-146a controls nuclear factor kappa B-dependent inflammatory responses in keratinocytes and chronic skin inflammation in AD. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights

Cyclocreatine treatment improves cognition in mice with creatine transporter deficiency

PubMed Central

Kurosawa, Yuko; DeGrauw, Ton J.; Lindquist, Diana M.; Blanco, Victor M.; Pyne-Geithman, Gail J.; Daikoku, Takiko; Chambers, James B.; Benoit, Stephen C.; Clark, Joseph F.

2012-01-01

The second-largest cause of X-linked mental retardation is a deficiency in creatine transporter (CRT; encoded by SLC6A8), which leads to speech and language disorders with severe cognitive impairment. This syndrome, caused by the absence of creatine in the brain, is currently untreatable because CRT is required for creatine entry into brain cells. Here, we developed a brain-specific Slc6a8 knockout mouse (Slc6a8–/y) as an animal model of human CRT deficiency in order to explore potential therapies for this syndrome. The phenotype of the Slc6a8–/y mouse was comparable to that of human patients. We successfully treated the Slc6a8–/y mice with the creatine analog cyclocreatine. Brain cyclocreatine and cyclocreatine phosphate were detected after 9 weeks of cyclocreatine treatment in Slc6a8–/y mice, in contrast to the same mice treated with creatine or placebo. Cyclocreatine-treated Slc6a8–/y mice also exhibited a profound improvement in cognitive abilities, as seen with novel object recognition as well as spatial learning and memory tests. Thus, cyclocreatine appears promising as a potential therapy for CRT deficiency. PMID:22751104

B-vitamin deficiency is protective against DSS-induced colitis in mice

PubMed Central

Benight, Nancy M.; Stoll, Barbara; Chacko, Shaji; da Silva, Vanessa R.; Marini, Juan C.; Gregory, Jesse F.; Stabler, Sally P.

2011-01-01

Vitamin deficiencies are common in patients with inflammatory bowel disease (IBD). Homocysteine (Hcys) is a thrombogenic amino acid produced from methionine (Met), and its increase in patients with IBD indicates a disruption of Met metabolism; however, the role of Hcys and Met metabolism in IBD is not well understood. We hypothesized that disrupted Met metabolism from a B-vitamin-deficient diet would exacerbate experimental colitis. Mice were fed a B6-B12-deficient or control diet for 2 wk and then treated with dextran sodium sulfate (DSS) to induce colitis. We monitored disease activity during DSS treatment and collected plasma and tissue for analysis of inflammatory tissue injury and Met metabolites. We also quantified Met cycle activity by measurements of in vivo Met kinetics using [1-13C-methyl-2H3]methionine infusion in similarly treated mice. Unexpectedly, we found that mice given the B-vitamin-deficient diet had improved clinical outcomes, including increased survival, weight maintenance, and reduced disease scores. We also found lower histological disease activity and proinflammatory gene expression (TNF-α and inducible nitric oxide synthase) in the colon in deficient-diet mice. Metabolomic analysis showed evidence that these effects were associated with deficient B6, as markers of B12 function were only mildly altered. In vivo methionine kinetics corroborated these results, showing that the deficient diet suppressed transsulfuration but increased remethylation. Our findings suggest that disrupted Met metabolism attributable to B6 deficiency reduces the inflammatory response and disease activity in DSS-challenged mice. These results warrant further human clinical studies to determine whether B6 deficiency and elevated Hcys in patients with IBD contribute to disease pathobiology. PMID:21596995

Neuregulin 1 Deficiency Modulates Adolescent Stress-Induced Dendritic Spine Loss in a Brain Region-Specific Manner and Increases Complement 4 Expression in the Hippocampus.

PubMed

Clarke, David J; Chohan, Tariq W; Kassem, Mustafa S; Smith, Kristie L; Chesworth, Rose; Karl, Tim; Kuligowski, Michael P; Fok, Sandra Y; Bennett, Maxwell R; Arnold, Jonathon C

2018-03-16

One neuropathological feature of schizophrenia is a diminished number of dendritic spines in the prefrontal cortex and hippocampus. The neuregulin 1 (Nrg1) system is involved in the plasticity of dendritic spines, and chronic stress decreases dendritic spine densities in the prefrontal cortex and hippocampus. Here, we aimed to assess whether Nrg1 deficiency confers vulnerability to the effects of adolescent stress on dendritic spine plasticity. We also assessed other schizophrenia-relevant neurobiological changes such as microglial cell activation, loss of parvalbumin (PV) interneurons, and induction of complement factor 4 (C4). Adolescent male wild-type (WT) and Nrg1 heterozygous mice were subjected to chronic restraint stress before their brains underwent Golgi impregnation or immunofluorescent staining of PV interneurons, microglial cells, and C4. Stress in WT mice promoted dendritic spine loss and microglial cell activation in the prefrontal cortex and the hippocampus. However, Nrg1 deficiency rendered mice resilient to stress-induced dendritic spine loss in the infralimbic cortex and the CA3 region of the hippocampus without affecting stress-induced microglial cell activation in these brain regions. Nrg1 deficiency and adolescent stress combined to trigger increased dendritic spine densities in the prelimbic cortex. In the hippocampal CA1 region, Nrg1 deficiency accentuated stress-induced dendritic spine loss. Nrg1 deficiency increased C4 protein and decreased C4 mRNA expression in the hippocampus, and the number of PV interneurons in the basolateral amygdala. This study demonstrates that Nrg1 modulates the impact of stress on the adolescent brain in a region-specific manner. It also provides first evidence of a link between Nrg1 and C4 systems in the hippocampus.

Epidermal cornification is preceded by the expression of a keratinocyte-specific set of pyroptosis-related genes.

PubMed

Lachner, Julia; Mlitz, Veronika; Tschachler, Erwin; Eckhart, Leopold

2017-12-12

The homeostasis of the epidermis depends on keratinocyte differentiation and cornification, a mode of programmed cell death that does not elicit inflammation. Here, we report that cornification is associated with the expression of specific genes that control multiple steps of pyroptosis, another form of cell death that involves the processing and release of interleukin-1 family (IL1F) cytokines. Expression levels of pro-inflammatory IL1A and IL1B and of the pyroptotic pore-forming gasdermin (GSDM) D were downregulated during terminal differentiation of human keratinocytes in vitro. By contrast, negative regulators of IL-1 processing, including NLR family pyrin domain containing 10 (NLRP10) and pyrin domain-containing 1 (PYDC1), the anti-inflammatory IL1F members IL-37 (IL1F7) and IL-38 (IL1F10), and GSDMA, were strongly induced in differentiated keratinocytes. In human tissues, these keratinocyte differentiation-associated genes are expressed in the skin at higher levels than in any other organ, and mammalian species, that have lost the epidermal cornification program during evolution, i.e. whales and dolphins, lack homologs of these genes. Together, our results suggest that human epidermal cornification is accompanied by a tight control of pyroptosis and warrant further studies of potential defects in the balance between cornification and pyroptosis in skin pathologies.

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway.

PubMed

Yang, Hongpeng; Guo, Yue; Wang, Dawei; Yang, Xiaofei; Ha, Chengzhi

2018-01-02

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.

Impact of PACAP and PAC1 Receptor Deficiency on the Neurochemical and Behavioral Effects of Acute and Chronic Restraint Stress in Male C57BL/6 Mice

PubMed Central

Mustafa, Tomris; Jiang, Sunny Zhihong; Eiden, Adrian M.; Weihe, Eberhard; Thistlethwaite, Ian; Eiden, Lee E.

2016-01-01

Acute restraint stress (ARS) for 3 hours causes CORT elevation in venous blood, which is accompanied by Fos up-regulation in the paraventricular nucleus (PVN) of male C57BL/6 mice. CORT elevation by ARS is attenuated in PACAP-deficient mice, but unaffected in PAC1-deficient mice. Correspondingly, Fos up-regulation by ARS is greatly attenuated in PACAP-deficient mice, but much less so in PAC1-deficient animals. We noted that both PACAP- and PAC1-deficiency greatly attenuate CORT elevation after ARS when CORT measurements are performed on trunk blood following euthanasia by abrupt cervical separation: this latter observation is of critical importance in assessing the role of PACAP neurotransmission in ARS, based on previous reports in which serum CORT was sampled from trunk blood. Seven days of chronic restraint stress (CRS) induces non-habituating CORT elevation, and weight loss consequent to hypophagia, in wild-type male C57BL/6 mice. Both CORT elevation and weight loss following seven day CRS are severely blunted in PACAP-deficient mice, but only slightly in PAC1 deficient mice. However, longer periods of daily restraint (14–21 days) resulted in sustained weight loss and elevated CORT in wild-type mice, and these effects of long-term chronic stress were attenuated or abolished in both PACAP- and PAC1-deficient mice. We conclude that while a PACAP receptor in addition to PAC1 may mediate some of the PACAP-dependent central effects of acute restraint stress and short-term (<7 days) chronic restraint stress on the HPA axis, the PAC1 receptor plays a prominent role in mediating PACAP-dependent HPA axis activation, and hypophagia, during long-term (>7 days) chronic restraint stress. PMID:25853791

Impact of PACAP and PAC1 receptor deficiency on the neurochemical and behavioral effects of acute and chronic restraint stress in male C57BL/6 mice.

PubMed

Mustafa, Tomris; Jiang, Sunny Zhihong; Eiden, Adrian M; Weihe, Eberhard; Thistlethwaite, Ian; Eiden, Lee E

2015-01-01

Acute restraint stress (ARS) for 3 h causes corticosterone (CORT) elevation in venous blood, which is accompanied by Fos up-regulation in the paraventricular nucleus (PVN) of male C57BL/6 mice. CORT elevation by ARS is attenuated in PACAP-deficient mice, but unaffected in PAC1-deficient mice. Correspondingly, Fos up-regulation by ARS is greatly attenuated in PACAP-deficient mice, but much less so in PAC1-deficient animals. We noted that both PACAP- and PAC1-deficiency greatly attenuate CORT elevation after ARS when CORT measurements are performed on trunk blood following euthanasia by abrupt cervical separation: this latter observation is of critical importance in assessing the role of PACAP neurotransmission in ARS, based on previous reports in which serum CORT was sampled from trunk blood. Seven days of chronic restraint stress (CRS) induces non-habituating CORT elevation, and weight loss consequent to hypophagia, in wild-type male C57BL/6 mice. Both CORT elevation and weight loss following 7-day CRS are severely blunted in PACAP-deficient mice, but only slightly in PAC1-deficient mice. However, longer periods of daily restraint (14-21 days) resulted in sustained weight loss and elevated CORT in wild-type mice, and these effects of long-term chronic stress were attenuated or abolished in both PACAP- and PAC1-deficient mice. We conclude that while a PACAP receptor in addition to PAC1 may mediate some of the PACAP-dependent central effects of ARS and short-term (<7 days) CRS on the hypothalamo-pituitary-adrenal (HPA) axis, the PAC1 receptor plays a prominent role in mediating PACAP-dependent HPA axis activation, and hypophagia, during long-term (>7 days) CRS.

Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice.

PubMed

Karshovska, Ela; Zhao, Zhen; Blanchet, Xavier; Schmitt, Martin M N; Bidzhekov, Kiril; Soehnlein, Oliver; von Hundelshausen, Philipp; Mattheij, Nadine J; Cosemans, Judith M E M; Megens, Remco T A; Koeppel, Thomas A; Schober, Andreas; Hackeng, Tilman M; Weber, Christian; Koenen, Rory R

2015-02-13

Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbβ3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity. This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbβ3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbβ3 signaling in vitro. Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease. © 2014 American Heart Association, Inc.

Entry mechanisms of herpes simplex virus 1 into murine epidermis: involvement of nectin-1 and herpesvirus entry mediator as cellular receptors.

PubMed

Petermann, Philipp; Thier, Katharina; Rahn, Elena; Rixon, Frazer J; Bloch, Wilhelm; Özcelik, Semra; Krummenacher, Claude; Barron, Martin J; Dixon, Michael J; Scheu, Stefanie; Pfeffer, Klaus; Knebel-Mörsdorf, Dagmar

2015-01-01

Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo. The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin and whether both nectin-1 and HVEM are involved are not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1-deficient epidermis, entry was strongly reduced. Almost no entry was observed, however, in nectin-1-deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1-deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis, while HVEM has a more limited role. For primary murine keratinocytes, on which nectin-1 acts as a single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we concluded that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, but the restricted presence of HVEM can potentially replace nectin-1 as a receptor, illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo. Herpes simplex virus (HSV) can cause a range of diseases in humans, from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV, and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing

Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice.

PubMed

Westerterp, Marit; Murphy, Andrew J; Wang, Mi; Pagler, Tamara A; Vengrenyuk, Yuliya; Kappus, Mojdeh S; Gorman, Darren J; Nagareddy, Prabhakara R; Zhu, Xuewei; Abramowicz, Sandra; Parks, John S; Welch, Carrie; Fisher, Edward A; Wang, Nan; Yvan-Charvet, Laurent; Tall, Alan R

2013-05-24

Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. To assess the role of macrophage cholesterol efflux pathways in atherogenesis. We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABC(DKO) mice) but not in hematopoietic stem or progenitor populations. MAC-ABC(DKO) bone marrow (BM) was transplanted into Ldlr(-/-) recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet-fed MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production. These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

Sulforaphane suppresses ultraviolet B-induced inflammation in HaCaT keratinocytes and HR-1 hairless mice.

PubMed

Shibata, Akira; Nakagawa, Kiyotaka; Yamanoi, Hiroko; Tsuduki, Tsuyoshi; Sookwong, Phumon; Higuchi, Ohki; Kimura, Fumiko; Miyazawa, Teruo

2010-08-01

Ultraviolet B (UVB) irradiation induces skin damage and inflammation. One way to reduce the inflammation is via the use of molecules termed photochemopreventive agents. Sulforaphane (4-methylsulfinylbutyl isothiocyanate, SF), which is found in cruciferous vegetables, is known for its potent physiological properties. This study was designed to evaluate the effect of SF on skin inflammation in vitro and in vivo. In in vitro study using immortalized human keratinocytes (HaCaT), UVB caused marked inflammatory responses [i.e., decrease of HaCaT viability and increase of production of an inflammatory marker interleukin-6 (IL-6)]. SF recovered the cell proliferation and suppressed the IL-6 production. These anti-inflammatory effects of SF were explained by its ability to reduce UVB-induced inflammatory gene expressions [IL-6, IL-1beta and cyclooxgenase-2 (COX-2)]. Because SF seems to have an impact on COX-2 expression, we focused on COX-2 and found that SF reduced UVB-induced COX-2 protein expression. In support of this, PGE(2) released from HaCaT was suppressed by SF. Western blot analysis revealed that SF inhibited p38, ERK and SAPK/JNK activation, indicating that the inhibition of mitogen-activated protein kinases (MAPK) by SF would attenuate the expression of inflammatory mediators (e.g., COX-2), thereby reducing inflammatory responses. Moreover, we conducted skin thickening assay using HR-1 hairless mice and found that UVB-induced skin thickness, COX-2 protein expression and hyperplasia were all suppressed by feeding SF to the mice. These results suggest that SF has a potential use as a compound for protection against UVB-induced skin inflammation. Copyright 2010 Elsevier Inc. All rights reserved.

Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.

PubMed

Brauweiler, Anne M; Bin, Lianghua; Kim, Byung Eui; Oyoshi, Michiko K; Geha, Raif S; Goleva, Elena; Leung, Donald Y M

2013-02-01

The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Anti-inflammatory and cytoprotective effects of a squalene synthase inhibitor, TAK-475 active metabolite-I, in immune cells simulating mevalonate kinase deficiency (MKD)-like condition.

PubMed

Suzuki, Nobutaka; Ito, Tatsuo; Matsui, Hisanori; Takizawa, Masayuki

2016-01-01

TAK-475 (lapaquistat acetate) and its active metabolite-I (TAK-475 M-I) inhibit squalene synthase, which catalyzes the conversion of farnesyl diphosphate (FPP) to squalene. FPP is a substrate for synthesis of other mevalonate-derived isoprenoids (MDIs) such as farnesol (FOH), geranlygeranyl diphosphate (GGPP), and geranylgeraniol. In patients with MKD, a rare autosomal recessive disorder, defective activity of mevalonate kinase leads to a shortage of MDIs. MDIs especially GGPP are required for prenylation of proteins, which is a posttranslation modification necessary for proper functioning of proteins like small guanosine triphosphatases. Malfunction of prenylation of proteins results in upregulation of the inflammatory cascade, leading to increased production of proinflammatory cytokines like interleukin-1β (IL-1β), eventually leading to episodic febrile attacks. In vitro, TAK-475 M-I incubation in a concentration dependent manner increased levels of FPP, GGPP, and FOH in human monocytic THP-1 cells. In subsequent experiments, THP-1 cells or human peripheral blood mononuclear cells (PBMCs) were incubated with simvastatin, which inhibits hydroxymethylglutaryl-coenzyme A reductase and thereby decreases levels of the precursors of MDIs, leading to the depletion of MDIs as expected in MKD patients. Increased levels of GGPP and FPP attenuated lipopolysaccharide (LPS)-induced IL-1β production in THP-1 cells and human PBMCs in statin-treated conditions. The MDIs also significantly reduced the damaged cell ratio in this active MKD-like condition. Moreover, TAK-475 M-I directly inhibited LPS-induced IL-1β production from statin-treated THP-1 cells. These results show anti-inflammatory and cytoprotective effects of MDIs via TAK-475 M-I treatment in statin-treated immune cells, suggesting that possible therapeutic effects of TAK-475 treatment in MKD patients.

Autophagy-deficient mice develop multiple liver tumors

PubMed Central

Takamura, Akito; Komatsu, Masaaki; Hara, Taichi; Sakamoto, Ayako; Kishi, Chieko; Waguri, Satoshi; Eishi, Yoshinobu; Hino, Okio; Tanaka, Keiji; Mizushima, Noboru

2011-01-01

Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7−/− mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7−/− liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression. PMID:21498569

Gender Dependent Evaluation of Autism like Behavior in Mice Exposed to Prenatal Zinc Deficiency

PubMed Central

Grabrucker, Stefanie; Boeckers, Tobias M.; Grabrucker, Andreas M.

2016-01-01

Zinc deficiency has recently been linked to the etiology of autism spectrum disorders (ASD) as environmental risk factor. With an estimated 17% of the world population being at risk of zinc deficiency, especially zinc deficiency during pregnancy might be a common occurrence, also in industrialized nations. On molecular level, zinc deficiency has been shown to affect a signaling pathway at glutamatergic synapses that has previously been identified through genetic mutations in ASD patients, the Neurexin-Neuroligin-Shank pathway, via altering zinc binding Shank family members. In particular, prenatal zinc deficient but not acute zinc deficient animals have been reported to display autism like behavior in some behavioral tests. However, a full behavioral analysis of a possible autism like behavior has been lacking so far. Here, we performed an extensive behavioral phenotyping of mice born from mothers with mild zinc deficiency during all trimesters of pregnancy. Prenatal zinc deficient animals were investigated as adults and gender differences were assessed. Our results show that prenatal zinc deficient mice display increased anxiety, deficits in nest building and various social interaction paradigm, as well as mild alterations in ultrasonic vocalizations. A gender specific analysis revealed only few sex specific differences. Taken together, given that similar behavioral abnormalities as reported here are frequently observed in ASD mouse models, we conclude that prenatal zinc deficient animals even without specific genetic susceptibility for ASD, already show some features of ASD like behavior. PMID:26973485

Keratinocyte response to immobilized growth factors for enhanced dermal wound healing

NASA Astrophysics Data System (ADS)

Stefonek-Puccinelli, Tracy Jane

Chronic wounds cost billions of dollars per year to treat and wound care is limited to ineffective and/or expensive options. Chronic wounds are characterized by a failure to reepithelialize, as well as deficiencies in growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1), normally present during wound healing. Our system described herein begins to tackle the problems associated with designing bioactive materials for chronic wound healing applications. We show that we can induce accelerated keratinocyte migration with photo-immobilized EGF and further control migration speed through the culture of cells on different types of gradient patterns of EGF. We also successfully immobilized IGF-1 while retaining its bioactivity, and further showed it induces directed keratinocyte migration, although not as potently as immobilized EGF. Potential synergy between co-immobilized IGF-1 and EGF was also investigated, although EGF continued to dominate the cellular response, and no significant increase in cell migration was achieved via the addition of IGF-1 to the system. To further understand cellular response to our immobilized growth factors, we investigated keratinocyte signaling and function in response to changes in EGF presentation. It was found that immobilized and soluble EGF can play different, yet complementary, roles in regulating keratinocyte function. Specifically, keratinocytes responded to immobilized EGF with high EGF receptor (EGFR) activation, accompanied by low proliferation and high migratory activity. In contrast, keratinocytes treated with soluble EGF displayed a highly proliferative, rather than migratory, phenotype. We then transitioned our photo-immobilization techniques to materials that may be more suitable as a wound dressing, such as silk fibroin films. Silk fibroin is a natural fiber with many desirable qualities for a biomaterial including high strength and elasticity, biocompatibility, a beta

Atp1a3-deficient heterozygous mice show lower rank in the hierarchy and altered social behavior.

PubMed

Sugimoto, H; Ikeda, K; Kawakami, K

2018-06-01

Atp1a3 is the Na-pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3-deficient heterozygous mice (Atp1a3 +/- ) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3 +/- and compared the rank and hierarchy structure between Atp1a3 +/- and wild-type mice within a housing cage using the round-robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3 +/- than wild-type mice, and Atp1a3 +/- approached Atp1a3 +/- mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3 +/- group than in the wild-type group, while no difference was observed in the mixed-genotype housing condition. (3) Hierarchy formation was not different between Atp1a3 +/- and wild type. (4) Atp1a3 +/- showed a lower rank in the mixed-genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3 +/- showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Inhibition of PTP1B Restores IRS1-Mediated Hepatic Insulin Signaling in IRS2-Deficient Mice

PubMed Central

González-Rodríguez, Águeda; Gutierrez, Jose A. Mas; Sanz-González, Silvia; Ros, Manuel; Burks, Deborah J.; Valverde, Ángela M.

2010-01-01

OBJECTIVE Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2−/− mice. RESEARCH DESIGN AND METHODS We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2−/− and IRS2−/−/PTP1B−/− mice. Additionally, hepatic insulin signaling was assessed in control and IRS2−/− mice treated with resveratrol, an antioxidant present in red wine. RESULTS In livers of hyperglycemic IRS2−/− mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2−/− mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action. PMID:20028942

Nitric oxide mediates aortic disease in mice deficient in the metalloprotease Adamts1 and in a mouse model of Marfan syndrome.

PubMed

Oller, Jorge; Méndez-Barbero, Nerea; Ruiz, E Josue; Villahoz, Silvia; Renard, Marjolijn; Canelas, Lizet I; Briones, Ana M; Alberca, Rut; Lozano-Vidal, Noelia; Hurlé, María A; Milewicz, Dianna; Evangelista, Arturo; Salaices, Mercedes; Nistal, J Francisco; Jiménez-Borreguero, Luis Jesús; De Backer, Julie; Campanero, Miguel R; Redondo, Juan Miguel

2017-02-01

Heritable thoracic aortic aneurysms and dissections (TAAD), including Marfan syndrome (MFS), currently lack a cure, and causative mutations have been identified for only a fraction of affected families. Here we identify the metalloproteinase ADAMTS1 and inducible nitric oxide synthase (NOS2) as therapeutic targets in individuals with TAAD. We show that Adamts1 is a major mediator of vascular homeostasis, given that genetic haploinsufficiency of Adamts1 in mice causes TAAD similar to MFS. Aortic nitric oxide and Nos2 levels were higher in Adamts1-deficient mice and in a mouse model of MFS (hereafter referred to as MFS mice), and Nos2 inactivation protected both types of mice from aortic pathology. Pharmacological inhibition of Nos2 rapidly reversed aortic dilation and medial degeneration in young Adamts1-deficient mice and in young or old MFS mice. Patients with MFS showed elevated NOS2 and decreased ADAMTS1 protein levels in the aorta. These findings uncover a possible causative role for the ADAMTS1-NOS2 axis in human TAAD and warrant evaluation of NOS2 inhibitors for therapy.

Attenuated behavioural responses to acute and chronic cocaine in GASP-1-deficient mice.

PubMed

Boeuf, Julien; Trigo, José Manuel; Moreau, Pierre-Henri; Lecourtier, Lucas; Vogel, Elise; Cassel, Jean-Cristophe; Mathis, Chantal; Klosen, Paul; Maldonado, Rafael; Simonin, Frédéric

2009-09-01

G protein-coupled receptor (GPCR) associated sorting protein 1 (GASP-1) interacts with GPCRs and is implicated in their postendocytic sorting. Recently, GASP-1 has been shown to regulate dopamine (D(2)) and cannabinoid (CB1) receptor signalling, suggesting that preventing GASP-1 interaction with GPCRs might provide a means to limit the decrease in receptor signalling upon sustained agonist treatment. In order to test this hypothesis, we have generated and behaviourally characterized GASP-1 knockout (KO) mice and have examined the consequences of the absence of GASP-1 on chronic cocaine treatments. GASP-1 KO and wild-type (WT) mice were tested for sensitization to the locomotor effects of cocaine. Additional mice were trained to acquire intravenous self-administration of cocaine on a fixed ratio 1 schedule of reinforcement, and the motivational value of cocaine was then assessed using a progressive ratio schedule of reinforcement. The dopamine and muscarinic receptor densities were quantitatively evaluated in the striatum of WT and KO mice tested for sensitization and self-administration. Acute and sensitized cocaine-locomotor effects were attenuated in KO mice. A decrease in the percentage of animals that acquired cocaine self-administration was also observed in GASP-1-deficient mice, which was associated with pronounced down-regulation of dopamine and muscarinic receptors in the striatum. These data indicate that GASP-1 participates in acute and chronic behavioural responses induced by cocaine and are in agreement with a role of GASP-1 in postendocytic sorting of GPCRs. However, in contrast to previous studies, our data suggest that upon sustained receptor stimulation GASP-1 stimulates recycling rather than receptor degradation.

Fractalkine receptor (CX3CR1) deficiency sensitizes mice to the behavioral changes induced by lipopolysaccharide

PubMed Central

2010-01-01

Background Interactions between fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) regulate microglial activation in the CNS. Recent findings indicate that age-associated impairments in CX3CL1 and CX3CR1 are directly associated with exaggerated microglial activation and an impaired recovery from sickness behavior after peripheral injection of lipopolysaccharide (LPS). Therefore, the purpose of this study was to determine the extent to which an acute LPS injection causes amplified and prolonged microglial activation and behavioral deficits in CX3CR1-deficient mice (CX3CR1-/-). Methods CX3CR1-/- mice or control heterozygote mice (CX3CR1+/-) were injected with LPS (0.5 mg/kg i.p.) or saline and behavior (i.e., sickness and depression-like behavior), microglial activation, and markers of tryptophan metabolism were determined. All data were analyzed using Statistical Analysis Systems General Linear Model procedures and were subjected to one-, two-, or three-way ANOVA to determine significant main effects and interactions. Results LPS injection caused a prolonged duration of social withdrawal in CX3CR1-/- mice compared to control mice. This extended social withdrawal was associated with enhanced mRNA expression of IL-1β, indolamine 2,3-dioxygenase (IDO) and kynurenine monooxygenase (KMO) in microglia 4 h after LPS. Moreover, elevated expression of IL-1β and CD14 was still detected in microglia of CX3CR1-/- mice 24 h after LPS. There was also increased turnover of tryptophan, serotonin, and dopamine in the brain 24 h after LPS, but these increases were independent of CX3CR1 expression. When submitted to the tail suspension test 48 and 72 h after LPS, an increased duration of immobility was evident only in CX3CR1-/- mice. This depression-like behavior in CX3CR1-/- mice was associated with a persistent activated microglial phenotype in the hippocampus and prefrontal cortex. Conclusions Taken together, these data indicate that a deficiency of CX3CR1 is permissive to

Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

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

Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir

2014-03-01

4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependentmore » increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

Inner ear dysfunction in caspase-3 deficient mice

PubMed Central

2011-01-01

Background Caspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (Casp3-/-) mouse strain. Results Average ABR thresholds of Casp3-/- mice were significantly elevated (P < 0.05) compared to Casp3+/- mice and Casp3+/+ mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (P < 0.05) in Casp3-/- mice, whereas Casp3+/- and Casp3+/+ mice showed normal and comparable values to each other. Casp3-/- mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, Casp3-/- mice had minimal response to any of the stimuli tested, whereas Casp3+/- mice had an intermediate response compared to Casp3+/+ mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in Casp3-/- mice whereas the Casp3+/- and Casp3+/+ mice had normal hair cell numbers. Conclusions These results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule. PMID:21988729

Aldose reductase deficiency protects from autoimmune- and endotoxin-induced uveitis in mice.

PubMed

Yadav, Umesh C S; Shoeb, Mohammed; Srivastava, Satish K; Ramana, Kota V

2011-10-17

To investigate the effect of aldose reductase (AR) deficiency in protecting the chronic experimental autoimmune (EAU) and acute endotoxin-induced uveitis (EIU) in c57BL/6 mice. The WT and AR-null (ARKO) mice were immunized with human interphotoreceptor retinoid-binding peptide (hIRPB-1-20), to induce EAU, or were injected subcutaneously with lipopolysaccharide (LPS; 100 μg) to induce EIU. The mice were killed on day 21 for EAU and at 24 hours for EIU, when the disease was at its peak, and the eyes were immediately enucleated for histologic and biochemical studies. Spleen-derived T-lymphocytes were used to study the antigen-specific immune response in vitro and in vivo. In WT-EAU mice, severe damage to the retinal wall, especially to the photoreceptor layer was observed, corresponding to a pathologic score of ∼2, which was significantly prevented in the ARKO or AR inhibitor-treated mice. The levels of cytokines and chemokines increased markedly in the whole-eye homogenates of WT-EAU mice, but not in ARKO-EAU mice. Further, expression of inflammatory marker proteins such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and vascular cell adhesion molecule (VCAM)-1 was increased in the WT-EIU mouse eyes but not in the ARKO-EIU eyes. The T cells proliferated vigorously when exposed to the hIRPB antigen in vitro and secreted various cytokines and chemokines, which were significantly inhibited in the T cells isolated from the ARKO mice. These findings suggest that AR-deficiency/inhibition protects against acute as well as chronic forms of ocular inflammatory complications such as uveitis.

The caspase-1 inhibitor CARD18 is specifically expressed during late differentiation of keratinocytes and its expression is lost in lichen planus.

PubMed

Qin, Haihong; Jin, Jiang; Fischer, Heinz; Mildner, Michael; Gschwandtner, Maria; Mlitz, Veronika; Eckhart, Leopold; Tschachler, Erwin

2017-08-01

CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease. Copyright © 2017 Japanese Society for Investigative

CALHM1 deficiency impairs cerebral neuron activity and memory flexibility in mice.

PubMed

Vingtdeux, Valérie; Chang, Eric H; Frattini, Stephen A; Zhao, Haitian; Chandakkar, Pallavi; Adrien, Leslie; Strohl, Joshua J; Gibson, Elizabeth L; Ohmoto, Makoto; Matsumoto, Ichiro; Huerta, Patricio T; Marambaud, Philippe

2016-04-12

CALHM1 is a cell surface calcium channel expressed in cerebral neurons. CALHM1 function in the brain remains unknown, but recent results showed that neuronal CALHM1 controls intracellular calcium signaling and cell excitability, two mechanisms required for synaptic function. Here, we describe the generation of Calhm1 knockout (Calhm1(-/-)) mice and investigate CALHM1 role in neuronal and cognitive functions. Structural analysis revealed that Calhm1(-/-) brains had normal regional and cellular architecture, and showed no evidence of neuronal or synaptic loss, indicating that CALHM1 deficiency does not affect brain development or brain integrity in adulthood. However, Calhm1(-/-) mice showed a severe impairment in memory flexibility, assessed in the Morris water maze, and a significant disruption of long-term potentiation without alteration of long-term depression, measured in ex vivo hippocampal slices. Importantly, in primary neurons and hippocampal slices, CALHM1 activation facilitated the phosphorylation of NMDA and AMPA receptors by protein kinase A. Furthermore, neuronal CALHM1 activation potentiated the effect of glutamate on the expression of c-Fos and C/EBPβ, two immediate-early gene markers of neuronal activity. Thus, CALHM1 controls synaptic activity in cerebral neurons and is required for the flexible processing of memory in mice. These results shed light on CALHM1 physiology in the mammalian brain.

Metallochaperone for Cu,Zn-superoxide dismutase (CCS) protein but not mRNA is higher in organs from copper-deficient mice and rats.

PubMed

Prohaska, Joseph R; Broderius, Margaret; Brokate, Bruce

2003-09-15

Cu,Zn-superoxide dismutase (SOD1) is an abundant metalloenzyme important in scavenging superoxide ions. Cu-deficient rats and mice have lower SOD1 activity and protein, possibly because apo-SOD1 is degraded faster than holo-SOD1. SOD1 interacts with and requires its metallochaperone CCS for donating copper. We produced dietary Cu deficiency in rodents to determine if the reduction in SOD1 was related to the level of its specific metallochaperone CCS. CCS levels determined by immunoblot were 2- to 3-fold higher in liver, heart, kidney, and brain from male Cu-deficient rats and mice under a variety of conditions. CCS was also higher in livers of Cu-deficient dams. Interestingly, CCS levels in brain of Cu-deficient mice were also higher even though SOD1 activity and protein were not altered, suggesting that the rise in CCS is correlated with altered Cu status rather than a direct result of lower SOD1. A DNA probe specific for rat CCS detected a single transcript by Northern blot hybridization with liver RNA. CCS mRNA levels in mouse and rat liver were not altered by dietary treatment. These results suggest a posttranscriptional mechanism for higher CCS protein when Cu is limiting in the cell, perhaps due to slower protein turnover. Elevation in CCS level is one of the most dramatic alterations in Cu binding proteins accompanying Cu deficiency and may be useful to assess Cu status.

Tumor cell apoptosis induces tumor-specific immunity in a CC chemokine receptor 1- and 5-dependent manner in mice.

PubMed

Iida, Noriho; Nakamoto, Yasunari; Baba, Tomohisa; Kakinoki, Kaheita; Li, Ying-Yi; Wu, Yu; Matsushima, Kouji; Kaneko, Shuichi; Mukaida, Naofumi

2008-10-01

The first step in the generation of tumor immunity is the migration of dendritic cells (DCs) to the apoptotic tumor, which is presumed to be mediated by various chemokines. To clarify the roles of chemokines, we induced apoptosis using suicide gene therapy and investigated the immune responses following tumor apoptosis. We injected mice with a murine hepatoma cell line, BNL 1ME A.7R.1 (BNL), transfected with HSV-thymidine kinase (tk) gene and then treated the animals with ganciclovir (GCV). GCV treatment induced massive tumor cell apoptosis accompanied with intratumoral DC infiltration. Tumor-infiltrating DCs expressed chemokine receptors CCR1 and CCR5, and T cells and macrophages expressed CCL3, a ligand for CCR1 and CCR5. Moreover, tumor apoptosis increased the numbers of DCs migrating into the draining lymph nodes and eventually generated a specific cytotoxic cell population against BNL cells. Although GCV completely eradicated HSV-tk-transfected BNL cells in CCR1-, CCR5-, or CCL3-deficient mice, intratumoral and intranodal DC infiltration and the subsequent cytotoxicity generation were attenuated in these mice. When parental cells were injected again after complete eradication of primary tumors by GCV treatment, the wild-type mice completely rejected the rechallenged cells, but the deficient mice exhibited impairment in rejection. Thus, we provide definitive evidence indicating that CCR1 and CCR5 and their ligand CCL3 play a crucial role in the regulation of intratumoral DC accumulation and the subsequent establishment of tumor immunity following induction of tumor apoptosis by suicide genes.

Combined Vitamin C and Vitamin E Deficiency Worsens Early Atherosclerosis in ApoE-Deficient Mice

PubMed Central

Babaev, Vladimir R.; Li, Liying; Shah, Sanket; Fazio, Sergio; Linton, MacRae F.; May, James M.

2010-01-01

Objective Atherosclerosis is an inflammatory condition associated with oxidative stress, but controversy persists regarding whether antioxidants such as vitamins C and E are preventative. To assess the role of combined deficiencies of vitamins C and E on the earliest stages of atherosclerosis, four combinations of vitamin supplementation (Low C/Low E, Low C/High E, High C/Low E, High C/High E) were studied in atherosclerosis-prone apolipoprotein E (apoE)-deficient mice also unable to synthesize their own vitamin C (gulo−/−). The effect of a more severe depletion of vitamin C alone was evaluated in a second experiment using gulo−/− mice carrying the hemizygous deletion of SVCT2, the vitamin C transporter. Methods and Results After 8 weeks on a high-fat diet (16% lard, 0.2% cholesterol), atherosclerosis developed in the aortic sinus areas of mice in all diet groups. Each vitamin-deficient diet significantly decreased liver and brain contents of the corresponding vitamin. Combined deficiency of both vitamins increased lipid peroxidation, doubled plaque size, and increased plaque macrophage content by 2-3-fold in males, although only plaque macrophage content was increased in females. A more severe deficiency of vitamin C in gulo−/− mice with defective cellular uptake of vitamin C increased both oxidative stress and atherosclerosis in apoE−/− mice compared to littermates on a diet replete in vitamin C, again most clearly in males. Conclusion Combined vitamin E and C deficiencies are required to worsen early atherosclerosis in an apoE-deficient mouse model. However, a more severe cellular deficiency of vitamin C alone promotes atherosclerosis when vitamin E is replete. PMID:20558818

γδ T-cell-deficient mice show alterations in mucin expression, glycosylation, and goblet cells but maintain an intact mucus layer

PubMed Central

Kober, Olivia I.; Ahl, David; Pin, Carmen; Holm, Lena; Carding, Simon R.

2014-01-01

Intestinal homeostasis is maintained by a hierarchy of immune defenses acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion. In this study, we used γδ T-cell-deficient (TCRδ−/−) mice to examine whether and how γδ T-cells modulate the properties of the intestinal mucus layer. Increased susceptibility of TCRδ−/− mice to dextran sodium sulfate (DSS)-induced colitis is associated with a reduced number of goblet cells. Alterations in the number of goblet cells and crypt lengths were observed in the small intestine and colon of TCRδ−/− mice compared with C57BL/6 wild-type (WT) mice. Addition of keratinocyte growth factor to small intestinal organoid cultures from TCRδ−/− mice showed a marked increase in crypt growth and in both goblet cell number and redistribution along the crypts. There was no apparent difference in the thickness or organization of the mucus layer between TCRδ−/− and WT mice, as measured in vivo. However, γδ T-cell deficiency led to reduced sialylated mucins in association with increased gene expression of gel-secreting Muc2 and membrane-bound mucins, including Muc13 and Muc17. Collectively, these data provide evidence that γδ T cells play an important role in the maintenance of mucosal homeostasis by regulating mucin expression and promoting goblet cell function in the small intestine. PMID:24503767

Attenuated EAN in TNF-α Deficient Mice Is Associated with an Altered Balance of M1/M2 Macrophages

PubMed Central

Zhang, Hong-Liang; Hassan, Mohammed Y.; Zheng, Xiang-Yu; Azimullah, Sheikh; Quezada, Hernan Concha; Amir, Naheed; Elwasila, Mohamed; Mix, Eilhard; Adem, Abdu; Zhu, Jie

2012-01-01

The role of tumor necrosis factor (TNF)-α and its receptors in neuroautoimmune and neuroinflammatory diseases has been controversial. On the basis of our previous studies, we hereby aimed to further clarify TNF-α’s mechanism of action and to explore the potential role of TNF-α receptor (TNFR)1 as a therapeutic target in experimental autoimmune neuritis (EAN). EAN was induced by immunization with P0 peptide 180–199 in TNF-α knockout (KO) mice and anti-TNFR1 antibodies were used to treat EAN. Particularly, the effects of TNF-α deficiency and TNFR1 blockade on macrophage functions were investigated. The onset of EAN in TNF-α KO mice was markedly later than that in wild type (WT) mice. From day 14 post immunization, the clinical signs of TNF-α KO mice were significantly milder than those of their WT counterparts. Further, we showed that the clinical severity of WT mice treated with anti-TNFR1 antibodies was less severe than that of the control WT mice receiving PBS. Nevertheless, no difference with regard to the clinical signs of EAN or inflammatory infiltration in cauda equina was seen between TNF-α KO and WT mice with EAN after blockade of TNFR1. Although TNF-α deficiency did not alter the proliferation of lymphocytes in response to either antigenic or mitogenic stimuli, it down-regulated the production of interleukin (IL)-12 and nitric oxide (NO), and enhanced the production of IL-10 in macrophages. Increased ratio of regulatory T cells (Tregs) and reduced production of interferon (IFN)-γ in cauda equina infiltrating cells, and elevated levels of IgG2b antibodies against P0 peptide 180–199 in sera were found in TNF-α KO mice with EAN. In conclusion, TNF-α deficiency attenuates EAN via altering the M1/M2 balance of macrophages. PMID:22666471

Topical application of glycolic acid suppresses the UVB induced IL-6, IL-8, MCP-1 and COX-2 inflammation by modulating NF-κB signaling pathway in keratinocytes and mice skin.

PubMed

Tang, Sheau-Chung; Liao, Pei-Yun; Hung, Sung-Jen; Ge, Jheng-Siang; Chen, Shiou-Mei; Lai, Ji-Ching; Hsiao, Yu-Ping; Yang, Jen-Hung

2017-06-01

Glycolic acid (GA), commonly present in fruits, has been used to treat dermatological diseases. Extensive exposure to solar ultraviolet B (UVB) irradiation plays a crucial role in the induction of skin inflammation. The development of photo prevention from natural materials represents an effective strategy for skin keratinocytes. The aim of this study was to investigate the molecular mechanisms underlying the glycolic acid (GA)-induced reduction of UVB-mediated inflammatory responses. We determined the effects of different concentrations of GA on the inflammatory response of human keratinocytes HaCaT cells and C57BL/6J mice dorsal skin. After GA was topically applied, HaCaT and mice skin were exposed to UVB irradiation. GA reduced the production of UVB-induced nuclear factor kappa B (NF-κB)-dependent inflammatory mediators [interleukin (IL)-1β, IL-6, IL-8, cyclooxygenase (COX)-2, tumor necrosis factor-α, and monocyte chemoattractant protein (MCP-1)] at both mRNA and protein levels. GA inhibited the UVB-induced promoter activity of NF-κB in HaCaT cells. GA attenuated the elevation of senescence associated with β-galactosidase activity but did not affect the wound migration ability. The topical application of GA inhibited the genes expression of IL-1β, IL-6, IL-8, COX-2, and MCP-1 in UVB-exposed mouse skin. The mice to UVB irradiation after GA was topically applied for 9 consecutive days and reported that 1-1.5% of GA exerted anti-inflammatory effects on mouse skin. We clarified the molecular mechanism of GA protection against UVB-induced inflammation by modulating NF-κB signaling pathways and determined the optimal concentration of GA in mice skin exposed to UVB irradiation. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Arginase-1 deficiency.

PubMed

Sin, Yuan Yan; Baron, Garrett; Schulze, Andreas; Funk, Colin D

2015-12-01

Arginase-1 (ARG1) deficiency is a rare autosomal recessive disorder that affects the liver-based urea cycle, leading to impaired ureagenesis. This genetic disorder is caused by 40+ mutations found fairly uniformly spread throughout the ARG1 gene, resulting in partial or complete loss of enzyme function, which catalyzes the hydrolysis of arginine to ornithine and urea. ARG1-deficient patients exhibit hyperargininemia with spastic paraparesis, progressive neurological and intellectual impairment, persistent growth retardation, and infrequent episodes of hyperammonemia, a clinical pattern that differs strikingly from other urea cycle disorders. This review briefly highlights the current understanding of the etiology and pathophysiology of ARG1 deficiency derived from clinical case reports and therapeutic strategies stretching over several decades and reports on several exciting new developments regarding the pathophysiology of the disorder using ARG1 global and inducible knockout mouse models. Gene transfer studies in these mice are revealing potential therapeutic options that can be exploited in the future. However, caution is advised in extrapolating results since the lethal disease phenotype in mice is much more severe than in humans indicating that the mouse models may not precisely recapitulate human disease etiology. Finally, some of the functions and implications of ARG1 in non-urea cycle activities are considered. Lingering questions and future areas to be addressed relating to the clinical manifestations of ARG1 deficiency in liver and brain are also presented. Hopefully, this review will spark invigorated research efforts that lead to treatments with better clinical outcomes.

MiR-143/145 deficiency attenuates the progression of atherosclerosis in Ldlr-/-mice.

PubMed

Sala, Federica; Aranda, Juan F; Rotllan, Noemi; Ramírez, Cristina M; Aryal, Binod; Elia, Leonardo; Condorelli, Gianluigi; Catapano, Alberico Luigi; Fernández-Hernando, Carlos; Norata, Giuseppe Danilo

2014-10-01

The miR-143/145 cluster regulates VSMC specific gene expression, thus controlling differentiation, plasticity and contractile function, and promoting the VSMC phenotypic switch from a contractile/non-proliferative to a migrating/proliferative state. More recently increased miR-145 expression was observed in human carotid atherosclerotic plaques from symptomatic patients. The goal of this study was to investigate the contribution of miR-143/145 during atherogenesis by generating mice lacking miR-143/145 on an Ldlr-deficient background. Ldlr-/- and Ldlr-/--miR-143/145-/- (DKO) were fed a Western diet (WD) for 16 weeks. At the end of the treatment, the lipid profile and the atherosclerotic lesions were assessed in both groups of mice. Absence of miR-143/145 significantly reduced atherosclerotic plaque size and macrophage infiltration. Plasma total cholesterol levels were lower in DKO and FLPC analysis showed decreased cholesterol content in VLDL and LDL fractions. Interestingly miR-143/145 deficiency per se resulted in increased hepatic and vascular ABCA1 expression. We further confirmed the direct regulation of miR-145 on ABCA1 expression by qRT-PCR, Western blotting and 3'UTR-luciferase reporter assays. In summary, miR-143/145 deficiency significantly reduces atherosclerosis in mice. Therapeutic inhibition of miR-145 might be useful for treating atherosclerotic vascular disease.

Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation.

PubMed

Mendias, Christopher L; Kayupov, Erdan; Bradley, Joshua R; Brooks, Susan V; Claflin, Dennis R

2011-07-01

Myostatin (MSTN) is a member of the transforming growth factor-β superfamily of cytokines and is a negative regulator of skeletal muscle mass. Compared with MSTN(+/+) mice, the extensor digitorum longus muscles of MSTN(-/-) mice exhibit hypertrophy, hyperplasia, and greater maximum isometric force production (F(o)), but decreased specific maximum isometric force (sF(o); F(o) normalized by muscle cross-sectional area). The reason for the reduction in sF(o) was not known. Studies in myotubes indicate that inhibiting myostatin may increase muscle mass by decreasing the expression of the E3 ubiquitin ligase atrogin-1, which could impact the force-generating capacity and size of muscle fibers. To gain a greater understanding of the influence of myostatin on muscle contractility, we determined the impact of myostatin deficiency on the contractility of permeabilized muscle fibers and on the levels of atrogin-1 and ubiquitinated myosin heavy chain in whole muscle. We hypothesized that single fibers from MSTN(-/-) mice have a greater F(o), but no difference in sF(o), and a decrease in atrogin-1 and ubiquitin-tagged myosin heavy chain levels. The results indicated that fibers from MSTN(-/-) mice have a greater cross-sectional area, but do not have a greater F(o) and have a sF(o) that is significantly lower than fibers from MSTN(+/+) mice. The extensor digitorum longus muscles from MSTN(-/-) mice also have reduced levels of atrogin-1 and ubiquitinated myosin heavy chain. These findings suggest that myostatin inhibition in otherwise healthy muscle increases the size of muscle fibers and decreases atrogin-1 levels, but does not increase the force production of individual muscle fibers.

SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

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

Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

2014-06-15

Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutatedmore » (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.« less

Abnormal Brain Iron Metabolism in Irp2 Deficient Mice Is Associated with Mild Neurological and Behavioral Impairments

PubMed Central

Zumbrennen-Bullough, Kimberly B.; Becker, Lore; Garrett, Lillian; Hölter, Sabine M.; Calzada-Wack, Julia; Mossbrugger, Ilona; Quintanilla-Fend, Leticia; Racz, Ildiko; Rathkolb, Birgit; Klopstock, Thomas; Wurst, Wolfgang; Zimmer, Andreas; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabě; Romney, Steven J.; Leibold, Elizabeth A.

2014-01-01

Iron Regulatory Protein 2 (Irp2, Ireb2) is a central regulator of cellular iron homeostasis in vertebrates. Two global knockout mouse models have been generated to explore the role of Irp2 in regulating iron metabolism. While both mouse models show that loss of Irp2 results in microcytic anemia and altered body iron distribution, discrepant results have drawn into question the role of Irp2 in regulating brain iron metabolism. One model shows that aged Irp2 deficient mice develop adult-onset progressive neurodegeneration that is associated with axonal degeneration and loss of Purkinje cells in the central nervous system. These mice show iron deposition in white matter tracts and oligodendrocyte soma throughout the brain. A contrasting model of global Irp2 deficiency shows no overt or pathological signs of neurodegeneration or brain iron accumulation, and display only mild motor coordination and balance deficits when challenged by specific tests. Explanations for conflicting findings in the severity of the clinical phenotype, brain iron accumulation and neuronal degeneration remain unclear. Here, we describe an additional mouse model of global Irp2 deficiency. Our aged Irp2−/− mice show marked iron deposition in white matter and in oligodendrocytes while iron content is significantly reduced in neurons. Ferritin and transferrin receptor 1 (TfR1, Tfrc), expression are increased and decreased, respectively, in the brain from Irp2−/− mice. These mice show impairments in locomotion, exploration, motor coordination/balance and nociception when assessed by neurological and behavioral tests, but lack overt signs of neurodegenerative disease. Ultrastructural studies of specific brain regions show no evidence of neurodegeneration. Our data suggest that Irp2 deficiency dysregulates brain iron metabolism causing cellular dysfunction that ultimately leads to mild neurological, behavioral and nociceptive impairments. PMID:24896637

GW501516, a PPARδ agonist, ameliorates tubulointerstitial inflammation in proteinuric kidney disease via inhibition of TAK1-NFκB pathway in mice.

PubMed

Yang, Xu; Kume, Shinji; Tanaka, Yuki; Isshiki, Keiji; Araki, Shin-ichi; Chin-Kanasaki, Masami; Sugimoto, Toshiro; Koya, Daisuke; Haneda, Masakazu; Sugaya, Takeshi; Li, Detian; Han, Ping; Nishio, Yoshihiko; Kashiwagi, Atsunori; Maegawa, Hiroshi; Uzu, Takashi

2011-01-01

Peroxisome proliferator-activated receptors (PPARs) are a nuclear receptor family of ligand-inducible transcription factors, which have three different isoforms: PPARα, δ and γ. It has been demonstrated that PPARα and γ agonists have renoprotective effects in proteinuric kidney diseases; however, the role of PPARδ agonists in kidney diseases remains unclear. Thus, we examined the renoprotective effect of GW501516, a PPARδ agonist, in a protein-overload mouse nephropathy model and identified its molecular mechanism. Mice fed with a control diet or GW501516-containing diet were intraperitoneally injected with free fatty acid (FFA)-bound albumin or PBS(-). In the control group, protein overload caused tubular damages, macrophage infiltration and increased mRNA expression of MCP-1 and TNFα. These effects were prevented by GW501516 treatment. In proteinuric kidney diseases, excess exposure of proximal tubular cells to albumin, FFA bound to albumin or cytokines such as TNFα is detrimental. In vitro studies using cultured proximal tubular cells showed that GW501516 attenuated both TNFα- and FFA (palmitate)-induced, but not albumin-induced, MCP-1 expression via direct inhibition of the TGF-β activated kinase 1 (TAK1)-NFκB pathway, a common downstream signaling pathway to TNFα receptor and toll-like receptor-4. In conclusion, we demonstrate that GW501516 has an anti-inflammatory effect in renal tubular cells and may serve as a therapeutic candidate to attenuate tubulointerstitial lesions in proteinuric kidney diseases.

GW501516, a PPARδ Agonist, Ameliorates Tubulointerstitial Inflammation in Proteinuric Kidney Disease via Inhibition of TAK1-NFκB Pathway in Mice

PubMed Central

Yang, Xu; Kume, Shinji; Tanaka, Yuki; Isshiki, Keiji; Araki, Shin-ichi; Chin-Kanasaki, Masami; Sugimoto, Toshiro; Koya, Daisuke; Haneda, Masakazu; Sugaya, Takeshi; Li, Detian; Han, Ping; Nishio, Yoshihiko; Kashiwagi, Atsunori; Maegawa, Hiroshi; Uzu, Takashi

2011-01-01

Peroxisome proliferator-activated receptors (PPARs) are a nuclear receptor family of ligand-inducible transcription factors, which have three different isoforms: PPARα, δ and γ. It has been demonstrated that PPARα and γ agonists have renoprotective effects in proteinuric kidney diseases; however, the role of PPARδ agonists in kidney diseases remains unclear. Thus, we examined the renoprotective effect of GW501516, a PPARδ agonist, in a protein-overload mouse nephropathy model and identified its molecular mechanism. Mice fed with a control diet or GW501516-containing diet were intraperitoneally injected with free fatty acid (FFA)-bound albumin or PBS(−). In the control group, protein overload caused tubular damages, macrophage infiltration and increased mRNA expression of MCP-1 and TNFα. These effects were prevented by GW501516 treatment. In proteinuric kidney diseases, excess exposure of proximal tubular cells to albumin, FFA bound to albumin or cytokines such as TNFα is detrimental. In vitro studies using cultured proximal tubular cells showed that GW501516 attenuated both TNFα- and FFA (palmitate)-induced, but not albumin-induced, MCP-1 expression via direct inhibition of the TGF-β activated kinase 1 (TAK1)-NFκB pathway, a common downstream signaling pathway to TNFα receptor and toll-like receptor-4. In conclusion, we demonstrate that GW501516 has an anti-inflammatory effect in renal tubular cells and may serve as a therapeutic candidate to attenuate tubulointerstitial lesions in proteinuric kidney diseases. PMID:21966476

Transplantation of bone marrow-derived mesenchymal stem cells rescues partially rachitic phenotypes induced by 1,25-Dihydroxyvitamin D deficiency in mice

PubMed Central

Zhang, Zengli; Yin, Shaomeng; Xue, Xian; Ji, Ji; Tong, Jian; Goltzman, David; Miao, Dengshun

2016-01-01

To determine whether the transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) can improve the 1,25(OH)2D deficiency-induced rachitic phenotype, 2×106 BM-MSCs from wild-type mice or vehicle were transplanted by tail vein injection into mice deficient in 1,25(OH)2D due to targeted deletion of 1α(OH)ase (1α(OH)ase-/-). Our results show that 1α(OH)ase mRNA was expressed in the BM-MSCs derived from wild-type mice, and was detected in long bone, kidney and intestine from BM-MSC-transplanted 1α(OH)ase-/- recipients. Serum calcium, 1,25(OH)2D3 levels and body weight were significantly increased in BM-MSC-transplanted 1α(OH)ase-/- recipients compared to vehicle-treated 1α(OH)ase-/- mice. Skeletal mineralization improved in 1α(OH)ase-/- recipients as demonstrated by BMD measurement, micro-CT analysis and von Kossa staining of undecalcified sections. Expression levels of type I collagen, osteocalcin, bone sialoprotein and vitronectin and the size of calcified nodules were decreased in BM-MSC cultures from 1α(OH)ase-/- mice compared with those from wild-type mice, however, these parameters were increased in those from BM-MSCs-transplanted 1α(OH)ase-/- recipients compared with those from vehicle-treated 1α(OH)ase-/- mice. This study indicates that donor BM-MSCs cells can relocate to multiple tissues where they synthesize 1α(OH)ase and produce 1,25(OH)2D that contributes to the improvement of serum calcium and skeletal mineralization. Results from this study suggest that BM-MSC transplantation may provide a therapeutic approach to treatment of pseudovitamin D-deficiency rickets. PMID:27830022

Transplantation of bone marrow-derived mesenchymal stem cells rescues partially rachitic phenotypes induced by 1,25-Dihydroxyvitamin D deficiency in mice.

PubMed

Zhang, Zengli; Yin, Shaomeng; Xue, Xian; Ji, Ji; Tong, Jian; Goltzman, David; Miao, Dengshun

2016-01-01

To determine whether the transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) can improve the 1,25(OH) 2 D deficiency-induced rachitic phenotype, 2×10 6 BM-MSCs from wild-type mice or vehicle were transplanted by tail vein injection into mice deficient in 1,25(OH) 2 D due to targeted deletion of 1α(OH)ase (1α(OH)ase -/- ). Our results show that 1α(OH)ase mRNA was expressed in the BM-MSCs derived from wild-type mice, and was detected in long bone, kidney and intestine from BM-MSC-transplanted 1α(OH)ase -/- recipients. Serum calcium, 1,25(OH) 2 D 3 levels and body weight were significantly increased in BM-MSC-transplanted 1α(OH)ase -/- recipients compared to vehicle-treated 1α(OH)ase -/- mice. Skeletal mineralization improved in 1α(OH)ase -/- recipients as demonstrated by BMD measurement, micro-CT analysis and von Kossa staining of undecalcified sections. Expression levels of type I collagen, osteocalcin, bone sialoprotein and vitronectin and the size of calcified nodules were decreased in BM-MSC cultures from 1α(OH)ase -/- mice compared with those from wild-type mice, however, these parameters were increased in those from BM-MSCs-transplanted 1α(OH)ase -/- recipients compared with those from vehicle-treated 1α(OH)ase -/- mice. This study indicates that donor BM-MSCs cells can relocate to multiple tissues where they synthesize 1α(OH)ase and produce 1,25(OH) 2 D that contributes to the improvement of serum calcium and skeletal mineralization. Results from this study suggest that BM-MSC transplantation may provide a therapeutic approach to treatment of pseudovitamin D-deficiency rickets.

Neurturin-deficient mice develop dry eye and keratoconjunctivitis sicca.

PubMed

Song, Xiu Jun; Li, De-Quan; Farley, William; Luo, Li Hui; Heuckeroth, Robert O; Milbrandt, Jeffrey; Pflugfelder, Stephen C

2003-10-01

Neurturin has been identified as a neurotrophic factor for parasympathetic neurons. Neurturin-deficient (NRTN(-/-)) mice have defective parasympathetic innervation of their lacrimal glands. This study was conducted to evaluate tear function and ocular surface phenotype in NRTN(-/-) mice. Determined by tail genomic DNA PCR, 25 NRTN(-/-) mice and 17 neurturin-normal (NRTN(+/+)) mice aged 6 weeks to 4 months were evaluated. Aqueous tear production, tear fluorescein clearance and corneal sensation were serially measured. Corneal permeability to AlexaFluor dextran (AFD; Molecular Probes, Eugene, OR) was measured by a fluorometric assay at 485 nm excitation and 530 nm emission. Histology was evaluated in PAS-stained sections. Mucin and HLA class II (IA) antigen were assessed by immunofluorescent staining. Tear IL-1beta was measured by ELISA, and tear matrix metalloproteinase (MMP)-9 by zymography. Gene expression in the corneal epithelia was analyzed by semiquantitative RT-PCR. In comparison to that in age-matched NRTN(+/+) mice, aqueous tear production, tear fluorescein clearance, and corneal sensation were significantly reduced in NRTN(-/-) mice, whereas corneal permeability to AFD was significantly increased. Immunoreactive MUC-4 and -5AC mucin and goblet cell density (P < 0.001) in the conjunctiva of NRTN(-/-) mice were lower than in NRTN(+/+) mice. The expression of MUC-1 and -4 mRNA by the corneal epithelium was reduced in NRTN(-/-) mice. There were a significantly greater number of IA antigen-positive conjunctival epithelial cells in NRTN(-/-) mice than NRTN(+/+) mice. Tear fluid IL-1beta and MMP-9 concentrations and the expression of IL-1beta, TNF-alpha, macrophage inflammatory protein (MIP)-2, cytokine-induced neutrophil chemoattractant (KC), and MMP-9 mRNA by the corneal epithelia were significantly increased in NRTN(-/-) mice, compared with NRTN(+/+) mice. Neurturin-deficient mice show phenotypic changes and ocular surface inflammation that mimic human

Strain Background Modifies Phenotypes in the ATP8B1-Deficient Mouse

PubMed Central

Vargas, Julie C.; Xu, Hongmei; Groen, Annamiek; Paulusma, Coen C.; Grenert, James P.; Pawlikowska, Ludmila; Sen, Saunak; Elferink, Ronald P. J. Oude; Bull, Laura N.

2010-01-01

Background Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. Methodology/Principal Findings We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. Conclusions/Significance Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease. PMID:20126555

Amelioration of Behavioral Abnormalities in BH4-deficient Mice by Dietary Supplementation of Tyrosine

PubMed Central

Kwak, Sang Su; Jeong, Mikyoung; Choi, Ji Hye; Kim, Daesoo; Min, Hyesun; Yoon, Yoosik; Hwang, Onyou; Meadows, Gary G.; Joe, Cheol O.

2013-01-01

This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH4)-deficient Spr −/− mice by the dietary supplementation of tyrosine. Since BH4 is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH4-deficient Spr −/− mice. We found that Spr −/− mice display variable ‘open-field’ behaviors, impaired motor functions on the ‘rotating rod’, and dystonic ‘hind-limb clasping’. In this study, we report that these aberrant motor deficits displayed by Spr −/− mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of Spr −/− mice may not be the biological feature of aberrant motor behaviors associated with BH4 deficiency. Brain levels of dopamine (DA) and its metabolites in Spr −/− mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of Spr −/− mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH4-deficiency. PMID:23577163

Evolutionary re-wiring of p63 and the epigenomic regulatory landscape in keratinocytes and its potential implications on species-specific gene expression and phenotypes

PubMed Central

Sethi, Isha; Gluck, Christian; Zhou, Huiqing

2017-01-01

Abstract Although epidermal keratinocyte development and differentiation proceeds in similar fashion between humans and mice, evolutionary pressures have also wrought significant species-specific physiological differences. These differences between species could arise in part, by the rewiring of regulatory network due to changes in the global targets of lineage-specific transcriptional master regulators such as p63. Here we have performed a systematic and comparative analysis of the p63 target gene network within the integrated framework of the transcriptomic and epigenomic landscape of mouse and human keratinocytes. We determined that there exists a core set of ∼1600 genomic regions distributed among enhancers and super-enhancers, which are conserved and occupied by p63 in keratinocytes from both species. Notably, these DNA segments are typified by consensus p63 binding motifs under purifying selection and are associated with genes involved in key keratinocyte and skin-centric biological processes. However, the majority of the p63-bound mouse target regions consist of either murine-specific DNA elements that are not alignable to the human genome or exhibit no p63 binding in the orthologous syntenic regions, typifying an occupancy lost subset. Our results suggest that these evolutionarily divergent regions have undergone significant turnover of p63 binding sites and are associated with an underlying inactive and inaccessible chromatin state, indicative of their selective functional activity in the transcriptional regulatory network in mouse but not human. Furthermore, we demonstrate that this selective targeting of genes by p63 correlates with subtle, but measurable transcriptional differences in mouse and human keratinocytes that converges on major metabolic processes, which often exhibit species-specific trends. Collectively our study offers possible molecular explanation for the observable phenotypic differences between the mouse and human skin and broadly

Human Keratinocytes Are Vanilloid Resistant

PubMed Central

Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

2008-01-01

Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

Attenuation of UVR-induced vitamin D3 synthesis in a mouse model deleted for keratinocyte lathosterol 5-desaturase.

PubMed

Makarova, Anastasia M; Pasta, Saloni; Watson, Gordon; Shackleton, Cedric; Epstein, Ervin H

2017-07-01

The lower risk of some internal cancers at lower latitudes has been linked to greater sun exposure and consequent higher levels of ultraviolet radiation (UVR)-produced vitamin D 3 (D 3 ). To separate the experimental effects of sunlight and of all forms of D 3 , a mouse in which UVR does not produce D 3 would be useful. To this end we have generated mice carrying a modified allele of sterol C5-desaturase (Sc5d), the gene encoding the enzyme that converts lathosterol to 7-dehydrocholesterol (7-DHC), such that Sc5d expression can be inactivated using the Cre/lox site-specific recombination system. By crossing to mice with tissue-specific expression of Cre or CreER 2 (Cre/estrogen receptor), we generated two lines of transgenic mice. One line has constitutive keratinocyte-specific inactivation of Sc5d (Sc5d k14KO ). The other line (Sc5d k14KOi ) has tamoxifen-inducible keratinocyte-specific inactivation of Sc5d. Mice deleted for keratinocyte Sc5d lose the ability to increase circulating D 3 following UVR exposure of the skin. Thus, unlike in control mice, acute UVR exposure did not affect circulating D 3 level in inducible Sc5d k14KOi mice. Keratinocyte-specific inactivation of Sc5d was proven by sterol measurement in hair - in control animals lathosterol and cholesta-7,24-dien-3β-ol, the target molecules of SC5D in the sterol biosynthetic pathways, together constituted a mean of 10% of total sterols; in the conditional knockout mice these sterols constituted a mean of 56% of total sterols. The constitutive knockout mice had an even greater increase, with lathosterol and cholesta-7,24-dien-3β-ol accounting for 80% of total sterols. In conclusion, the dominant presence of the 7-DHC precursors in hair of conditional animals and the lack of increased circulating D 3 following exposure to UVR reflect attenuated production of the D 3 photochemical precursor 7-DHC and, consequently, of D 3 itself. These animals provide a useful new tool for investigating the role of D 3

Immunity to sporozoite-induced malaria infection in mice. I. The effect of immunization of T and B cell-deficient mice. [X Radiation

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

Chen, D.H.; Tigelaar, R.E.; Weinbaum, F.I.

1977-04-01

The cellular basis of immunity to sporozoites was investigated by examining the effect of immunization of T and B cell-deficient C57BL/6N x BALB/c AnN F/sub 1/ (BLCF/sub 1/) mice compared to immunocompetent controls. Immunization of T cell-deficient (ATX-BM-ATS) BLCF/sub 1/ mice with x-irradiated sporozoites did not result in the generation of protective immunity. The same immunization protocols protected all immunocompetent controls. In contrast, B cell-deficient (..mu..-suppressed) BLCF/sub 1/ mice were protected by immunization in the majority of cases. The absence of detectable serum circumsporozoite precipitins or sporozoite neutralizing activity in the ..mu..-suppressed mice that resisted a sporozoite challenge suggests amore » minor role for these humoral factors in protection. These data demonstrate a preeminent role for T cells in the induction of protective immunity in BLCF/sub 1/ mice against a P. berghei sporozoite infection.« less

AKT1 provides an essential survival signal required for differentiation and stratification of primary human keratinocytes.

PubMed

Thrash, Barry R; Menges, Craig W; Pierce, Robert H; McCance, Dennis J

2006-04-28

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

Circadian clock-deficient mice as a tool for exploring disease etiology.