The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response1[OPEN

PubMed Central

Brauer, Elizabeth K.; Ahsan, Nagib; Kato, Naohiro; Coluccio, Alison E.; Thelen, Jay J.

2016-01-01

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca2+ uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H+)/K+ symporter that mediates a high-affinity uptake during K+ deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K+ efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K+ loss. Taken together, our results position ILK1 as a link between plant defense pathways and K+ homeostasis. PMID:27208244

Emodin Inhibits the Epithelial to Mesenchymal Transition of Epithelial Ovarian Cancer Cells via ILK/GSK-3β/Slug Signaling Pathway

PubMed Central

Lu, Jingjing; Xu, Ying; Wei, Xuan; Zhao, Zhe; Xue, Jing

2016-01-01

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite the anticancer capabilities of emodin observed in many cancers, including EOC, the underlying molecular mechanism remains to be elucidated. A crucial link has been discovered between the acquisition of metastatic traits and the epithelial-mesenchymal transition (EMT). The present study aimed to determine whether emodin could inhibit the EMT of EOC cells and explore the underlying mechanism. The CCK-8 assay and transwell assay showed that emodin effectively repressed the abilities of proliferation, invasion, and migration in A2780 and SK-OV-3 cells. The Western blot showed that emodin upregulated epithelial markers (E-cadherin and Claudin) while it downregulated mesenchymal markers (N-cadherin and Vimentin) and transcription factor (Slug) in a dose-dependent fashion. After transfection of siRNA-Slug, both Slug and N-cadherin were downregulated in EOC cells while E-cadherin was upregulated, which was intensified by emodin. Besides, emodin decreased the expression of ILK, p-GSK-3β, β-catenin, and Slug. Transfection of siRNA-ILK also achieved the same effects, which was further strengthened by following emodin treatment. Nevertheless, SB216763, an inhibitor of GSK-3β, could reverse the effects of emodin except for ILK expression. These findings suggest that emodin inhibited the EMT of EOC cells via ILK/GSK-3β/Slug signaling pathway. PMID:28097141

Emodin Inhibits the Epithelial to Mesenchymal Transition of Epithelial Ovarian Cancer Cells via ILK/GSK-3β/Slug Signaling Pathway.

PubMed

Lu, Jingjing; Xu, Ying; Wei, Xuan; Zhao, Zhe; Xue, Jing; Liu, Peishu

2016-01-01

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite the anticancer capabilities of emodin observed in many cancers, including EOC, the underlying molecular mechanism remains to be elucidated. A crucial link has been discovered between the acquisition of metastatic traits and the epithelial-mesenchymal transition (EMT). The present study aimed to determine whether emodin could inhibit the EMT of EOC cells and explore the underlying mechanism. The CCK-8 assay and transwell assay showed that emodin effectively repressed the abilities of proliferation, invasion, and migration in A2780 and SK-OV-3 cells. The Western blot showed that emodin upregulated epithelial markers (E-cadherin and Claudin) while it downregulated mesenchymal markers (N-cadherin and Vimentin) and transcription factor (Slug) in a dose-dependent fashion. After transfection of siRNA-Slug, both Slug and N-cadherin were downregulated in EOC cells while E-cadherin was upregulated, which was intensified by emodin. Besides, emodin decreased the expression of ILK, p-GSK-3 β , β -catenin, and Slug. Transfection of siRNA-ILK also achieved the same effects, which was further strengthened by following emodin treatment. Nevertheless, SB216763, an inhibitor of GSK-3 β , could reverse the effects of emodin except for ILK expression. These findings suggest that emodin inhibited the EMT of EOC cells via ILK/GSK-3 β /Slug signaling pathway.

Activated PAR-2 regulates pancreatic cancer progression through ILK/HIF-α-induced TGF-α expression and MEK/VEGF-A-mediated angiogenesis.

PubMed

Chang, Li-Hsun; Pan, Shiow-Lin; Lai, Chin-Yu; Tsai, An-Chi; Teng, Che-Ming

2013-08-01

Tissue factor initiates the process of thrombosis and activates cell signaling through protease-activated receptor-2 (PAR-2). The aim of this study was to investigate the pathological role of PAR-2 signaling in pancreatic cancer. We first demonstrated that activated PAR-2 up-regulated the protein expression of both hypoxia-inducible factor-1α (HIF-1α) and HIF-2α, resulting in enhanced transcription of transforming growth factor-α (TGF-α). Down-regulation of HIFs-α by siRNA or YC-1, an HIF inhibitor, resulted in depleted levels of TGF-α protein. Furthermore, PAR-2, through integrin-linked kinase (ILK) signaling, including the p-AKT, promoted HIF protein expression. Diminishing ILK by siRNA decreased the levels of PAR-2-induced p-AKT, HIFs-α, and TGF-α; our results suggest that ILK is involved in the PAR-2-mediated TGF-α via an HIF-α-dependent pathway. Furthermore, the culture medium from PAR-2-treated pancreatic cancer cells enhanced human umbilical vein endothelial cell proliferation and tube formation, which was blocked by the MEK inhibitor, PD98059. We also found that activated PAR-2 enhanced tumor angiogenesis through the release of vascular endothelial growth factor-A (VEGF-A) from cancer cells, independent of the ILK/HIFs-α pathways. Consistent with microarray analysis, activated PAR-2 induced TGF-A and VEGF-A gene expression. In conclusion, the activation of PAR-2 signaling induced human pancreatic cancer progression through the induction of TGF-α expression by ILK/HIFs-α, as well as through MEK/VEGF-A-mediated angiogenesis, and it plays a role in the interaction between cancer progression and cancer-related thrombosis. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Balance between apoptosis or survival induced by changes in extracellular-matrix composition in human mesangial cells: a key role for ILK-NFκB pathway.

PubMed

del Nogal, María; Luengo, Alicia; Olmos, Gemma; Lasa, Marina; Rodriguez-Puyol, Diego; Rodriguez-Puyol, Manuel; Calleros, Laura

2012-12-01

Renal fibrosis is the final outcome of many clinical conditions that lead to chronic renal failure, characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, in particular collagen type I. The aim of this study was to identify the mechanisms responsible for human mesangial cell survival, conditioned by changes in extracellular-matrix composition. Our results indicate that collagen I induces apoptosis in cells but only after inactivation of the pro-survival factor NFκB by either the super-repressor IκBα or the PDTC inhibitor. Collagen I activates a death pathway, through ILK/GSK-3β-dependent Bim expression. Moreover, collagen I significantly increases NFκB-dependent transcription, IκBα degradation and p65/NFκB translocation to the nucleus; it activates β1 integrin and this is accompanied by increased activity of ILK which leads to AKT activation. Knockdown of ILK or AKT with small interfering RNA suppresses the increase in NFκB activity. NFκB mediates cell survival through the antiapoptotic protein Bcl-xL. Our data suggest that human mesangial cells exposed to abnormal collagen I are protected against apoptosis by a complex mechanism involving integrin β1/ILK/AKT-dependent NFκB activation with consequent Bcl-xL overexpression, that opposes a simultaneously activated ILK/GSK-3β-dependent Bim expression and this dual mechanism may play a role in the progression of glomerular dysfunction.

SiO2@antisense molecules covered by nepetalactone, extracted from Nepeta gloeocephala, inhibits ILK phosphorylation and downstream PKB/AKT signaling in HeLa cells.

PubMed

Dehghany Ashkezary, M; Aboee-Mehrizi, F; Moradi, P

2017-01-01

In this study, the anticancer property of SiO 2 @antisense molecules (SiO 2 @AMs) and SiO 2 @AM covered by nepetalactone (SiO 2 @AM/CN), extracted from Nepeta gloeocephala, was investigated. Here integrin-linked kinase (ILK) phosphorylation and protein kinase B/AKT (PKB/AKT) signaling was studied when HeLa cells were exposed to SiO 2 @AM and SiO 2 @AM/CN. First, N. gloeocephala was identified at the Iranian National Herbarium. Then, its essential oil (EO) was obtained by the hydrodistillation method. In the next step, 4aα,7α,7aα-nepetalactone was extracted from the EO, based on the spectroscopic data. To obtain SiO 2 @AM/CN, 1 ml of SiO 2 @AM was mixed with extracted nepetalactone and then strongly shaken for 30 min. Finally, serial concentrations (100, 50, 25 and 12.5 μg ml -1 ) of SiO 2 @AM and SiO 2 @AM/CN were prepared and then exposed to HeLa cells (2 × 10 5 cells per ml) for 24 h at 37 °C. After incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell-cycle analysis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and western blots were carried out. To find ILK phosphorylation and PKB/AKT signaling, the expression of threonine-173 (Thr-173), serine-246 (Ser-246), total ILK, AKT-Ser473, AKT-Thr308 and total AKT was investigated. HeLa cells that were treated with SiO 2 @AM/CN had G2/M arrest. Based on the TUNEL assay, many apoptotic cells have been shown when they were exposed to SiO 2 @AM/CN. Importantly, SiO 2 @AM/CN decreased ILK phosphorylation at Thr-173 and Ser-246 without affecting total ILK levels. Moreover, SiO 2 @AM/CN decreased AKT-Ser473 and AKT-Thr308 phosphorylation without affecting total PKB/AKT protein.

Thymosin β4 induces invasion and migration of human colorectal cancer cells through the ILK/AKT/β-catenin signaling pathway

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

Piao, Zhengri; Center for Creative Biomedical Scientists; Hong, Chang-Soo

2014-09-26

Highlights: • Tβ4 is overexpressed in human colorectal cancer cells. • The overexpression of Tβ4 is correlated with stage of colorectal cancer. • Tβ4 stimulates cell adhesion, invasion, migration and EMT. • Tβ4 activates the ILK/AKT/β-catenin signaling pathway. - Abstract: Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. In this study, we show that Tβ4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tβ4 were correlated with stage of colorectal cancer, and thatmore » Tβ4 expression was associated with morphogenesis and EMT. Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cells showed decreased activities. We also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. These results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment.« less

Involvement of the TGFβ1- ILK-Akt signaling pathway in the effects of hesperidin in type 2 diabetic nephropathy.

PubMed

Zhang, YingHui; Wang, Bing; Guo, Feng; Li, ZhiZhen; Qin, GuiJun

2018-06-14

Diabetic nephropathy is one of the manifestations of systemic microangiopathy in diabetes. Hesperetin, a natural flavanone glycoside compound in citrus fruits, has been demonstrated to exert hypoglycemic effects and protect kidney in experimental diabetic animals. The current study was aimed to investigate the mechanisms underlying the hypoglycemic effects of hesperetin in high-fat/streptozocin (STZ)-induced diabetic nephropathy. The results showed that mice in whom hesperetin was administered for 4 weeks attenuated the increased fasting blood glucose and impaired glucose tolerance ability that was observed in high-fat/STZ mice. In addition, we found that hesperetin ameliorated the abnormalities of biochemical parameters in serum, liver, and kidney of mice with diabetic nephropathy. Hesperetin also rescued the irregular distortions in glomerular basement membrane and expanded mesangial regions. Moreover, hesperetin repaired the function of podocyte by increasing renal nephrin expression and decreasing renal alpha-smooth muscle actin expression. Furthermore, hesperetin inhibited the expression of transforming growth factor-β1 (TGF-β1) and its downstream effectors integrin-linked kinase (ILK) and Akt. In conclusion, our study implies that hesperetin produced protective effects in diabetic nephropathy possibly by suppressing TGF-β1-ILK-Akt signaling. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Shenqi detoxification granule combined with P311 inhibits epithelial-mesenchymal transition in renal fibrosis via TGF-β1-Smad-ILK pathway.

PubMed

Cai, Pingping; Liu, Xiang; Xu, Yuan; Qi, Fanghua; Si, Guomin

2017-01-01

Shenqi detoxification granule (SDG), a traditional Chinese herbal formula, has been shown to have nephroprotective and anti-fibrotic activities in patients with chronic kidney disease (CKD). However, its mechanisms in renal fibrosis and the progression of CKD remain largely unknown. P311, a highly conserved 8-kDa intracellular protein, plays a key role in renal fibrosis by regulating epithelial-mesenchymal transition (EMT). Previously, we found P311 might be involved in the pathogenesis of renal fibrosis by inhibiting EMT via the TGF-β1-Smad-ILK pathway. We also found SDG combined with P311 could ameliorate renal fibrosis by regulating the expression of EMT markers. Here we further examined the effect and mechanism of SDG combined with P311 on TGF-β1-mediated EMT in a rat model of unilateral ureteral occlusion (UUO) renal fibrosis. After establishment of the UUO model successfully, the rats were gavaged with SDG daily and/or injected with recombinant adenovirus p311 (also called Ad-P311) through the tail vein each week for 4 weeks. Serum creatinine (Cr), blood urea nitrogen (BUN) and albumin (ALB) levels were tested to observe renal function, and hematoxylin eosin (HE) and Masson staining were performed to observe kidney histopathology. Furthermore, the expression of EMT markers (E-cadherin and α-smooth muscle actin (α-SMA)) and EMT-related molecules TGF-β1, pSmad2/3, Smad7 and ILK were observed using immunohistochemical staining and Western blot analysis. Treatment with SDG and P311 improved renal function and histopathological abnormalities, as well as reversing the changes of EMT markers and EMT-related molecules, which indicated SDG combined with P311 could attenuate renal fibrosis in UUO rats, and the underlying mechanism might involve TGF-β1-mediated EMT and the TGF-β1-Smad-ILK signaling pathway. Therefore, SDG might be a novel alternative therapy for treating renal fibrosis and delaying the progression of CKD. Furthermore, SDG combined with P311 might

Nephrin phosphorylation regulates podocyte adhesion through the PINCH-1-ILK-α-parvin complex

PubMed Central

Zha, Dongqing; Chen, Cheng; Liang, Wei; Chen, Xinghua; Ma, Tean; Yang, Hongxia; van Goor, Harry; Ding, Guohua

2013-01-01

Nephrin, a structural molecule, is also a signaling molecule after phosphorylation. Inhibition of nephrin phosphorylation is correlated with podocyte injury. The PINCH-1-ILK-α-parvin (PIP) complex plays a crucial role in cell adhesion and cytoskeleton formation. We hypothesized that nephrin phosphorylation influenced cytoskeleton and cell adhesion in podocytes by regulating the PIP complex. The nephrin phosphorylation, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PIP complex knockdown resulted in cytoskeleton reorganization and decreased cell adhesion and spreading. Nephrin and its phosphorylation were unaffected after PIP complex knockdown. Furthermore, inhibition of nephrin phosphorylation suppressed PIP complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism. [BMB Reports 2013; 46(4): 230-235] PMID:23615266

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.

Integrin-linked kinase interactions with ELMO2 modulate cell polarity.

PubMed

Ho, Ernest; Irvine, Tames; Vilk, Gregory J A; Lajoie, Gilles; Ravichandran, Kodi S; D'Souza, Sudhir J A; Dagnino, Lina

2009-07-01

Cell polarization is a key prerequisite for directed migration during development, tissue regeneration, and metastasis. Integrin-linked kinase (ILK) is a scaffold protein essential for cell polarization, but very little is known about the precise mechanisms whereby ILK modulates polarization in normal epithelia. Elucidating these mechanisms is essential to understand tissue morphogenesis, transformation, and repair. Here we identify a novel ILK protein complex that includes Engulfment and Cell Motility 2 (ELMO2). We also demonstrate the presence of RhoG in ILK-ELMO2 complexes, and the localization of this multiprotein species specifically to the leading lamellipodia of polarized cells. Significantly, the ability of RhoG to bind ELMO is crucial for ILK induction of cell polarization, and the joint expression of ILK and ELMO2 synergistically promotes the induction of front-rear polarity and haptotactic migration. This places RhoG-ELMO2-ILK complexes in a key position for the development of cell polarity and forward movement. Although ILK is a component of many diverse multiprotein species that may contribute to cell polarization, expression of dominant-negative ELMO2 mutants is sufficient to abolish the ability of ILK to promote cell polarization. Thus, its interaction with ELMO2 and RhoG is essential for the ability of ILK to induce front-rear cell polarity.

Rictor and integrin-linked kinase interact and regulate Akt phosphorylation and cancer cell survival.

PubMed

McDonald, Paul C; Oloumi, Arusha; Mills, Julia; Dobreva, Iveta; Maidan, Mykola; Gray, Virginia; Wederell, Elizabeth D; Bally, Marcel B; Foster, Leonard J; Dedhar, Shoukat

2008-03-15

An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. This finding was interesting because rictor, originally identified as a regulator of cytoskeletal dynamics, is also a component of mammalian target of rapamycin complex 2 (mTORC2), a complex implicated in Akt phosphorylation. These functions overlap with known ILK functions. Coimmunoprecipitation analyses confirmed this interaction, and ILK and rictor colocalized in membrane ruffles and leading edges of cancer cells. Yeast two-hybrid assays showed a direct interaction between the NH(2)- and COOH-terminal domains of rictor and the ILK kinase domain. Depletion of ILK and rictor in breast and prostate cancer cell lines resulted in inhibition of Akt Ser(473) phosphorylation and induction of apoptosis, whereas, in several cell lines, depletion of mTOR increased Akt phosphorylation. Akt and Ser(473)P-Akt were detected in ILK immunoprecipitates and small interfering RNA-mediated depletion of rictor, but not mTOR, inhibited the amount of Ser(473)P-Akt in the ILK complex. Expression of the NH(2)-terminal (1-398 amino acids) rictor domain also resulted in the inhibition of ILK-associated Akt Ser(473) phosphorylation. These data show that rictor regulates the ability of ILK to promote Akt phosphorylation and cancer cell survival.

A Novel Role for Integrin-linked Kinase in Epithelial Sheet Morphogenesis

PubMed Central

Vespa, Alisa; D'Souza, Sudhir J.A.; Dagnino, Lina

2005-01-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration. PMID:15975904

Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1.

PubMed

Sayedyahossein, Samar; Xu, Stacey X; Rudkouskaya, Alena; McGavin, Martin J; McCormick, John K; Dagnino, Lina

2015-02-01

Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5-fold. ILK-deficient mouse keratinocytes internalized bacteria 2- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was 2.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection. © FASEB.

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

PubMed

Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

2005-09-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.

PubMed

Nakrieko, Kerry-Ann; Vespa, Alisa; Mason, David; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

2008-07-15

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

A dual role for integrin-linked kinase in platelets: regulating integrin function and α-granule secretion

PubMed Central

Sage, Tanya; Stevens, Joanne M.; Jordan, Peter A.; Jones, Sarah; Barrett, Natasha E.; St-Arnaud, Rene; Frampton, Jonathan; Dedhar, Shoukat; Gibbins, Jonathan M.

2008-01-01

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for α-granule secretion and therefore may play a central role in the regulation of platelet function. PMID:18772455

Integrin-linked Kinase Interactions with ELMO2 Modulate Cell Polarity

PubMed Central

Ho, Ernest; Irvine, Tames; Vilk, Gregory J.A.; Lajoie, Gilles; Ravichandran, Kodi S.; D'Souza, Sudhir J.A.

2009-01-01

Cell polarization is a key prerequisite for directed migration during development, tissue regeneration, and metastasis. Integrin-linked kinase (ILK) is a scaffold protein essential for cell polarization, but very little is known about the precise mechanisms whereby ILK modulates polarization in normal epithelia. Elucidating these mechanisms is essential to understand tissue morphogenesis, transformation, and repair. Here we identify a novel ILK protein complex that includes Engulfment and Cell Motility 2 (ELMO2). We also demonstrate the presence of RhoG in ILK–ELMO2 complexes, and the localization of this multiprotein species specifically to the leading lamellipodia of polarized cells. Significantly, the ability of RhoG to bind ELMO is crucial for ILK induction of cell polarization, and the joint expression of ILK and ELMO2 synergistically promotes the induction of front-rear polarity and haptotactic migration. This places RhoG–ELMO2–ILK complexes in a key position for the development of cell polarity and forward movement. Although ILK is a component of many diverse multiprotein species that may contribute to cell polarization, expression of dominant-negative ELMO2 mutants is sufficient to abolish the ability of ILK to promote cell polarization. Thus, its interaction with ELMO2 and RhoG is essential for the ability of ILK to induce front-rear cell polarity. PMID:19439446

mda-9/Syntenin protein positively regulates the activation of Akt protein by facilitating integrin-linked kinase adaptor function during adhesion to type I collagen.

PubMed

Hwangbo, Cheol; Park, Juhee; Lee, Jeong-Hyung

2011-09-23

The integrin-linked kinase (ILK)-PINCH1-α-parvin (IPP) complex functions as a signaling platform for integrins that modulates various cellular processes. ILK functions as a central adaptor for the assembly of IPP complex. We report here that mda-9/syntenin, a positive regulator of cancer metastasis, regulates the activation of Akt (also known as protein kinase B) by facilitating ILK adaptor function during adhesion to type I collagen (COL-I) in human breast cancer cells. COL-I stimulation induced the phosphorylation and plasma membrane translocation of Akt. Inhibition of mda-9/syntenin or expression of mutant ILK (E359K) significantly blocked the translocation of both ILK and Akt to the plasma membrane. mda-9/syntenin associated with ILK, and this association was increased at the plasma membrane by COL-I stimulation. Knockdown of mda-9/syntenin impaired COL-I-induced association of ILK with Akt and plasma membrane targeting of ILK-Akt complex. These results demonstrated that mda-9/syntenin regulates the activation of Akt by controlling the plasma membrane targeting of Akt via a mechanism that facilitates the association of Akt with ILK at the plasma membrane during adhesion to COL-I. On a striking note, inhibition of mda-9/syntenin impaired COL-I-induced plasma membrane translocation of the IPP complex and assembly of integrin β1-IPP signaling complexes. Thus, our study defines the role of mda-9/syntenin in ILK adaptor function and describes a new mechanism of mda-9/syntenin for regulation of cell migration.

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

Choi, Yoon Pyo; Kim, Baek Gil; Department of Pathology, Yonsei University College of Medicine, Seoul

Highlights: Black-Right-Pointing-Pointer The potential of targeting ILK and integrins for highly aggressive ovarian cancer. Black-Right-Pointing-Pointer Unanticipated synergistic effect for the combination of ILK/{beta}4 integrin. Black-Right-Pointing-Pointer Combination of ILK/{beta}4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. Black-Right-Pointing-Pointer Targeting of {beta}4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of {beta}1 and {beta}4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expressionmore » of {beta}1 and {beta}4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of {beta}4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of {beta}4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting {beta}4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.« less

Different exercise modalities have distinct effects on the integrin-linked kinase (ILK) and Ca2+ signaling pathways in the male rat bone

PubMed Central

Sontam, Dharani M; Firth, Elwyn C; Tsai, Peter; Vickers, Mark H; O’Sullivan, Justin M

2015-01-01

Mechanical loading is essential to maintain optimal skeletal health. Despite the fact that early-life exercise has positive, long-lasting effects on the musculo-skeletal system, the response of the musculo-skeletal system to spontaneous low-impact exercise has been poorly studied. Previously, we identified subtle morphological changes in the femoral diaphysis of exercised animals compared to nonexercised controls. We hypothesized that significant changes in gene expression of cells should precede significant measurable phenotypic changes in the tissues of which they are part. Here, we employed RNA-Seq to analyse the transcriptome of the cortical bone from the femoral mid-diaphysis of prepubertal male Sprague-Dawley rats that were assigned to control (CON); bipedal stance (BPS); or wheel exercise (WEX) groups for 15 days. We identified 808 and 324 differentially expressed transcripts in the BPS and WEX animals respectively. While a number of transcripts change their levels in an exercise-specific manner, we identified 191 transcripts that were differentially expressed in both BPS and WEX. Importantly, we observed that the exercise mode had diametrically opposite effects on transcripts for multiple genes within the integrin-linked kinase (ILK) and Ca2+ signaling pathways such that they were up-regulated in BPS and down-regulated in WEX. The findings are important for our understanding of possible ways in which different exercise regimens might affect bone when normal activities apply mechanical stimuli during postnatal growth and development. PMID:26471755

Inhibition of integrin-linked kinase blocks podocyte epithelial-mesenchymal transition and ameliorates proteinuria.

PubMed

Kang, Young Sun; Li, Yingjian; Dai, Chunsun; Kiss, Lawrence P; Wu, Chuanyue; Liu, Youhua

2010-08-01

Proteinuria is a primary clinical symptom of a large number of glomerular diseases that progress to end-stage renal failure. Podocyte dysfunctions play a fundamental role in defective glomerular filtration in many common forms of proteinuric kidney disorders. Since binding of these cells to the basement membrane is mediated by integrins, we determined the role of integrin-linked kinase (ILK) in podocyte dysfunction and proteinuria. ILK expression was induced in mouse podocytes by various injurious stimuli known to cause proteinuria including TGF-beta1, adriamycin, puromycin, and high ambient glucose. Podocyte ILK was also found to be upregulated in human proteinuric glomerular diseases. Ectopic expression of ILK in podocytes decreased levels of the epithelial markers nephrin and ZO-1, induced mesenchymal markers such as desmin, fibronectin, matrix metalloproteinase-9 (MMP-9), and alpha-smooth muscle actin (alpha-SMA), promoted cell migration, and increased the paracellular albumin flux across podocyte monolayers. ILK also induced Snail, a key transcription factor mediating epithelial-mesenchymal transition (EMT). Blockade of ILK activity with a highly selective small molecule inhibitor reduced Snail induction and preserved podocyte phenotypes following TGF-beta1 or adriamycin stimulation. In vivo, this ILK inhibitor ameliorated albuminuria, repressed glomerular induction of MMP-9 and alpha-SMA, and preserved nephrin expression in murine adriamycin nephropathy. Our results show that upregulation of ILK is a convergent pathway leading to podocyte EMT, migration, and dysfunction. ILK may be an attractive target for therapeutic intervention of proteinuric kidney diseases.

The mammalian target of rapamycin signaling pathway regulates myocyte enhancer factor-2C phosphorylation levels through integrin-linked kinase in goat skeletal muscle satellite cells.

PubMed

Wu, Haiqing; Ren, Yu; Pan, Wei; Dong, Zhenguo; Cang, Ming; Liu, Dongjun

2015-11-01

Mammalian target of rapamycin (mTOR) signaling pathway plays a key role in muscle development and is involved in multiple intracellular signaling pathways. Myocyte enhancer factor-2 (MEF2) regulates muscle cell proliferation and differentiation. However, how the mTOR signaling pathway regulates MEF2 activity remains unclear. We isolated goat skeletal muscle satellite cells (gSSCs) as model cells to explore mTOR signaling pathway regulation of MEF2C. We inhibited mTOR activity in gSSCs with PP242 and found that MEF2C phosphorylation was decreased and that muscle creatine kinase (MCK) expression was suppressed. Subsequently, we detected integrin-linked kinase (ILK) using MEF2C coimmunoprecipitation; ILK and MEF2C were colocalized in the gSSCs. We found that inhibiting mTOR activity increased ILK phosphorylation levels and that inhibiting ILK activity with Cpd 22 and knocking down ILK with small interfering RNA increased MEF2C phosphorylation and MCK expression. In the presence of Cpd 22, mTOR activity inhibition did not affect MEF2C phosphorylation. Moreover, ILK dephosphorylated MEF2C in vitro. These results suggest that the mTOR signaling pathway regulates MEF2C positively and regulates ILK negatively and that ILK regulates MEF2C negatively. It appears that the mTOR signaling pathway regulates MEF2C through ILK, further regulating the expression of muscle-related genes in gSSCs. © 2015 International Federation for Cell Biology.

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

PubMed

Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes.

PubMed

Terpstra, Leonieke; Prud'homme, Josée; Arabian, Alice; Takeda, Shu; Karsenty, Gérard; Dedhar, Shoukat; St-Arnaud, René

2003-07-07

Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

Modulation of type II TGF-β receptor degradation by integrin-linked kinase.

PubMed

Vi, Linda; Boo, Stellar; Sayedyahossein, Samar; Singh, Randeep K; McLean, Sarah; Di Guglielmo, Gianni M; Dagnino, Lina

2015-03-01

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.

Interaction of infectious spleen and kidney necrosis virus ORF119L with PINCH leads to dominant-negative inhibition of integrin-linked kinase and cardiovascular defects in zebrafish.

PubMed

Yuan, Ji-Min; He, Bai-Liang; Yang, Lu-Yun; Guo, Chang-Jun; Weng, Shao-Ping; Li, Shengwen Calvin; He, Jian-Guo

2015-01-01

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus, Iridoviridae family, causing a severe systemic disease with high mortality in mandarin fish (Siniperca chuatsi) in China and Southeast Asia. At present, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three-ankyrin-repeat (3ANK)-domain-containing protein, which shows high similarity to the dominant negative form of integrin-linked kinase (ILK); i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interaction in vitro in fathead minnow (FHM) cells. In vivo ORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of the sarcomeric Z disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by overexpressing a dominant negative form of ILK or by injecting an ILK antisense morpholino oligonucleotide. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z disks in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L-overexpressing zebrafish embryos. With these results, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the megalocytivirus pathogenesis. Our work is the first to show the role of a dominant negative inhibitor of the host ILK from ISKNV (an iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs ILK signaling. Intriguingly

Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1).

PubMed

Zhu, Xiang-Yu; Liu, Ning; Liu, Wei; Song, Shao-Wei; Guo, Ke-Jian

2012-04-01

Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.

Integrin-linked kinase modulates longevity and thermotolerance in C. elegans through neuronal control of HSF-1

PubMed Central

Kumsta, Caroline; Ching, Tsui-Ting; Nishimura, Mayuko; Davis, Andrew E; Gelino, Sara; Catan, Hannah H; Yu, Xiaokun; Chu, Chu-Chiao; Ong, Binnan; Panowski, Siler H; Baird, Nathan; Bodmer, Rolf; Hsu, Ao-Lin; Hansen, Malene

2014-01-01

Integrin-signaling complexes play important roles in cytoskeletal organization and cell adhesion in many species. Components of the integrin-signaling complex have been linked to aging in both Caenorhabditis elegans and Drosophila melanogaster, but the mechanism underlying this function is unknown. Here, we investigated the role of integrin-linked kinase (ILK), a key component of the integrin-signaling complex, in lifespan determination. We report that genetic reduction of ILK in both C. elegans and Drosophila increased resistance to heat stress, and led to lifespan extension in C. elegans without majorly affecting cytoskeletal integrity. In C. elegans, longevity and thermotolerance induced by ILK depletion was mediated by heat-shock factor-1 (HSF-1), a major transcriptional regulator of the heat-shock response (HSR). Reduction in ILK levels increased hsf-1 transcription and activation, and led to enhanced expression of a subset of genes with roles in the HSR. Moreover, induction of HSR-related genes, longevity and thermotolerance caused by ILK reduction required the thermosensory neurons AFD and interneurons AIY, which are known to play a critical role in the canonical HSR. Notably, ILK was expressed in neighboring neurons, but not in AFD or AIY, implying that ILK reduction initiates cell nonautonomous signaling through thermosensory neurons to elicit a noncanonical HSR. Our results thus identify HSF-1 as a novel effector of the organismal response to reduced ILK levels and show that ILK inhibition regulates HSF-1 in a cell nonautonomous fashion to enhance stress resistance and lifespan in C. elegans. PMID:24314125

Integrin linked kinase regulates the transcription of AQP2 by NFATC3.

PubMed

Hatem-Vaquero, Marco; Griera, Mercedes; Giermakowska, Wieslawa; Luengo, Alicia; Calleros, Laura; Gonzalez Bosc, Laura V; Rodríguez-Puyol, Diego; Rodríguez-Puyol, Manuel; De Frutos, Sergio

2017-09-01

Two processes are associated with progressive loss of renal function: 1) decreased aquaporin-2 (AQP2) expression and urinary concentrating capacity (Nephrogenic Diabetes Insipidus, NDI); and 2) changes in extracellular matrix (ECM) composition, e.g. increased collagen I (Col I) deposition, characteristic of tubule-interstitial fibrosis. AQP2 expression is regulated by both the ECM-to-intracellular scaffold protein integrin-linked kinase (ILK) by NFATc/AP1 and other transcription factors. In the present work, we used in vivo and in vitro approaches to examine ILK participation in NFATc3/AP-1-mediated increases in AQP2 gene expression. Both NFATc3 knock-out mice and ILK conditional-knockdown mice (cKD-ILK) display symptoms of NDI (polyuria and reduced AQP2 expression). NFATc3 is upregulated in the renal medulla tubular cells of cKD-ILK mice but with reduced nuclear localization. Inner medullary collecting duct mIMCD3 cells were subjected to ILK depletion and transfected with reporter plasmids. Pharmacological activators or inhibitors determined the effect of ILK activity on NFATc/AP-1-dependent increases in transcription of AQP2. Finally, mIMCD3 cultured on Col I showed reduced activity of the ILK/GSK3β/NFATc/AQP2 axis, suggesting this pathway is a potential target for therapeutic treatment of NDI. Copyright © 2017 Elsevier B.V. All rights reserved.

Elevating Integrin-linked Kinase Expression has Rescued Hippocampal Neurogenesis and Memory Deficits in an AD animal Model.

PubMed

Xu, Xu-Feng; Wang, You-Cui; Zong, Liang; Chen, Zhe-Yu; Li, Yan

2018-05-19

Alterations in adult neurogenesis have been regarded as a major cause of cognitive impairment in Alzheimer's disease (AD). The underlying mechanism of neurogenesis deficiency in AD remains unclear. In this study, we reported that Integrin-linked Kinase (ILK) protein levels and phosphorylation were significantly decreased in the hippocampus of APP/PS1 mice. Increased ILK expression of dentate gyrus (DG) rescued the hippocampus-dependent neurogenesis and memory deficits in APP/PS1 mice. Moreover, we demonstrated that the effect of ILK overexpression in the hippocampus was exerted via AKT-GSK3β pathway. Finally, we found that Fluoxetine, a selective serotonin reuptake inhibitor, could improve the impaired hippocampal neurogenesis and memory by enhancing ILK-AKT-GSK3β pathway activity in APP/PS1 mice. Thus, these findings demonstrated the effects of ILK on neurogenesis and memory recovery, suggesting that ILK is an important therapeutic target for AD prevention and treatment. Copyright © 2018. Published by Elsevier B.V.

Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury.

PubMed

Nakrieko, Kerry-Ann; Rudkouskaya, Alena; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

2011-07-15

Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15--expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury.

Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury

PubMed Central

Nakrieko, Kerry-Ann; Rudkouskaya, Alena; Irvine, Timothy S.; D'souza, Sudhir J. A.; Dagnino, Lina

2011-01-01

Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15–expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury. PMID:21593206

Shepherds' local knowledge and scientific data on the scavenging ecosystem service: Insights for conservation.

PubMed

Morales-Reyes, Zebensui; Martín-López, Berta; Moleón, Marcos; Mateo-Tomás, Patricia; Olea, Pedro P; Arrondo, Eneko; Donázar, José A; Sánchez-Zapata, José A

2018-05-05

Integrating indigenous and local knowledge (ILK) and scientific knowledge (SK) in the evaluation of ecosystem services has been recommended by the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. We examined the similarities and contradictions between shepherds' ILK and SK on the scavenging service provided by vertebrates in Spain. We conducted 73 face-to-face surveys with shepherds to evaluate their ILK. We collected scientific information on 20 scavenger species by monitoring the consumption of 45 livestock carcasses with camera traps. We found a high consistency between ILK and SK regarding the provision of the scavenging service by vertebrates, which was also consistent over the range of shepherd ages and experience. Our findings support the importance of ILK held by shepherds to better understand and to collect information on the scavenging service, particularly at the species level. The integration of ILK and SK into the management strategies of scavengers can benefit the conservation of globally endangered scavengers and the ecosystem services they provide.

Renal Integrin-Linked Kinase Depletion Induces Kidney cGMP-Axis Upregulation: Consequences on Basal and Acutely Damaged Renal Function

PubMed Central

Cano-Peñalver, José Luis; Griera, Mercedes; García-Jerez, Andrea; Hatem-Vaquero, Marco; Ruiz-Torres, María Piedad; Rodríguez-Puyol, Diego; de Frutos, Sergio; Rodríguez-Puyol, Manuel

2015-01-01

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and produces cGMP, which activates cGMP-dependent protein kinases (PKG) and is hydrolyzed by specific phosphodiesterases (PDE). The vasodilatory and cytoprotective capacity of cGMP-axis activation results in a therapeutic strategy for several pathologies. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix and intracellular signaling pathways, may modulate the expression and functionality of the cGMP-axis–related proteins. We introduce ILK as a novel modulator in renal homeostasis as well as a potential target for cisplatin (CIS)-induced acute kidney injury (AKI) improvement. We used an adult mice model of depletion of ILK (cKD-ILK), which showed basal increase of sGC and PKG expressions and activities in renal cortex when compared with wildtype (WT) littermates. Twenty-four h activation of sGC activation with NO enhanced the filtration rate in cKD-ILK. During AKI, cKD-ILK maintained the cGMP-axis upregulation with consequent filtration rates enhancement and ameliorated CIS-dependent tubular epithelial-to-mesenchymal transition and inflammation and markers. To emphasize the role of cGMP-axis upregulation due to ILK depletion, we modulated the cGMP axis under AKI in vivo and in renal cultured cells. A suboptimal dose of the PDE inhibitor ZAP enhanced the beneficial effects of the ILK depletion in AKI mice. On the other hand, CIS increased contractility-related events in cultured glomerular mesangial cells and necrosis rates in cultured tubular cells; ILK depletion protected the cells while sGC blockade with ODQ fully recovered the damage. PMID:26562149

Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway.

PubMed

Nho, Richard Seonghun; Xia, Hong; Kahm, Judy; Kleidon, Jill; Diebold, Deanna; Henke, Craig A

2005-07-15

A beta1 integrin phosphatidylinositol 3-kinase/Akt pathway regulates fibroblast survival in collagen matrices. When fibroblasts attach to collagen, Akt becomes phosphorylated, providing a survival signal. In contrast, in response to mechanical forces generated during collagen contraction, Akt is dephosphorylated and fibroblasts undergo apoptosis. The kinase(s) responsible for regulating Akt phosphorylation in response to matrix-derived mechanical signals are unclear. Integrin-linked kinase (ILK) is associated with the beta1 integrin in the focal adhesion complex and as such is a candidate kinase that may regulate Akt phosphorylation and fibroblast viability. Nevertheless, there is no direct evidence that matrix-derived mechanical forces regulate cell viability by modulating ILK activity. Here, we show that ILK activity decreased in response to collagen matrix contraction, which correlated with Akt dephosphorylation and induction of fibroblast apoptosis. In contrast, enforced activation of beta1 integrin by activating antibody preserved ILK and Akt activity during collagen matrix contraction, and this is associated with protection from collagen contraction-induced apoptosis. Knock-down of ILK by small, interfering RNA (siRNA) attenuated Akt phosphorylation in response to ligation of beta1 integrin by collagen or activating antibody and enhanced fibroblast apoptosis in response to collagen contraction. Kinase dead ILK attenuated Akt phosphorylation and enhanced fibroblast apoptosis, whereas hyperactive and wild type ILK augmented Akt phosphorylation and protected fibroblasts from apoptosis. Constitutively active Akt preserved Akt activity and rescued ILK siRNA-treated fibroblasts from collagen contraction-induced apoptosis. These data establish that matrix-derived mechanical forces sensed by beta1 integrin are capable of modulating ILK activity which regulates fibroblast viability via an Akt-dependent mechanism.

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity

PubMed Central

Im, Michelle; Dagnino, Lina

2018-01-01

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity. PMID:29568383

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity.

PubMed

Im, Michelle; Dagnino, Lina

2018-03-02

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity.

The roles of connective tissue growth factor and integrin-linked kinase in high glucose-induced phenotypic alterations of podocytes.

PubMed

Dai, Hou-Yong; Zheng, Min; Lv, Lin-Li; Tang, Ri-Ning; Ma, Kun-Ling; Liu, Dan; Wu, Min; Liu, Bi-Cheng

2012-01-01

Emerging evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). Connective tissue growth factor (CTGF) and integrin-linked kinase (ILK) are involved in the progression of DN. However, the underlying mechanisms of EMT are not well understood. The study aimed to investigate the roles of CTGF and ILK in high glucose-induced phenotypic alterations of podocytes and determine whether ILK signaling is downstream of CTGF. The epithelial marker of nephrin and the mesenchymal marker of desmin were investigated by real-time RT-PCR and Western blotting. The results demonstrated that podocytes displayed a spreading, arborized morphology in normal glucose, whereas they had a cobblestone morphology in high glucose conditions, accompanied by decreased nephrin expression and increased desmin expression, suggesting podocytes underwent EMT. In response to high glucose, CTGF and ILK expression in podocytes were increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Furthermore, the inhibition of CTGF with anti-CTGF antibody prevented the phenotypic transition, as demonstrated by the preservation of epithelial morphology, the suppression of high glucose-induced desmin overexpression and the restoration of nephrin. Of note, the upregulation of ILK induced by high glucose was partially blocked by the inhibition of CTGF. In summary, these findings suggested that CTGF and ILK were involved in high glucose-induced phenotypic alterations of podocytes. ILK acted as a downstream kinase of CTGF and high glucose-induced ILK expression might occur through CTGF-dependent and -independent pathways. Copyright © 2011 Wiley Periodicals, Inc.

Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier Function.

PubMed

Sayedyahossein, Samar; Rudkouskaya, Alena; Leclerc, Valerie; Dagnino, Lina

2016-02-01

A functional permeability barrier is essential to prevent the passage of water and electrolytes, macromolecules, and pathogens through the epidermis. This is accomplished in terminally differentiated keratinocytes through formation of a cornified envelope and the assembly of tight intercellular junctions. Integrin-linked kinase (ILK) is a scaffold protein essential for hair follicle morphogenesis and epidermal attachment to the basement membrane. However, the biological functions of ILK in differentiated keratinocytes remain poorly understood. Furthermore, whether ILK is implicated in keratinocyte differentiation and intercellular junction formation has remained an unresolved issue. Here we describe a pivotal role for ILK in keratinocyte differentiation responses to increased extracellular Ca(2+), regulation of adherens and tight junction assembly, and the formation of an outside-in permeability barrier toward macromolecules. In the absence of ILK, the calcium sensing receptor, E-cadherin, and ZO-1 fail to translocate to the cell membrane, through mechanisms that involve abnormalities in microtubules and in RhoA activation. In situ, ILK-deficient epidermis exhibits reduced tight junction formation and increased outside-in permeability to a dextran tracer, indicating reduced barrier properties toward macromolecules. Therefore, ILK is an essential component of keratinocyte differentiation programs that contribute to epidermal integrity and the establishment of its barrier properties. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Integrin-Linked Kinase Deletion in the Developing Lens Leads to Capsule Rupture, Impaired Fiber Migration and Non-Apoptotic Epithelial Cell Death

PubMed Central

Cammas, Laura; Wolfe, Jordan; Choi, Sue-Yeon; Dedhar, Shoukat; Beggs, Hilary E

2012-01-01

Purpose. The lens is a powerful model system to study integrin-mediated cell-matrix interaction in an in vivo context, as it is surrounded by a true basement membrane, the lens capsule. To characterize better the function of integrin-linked kinase (ILK), we examined the phenotypic consequences of its deletion in the developing mouse lens. Methods. ILK was deleted from the embryonic lens either at the time of placode invagination using the Le-Cre line or after initial lens formation using the Nestin-Cre line. Results. Early deletion of ILK leads to defects in extracellular matrix deposition that result in lens capsule rupture at the lens vesicle stage (E13.5). If ILK was deleted at a later time-point after initial establishment of the lens capsule, rupture was prevented. Instead, ILK deletion resulted in secondary fiber migration defects and, most notably, in cell death of the anterior epithelium (E18.5 − P0). Remarkably, dying cells did not stain positively for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or activated-caspase 3, suggesting that they were dying from a non-apoptotic mechanism. Moreover, cross to a Baxfl/fl/Bak−/− mouse line that is resistant to most forms of apoptosis failed to promote cell survival in the ILK-deleted lens epithelium. Electron microscopy revealed the presence of numerous membranous vacuoles containing degrading cellular material. Conclusions. Our study reveals a role for ILK in extracellular matrix organization, fiber migration, and cell survival. Furthermore, to our knowledge we show for the first time that ILK disruption results in non-apoptotic cell death in vivo. PMID:22491404

Multiple Roles of Integrin-Linked Kinase in Epidermal Development, Maturation and Pigmentation Revealed by Molecular Profiling

PubMed Central

Judah, David; Rudkouskaya, Alena; Wilson, Ryan; Carter, David E.; Dagnino, Lina

2012-01-01

Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function. PMID:22574216

Multiple roles of integrin-linked kinase in epidermal development, maturation and pigmentation revealed by molecular profiling.

PubMed

Judah, David; Rudkouskaya, Alena; Wilson, Ryan; Carter, David E; Dagnino, Lina

2012-01-01

Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function.

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

Epigenetic regulation of integrin-linked kinase expression depending on adhesion of gastric carcinoma cells.

PubMed

Kim, Yong-Bae; Lee, Sung-Yul; Ye, Sang-Kyu; Lee, Jung Weon

2007-02-01

Cell adhesion to the extracellular matrix (ECM) regulates gene expressions in diverse dynamic environments. However, the manner in which gene expressions are regulated by extracellular cues is largely unknown. In this study, suspended gastric carcinoma cells showed higher basal and transforming growth factor-beta1 (TGFbeta1)-mediated acetylations of histone 3 (H3) and Lys(9) of H3 and levels of integrin-linked kinase (ILK) mRNA and protein than did fibronectin-adherent cells did. Moreover, the insignificant acetylation and ILK expression in adherent cells were recovered by alterations of integrin signaling and actin organization, indicating a connection between cytoplasmic and nuclear changes. Higher acetylations in suspended cells were correlated with associations between Smad4, p300/CBP, and Lys(9)-acetylated H3. Meanwhile, adherent cells showed more associations between HDAC3, Ski, and MeCP2. Chromatin immunoprecipitations with anti-acetylated H3, Lys(9)-acetylated H3, or p300/CBP antibody resulted in more coprecipitated ILK promoter, correlated with enhanced ILK mRNA and protein levels, in suspended cells. Moreover, ILK expression inversely regulated cell adhesion to ECM proteins, and its overexpression enhanced cell growth in soft agar. These observations indicate that cell adhesion and/or its related molecular basis regulate epigenetic mechanisms leading to a loss of ILK transcription, which in turn regulates cell adhesion property in a feedback linkage.

Inhibition of integrin-linked kinase expression by emodin through crosstalk of AMPKα and ERK1/2 signaling and reciprocal interplay of Sp1 and c-Jun.

PubMed

Tang, Qing; Zhao, Shunyu; Wu, Jingjing; Zheng, Fang; Yang, LiJun; Hu, JingHeng; Hann, Swei Sunny

2015-07-01

Despite the anti-cancer effect of emodin observed in several cancers, the underlying molecular mechanism remains to be elucidated. In this study, we showed that emodin-inhibited NSCLC cell growth and increased phosphorylation of AMPKα and ERK1/2. In addition, emodin-inhibited ILK protein expression. The overexpression of ILK reversed the effect of emodin on cell growth inhibition. Furthermore, the blockade of AMPK by compound C abrogated, while metformin, an activator of AMPK, strengthened the effect of emodin on the inhibition of ILK expression. Interestingly, the inhibitor of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK)/ERK1/2 (PD98059) attenuated emodin-induced phosphorylation of AMPKα. Moreover, emodin reduced the protein expression of Sp1 and AP-1 subunit c-Jun. Exogenous expression of Sp1 and c-Jun diminished emodin-reduced ILK protein expression. Emodin suppressed ILK promoter activity, which was not observed in cells overexpression of Sp1 and treated with compound C. Intriguingly, exogenous expression of c-Jun overcame the emodin-inhibited Sp1 protein expression. Collectively, our results demonstrate that emodin inhibits ILK expression through AMPKα-mediated reduction of Sp1 and c-Jun. Metformin enhances the effects of emodin. Exogenous expression of Sp1 and c-Jun resists emodin-inhibited ILK promoter activity and protein expression. In addition, the overexpression of c-Jun diminishes emodin-induced AMPKα signaling. Thus, the crosstalk of AMPKα and MEK/ERK1/2 signaling and the reciprocal interaction between Sp1 and c-Jun proteins contribute to the overall responses of emodin. This novel signaling axis may be a therapeutic potential for prevention and treatment of NSCLC. Copyright © 2015 Elsevier Inc. All rights reserved.

Training Program in Breast Cancer Research at the University of Texas M.D. Anderson Cancer Center

DTIC Science & Technology

2004-09-01

treated with the RTK lated in 3T3-HER2 cells (Figure la, lane 3), while there inhibitor emodin and PI-3K-specific inhibitor wortman- was no...increasing its expression. Since it has been shown that emodin inhibited tyrosine phosphorylation (PY20) of PI-3K activates ILK (Delcommenne et al...IP C Wo Em in the cell survival pathway. As expected, Akt phos- ILK lgG ILK 19G p-MBP ft phorylation (p-Akt) was also blocked by emodin 4~’BP

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

Yang, Yanwu; Wang, Xiaoxia; Hawkins, Cheryl A.

The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK {center_dot} PINCH complex (28 kDa, K{sub D} {approx} 68 nm) involving the N-terminal ankyrin repeatmore » domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.« less

Integrin-linked kinase is required for TGF-β1 induction of dermal myofibroblast differentiation.

PubMed

Vi, Linda; de Lasa, Cristina; DiGuglielmo, Gianni M; Dagnino, Lina

2011-03-01

Cutaneous repair after injury requires activation of resident dermal fibroblasts and their transition to myofibroblasts. The key stimuli for myofibroblast formation are activation of transforming growth factor-β (TGF-β) receptors and mechanotransduction mediated by integrins and associated proteins. We investigated the role of integrin-linked kinase (ILK) in TGF-β1 induction of dermal fibroblast transition to myofibroblasts. ILK-deficient fibroblasts treated with TGF-β1 exhibited attenuation of Smad 2 and 3 phosphorylation, accompanied by impaired transcriptional activation of Smad targets, such as α-smooth muscle actin. These alterations were not limited to Smad-associated TGF-β1 responses, as stimulation of noncanonical mitogen-activated protein kinase pathways by this growth factor was also diminished in the absence of ILK. ILK-deficient fibroblasts exhibited abnormalities in the actin cytoskeleton, and did not form supermature focal adhesions or contractile F-actin stress fibers, indicating a severe impairment in their capacity to differentiate into myofibroblasts. These defects extended to the inability of cells to contract extracellular matrices when embedded in collagen lattices. We conclude that ILK is necessary to transduce signals implicated in the transition of dermal fibroblasts to myofibroblasts originating from matrix substrates and TGF-β1.

Emodin ameliorates high glucose induced-podocyte epithelial-mesenchymal transition in-vitro and in-vivo.

PubMed

Chen, Tingfang; Zheng, Li Yang; Xiao, Wenzhen; Gui, Dingkun; Wang, Xiaoxia; Wang, Niansong

2015-01-01

Epithelial-to-mesenchymal transition (EMT) is a potential pathway leading to podocyte depletion and proteinuria in diabetic kidney disease (DKD). Here, we investigated the protective effects of Emodin (EMO) on high glucose (HG) induced-podocyte EMT in-vitro and in-vivo. Conditionally immortalized mouse podocytes were exposed to HG with 30 μg /ml of EMO and 1 μmol/ml of integrin-linked kinase (ILK) inhibitor QLT0267 for 24 h. Streptozotocin (STZ)-induced diabetic rats were treated with EMO at 20 mg· kg(-1)· d(-1) and QLT0267 at 10 mg· kg(-1)· w(-1) p.o., for 12 weeks. Albuminuria and blood glucose level were measured. Immunohistochemistry, immunofluorescence, western blotting and real-time PCR were used to detect expression of ILK, the epithelial marker of nephrin and the mesenchymal marker of desmin in-vitro and in-vivo. HG increased podocyte ILK and desmin expression while decreased nephrin expression. However, EMO significantly inhibited ILK and desmin expression and partially restored nephrin expression in HG-stimulated podocytes. These in-vitro observations were further confirmed in-vivo. Treatment with EMO for 12 weeks attenuated albuminuria, renal histopathology and podocyte foot process effacement in diabetic rats. EMO also repressed renal ILK and desmin expression, preserved nephrin expression, as well as ameliorated albuminuria in STZ-induced diabetic rats. EMO ameliorated glucose-induced EMT and subsequent podocyte dysfunction partly through ILK and desmin inhibition as well as nephrin upregulatiotion, which might provide a potential novel therapeutic option for DKD. © 2015 S. Karger AG, Basel.

Estimation of pH effect on the structure and stability of kinase domain of human integrin-linked kinase.

PubMed

Syed, Sunayana Begum; Shahbaaz, Mohd; Khan, Sabab Hassan; Srivastava, Saurabha; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2018-01-07

Integrin-linked kinase (ILK) is an evolutionarily conserved Ser/Thr protein kinase, involved in many physiological functions such as signal transduction, actin rearrangement, cell proliferation, migration, polarisation, angiogenesis and apoptosis. An increased expression of ILK is associated with different cancers and thus considered as an attractive target for cancer therapy. We have successfully cloned, expressed and purified the kinase domain (193-446 residues) of ILK. To see the effect of pH on the structure and conformation, we performed circular diachroism, fluorescence and absorbance measurements in a wide range of pH conditions. We observed that within the range of pH 7.5-11.0, ILK 193-446 maintains its both secondary and tertiary structures. While visible aggregates were observed under the acidic pH 2.0-5.5 conditions, in order to complement these observations, we have performed molecular dynamics simulations of this kinase domain by mimicking diverse pH conditions which enabled us to see conformational preferences of the protein under such conditions. A significant correlation between the spectroscopic and molecular dynamics simulation was observed. These findings are useful to understand the conformation of ILK protein under certain pH condition which may be further implicated in the drug design and discovery.

Mechanistic insights into the urea-induced denaturation of kinase domain of human integrin linked kinase.

PubMed

Syed, Sunayana Begum; Khan, Faez Iqbal; Khan, Sabab Hasan; Srivastava, Saurabha; Hasan, Gulam Mustafa; Lobb, Kevin A; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2018-05-01

Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK 193-446 ) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ] 222 (mean residue ellipticity at 222nm), difference absorption coefficient at 292nm (Δε 292 ) and intrinsic fluorescence emission intensity at pH7.5 and 25±0.1°C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0-7.0M. A coincidence of normalized denaturation curves of optical properties ([θ] 222 , Δε 292 and λ max , the wavelength of maximum emission intensity) suggested that urea-induced denaturation of kinase domain of ILK is a two-state process. We further performed molecular dynamics simulation for 100ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies. Copyright © 2018 Elsevier B.V. All rights reserved.

EPA and DHA increased PPARγ expression and deceased integrin-linked kinase and integrin β1 expression in rat glomerular mesangial cells treated with lipopolysaccharide.

PubMed

Han, Wenchao; Zhao, Hui; Jiao, Bo; Liu, Fange

2014-04-01

Fish oil containing n-3 polyunsaturated fatty acids (n-3 PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is known to prevent the progression of nephropathy and retard the progression of kidney disease. This study sought to investigate the underlying mechanisms of EPA and DHA in terms of peroxisome proliferator-activated receptor γ (PPARγ), integrin-linked kinase (ILK), and integrin β1 expression in glomerular mesangial cells (GMCs) because of their critical roles in the development and progression of nephropathy. Lipopolysaccharide (LPS) significantly reduced the expression of PPARγand increased the expression of ILK at the mRNA level and at the protein level in GMCs as indicated by real-time PCR and Western blotting. In addition, LPS increased integrin β1 expression in GMCs at the mRNA level. Treatment with EPA and DHA significantly increased the expression of PPARγ and decreased the expression of ILK and integrin β1 in GMCs. These data suggest that the renoprotective effects of EPA and DHA may be related to their potential to increase the expression of PPARγ and decrease the expression of ILK and integrin β1.

Dermal fibroblast-to-myofibroblast transition sustained by αvß3 integrin-ILK-Snail1/Slug signaling is a common feature for hypermobile Ehlers-Danlos syndrome and hypermobility spectrum disorders.

PubMed

Zoppi, Nicoletta; Chiarelli, Nicola; Binetti, Silvia; Ritelli, Marco; Colombi, Marina

2018-04-01

Hypermobile Ehlers-Danlos syndrome (hEDS) is a heritable connective tissue disorder with unknown molecular basis mainly characterized by generalized joint hypermobility, joint instability complications, and minor skin changes. The phenotypic spectrum is broad and includes multiple associated symptoms shared with chronic inflammatory systemic diseases. The stricter criteria defined in the 2017 EDS nosology leave without an identity many individuals with symptomatic joint hypermobility and/or features of hEDS; for these patients, the term Hypermobility Spectrum Disorders (HSD) was introduced. We previously reported that in vitro cultured hEDS and HSD patients' skin fibroblasts show a disarray of several extracellular matrix (ECM) components and dysregulated expression of genes involved in connective tissue homeostasis and inflammatory/pain/immune responses. Herein, we report that hEDS and HSD skin fibroblasts exhibit in vitro a similar myofibroblast-like phenotype characterized by the organization of α-smooth muscle actin cytoskeleton, expression of OB-cadherin/cadherin-11, enhanced migratory capability associated with augmented levels of the ECM-degrading metalloproteinase-9, and altered expression of the inflammation mediators CCN1/CYR61 and CCN2/CTGF. We demonstrate that in hEDS and HSD cells this fibroblast-to-myofibroblast transition is triggered by a signal transduction pathway that involves αvβ3 integrin-ILK complexes, organized in focal adhesions, and the Snail1/Slug transcription factor, thus providing insights into the molecular mechanisms related to the pathophysiology of these protean disorders. The indistinguishable phenotype identified in hEDS and HSD cells resembles an inflammatory-like condition, which correlates well with the systemic phenotype of patients, and suggests that these multisystemic disorders might be part of a phenotypic continuum rather than representing distinct clinical entities. Copyright © 2018 Elsevier B.V. All rights reserved.

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus.

PubMed

Kanasaki, Keizo; Kanda, Yoshiko; Palmsten, Kristin; Tanjore, Harikrishna; Lee, Soo Bong; Lebleu, Valerie S; Gattone, Vincent H; Kalluri, Raghu

2008-01-15

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

PubMed

Ho, Ernest; Ivanova, Iordanka A; Dagnino, Lina

2016-12-01

The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions. Although the mechanisms that mediate E-cadherin delivery to the plasma membrane have been widely studied in simple epithelia, this process is less well understood in the stratified epidermis. In this study, we have investigated the role of Engulfment and Cell Motility 2 (ELMO2) and integrin-linked kinase (ILK) in the positioning of E-cadherin-containing recycling endosomes during establishment of cell-cell contacts in differentiating keratinocytes. We now show that induction of keratinocyte differentiation by Ca 2+ is accompanied by localization of ELMO2 and ILK to Rab4- and Rab11a-containing recycling endosomes. The positioning of long-loop Rab11a-positive endosomes at areas adjacent to cell-cell contacts is disrupted in ELMO2- or ILK-deficient keratinocytes, and is associated with impaired localization of E-cadherin to cell borders. Our studies show a previously unrecognized role for ELMO2 and ILK in modulation of endosomal positioning, which may play key roles in epidermal sheet maintenance and permeability barrier function. Copyright © 2016 Elsevier B.V. All rights reserved.

The psychosocial situation of obese children: Psychological factors and quality of life

PubMed Central

Korsten-Reck, U; Korsten, K; Haeberle, K; Kromeyer-Hauschild, K; Dickhuth, H H; Schulz, E

2009-01-01

The psychosocial situation of obese children at the beginning of the Freiburg Intervention Trial for Obese Children (FITOC) program influences the course and outcome of intervention therapy. At the beginning of FITOC, mothers of 30 children (12 ♂ age 10.5 ± 1.4, 18 ♀; 10.2 ± 1.3) rated the psychopathological symptoms using the Child Behavior Checklist (CBCL) and the quality of life of their children (Inventar zur Erfassung der Lebensqualität [ILK]). 46.6% of mothers rated their children to be disturbed (normal population group, 2% disturbed). There was no correlation between the body mass index of the child and the CBCL score. On the ILK, the obese children themselves selected “good” while the mother’s view of their childrens’ quality of life varied around “poor”. The stress on the mother increased with the degree of the child’s obesity and number of critical life events (p = 0.030). The CBCL and the ILK at the beginning of treatment serve as a good screening instrument for psychopathology and distress in children with obesity. PMID:22110318

Galectin-3 enhances angiogenic and migratory potential of microglial cells via modulation of integrin linked kinase signaling

PubMed Central

Wesley, Umadevi V.; Vemuganti, Raghu; Ayvaci, Rabia; Dempsey, Robert J.

2013-01-01

Focal cerebral ischemia initiates self-repair mechanisms that include the production of neurotrophic factors and cytokines. Galectin-3 is an important angiogenic cytokine. We have previously demonstrated that expression of galectin 3 (Gal-3), a carbohydrate binding protein is significantly upregulated in activated microglia in the brains of rats subjected to focal ischemia. Further blocking of Gal-3 function with Gal-3 neutralizing antibody decreased the microvessel density in ischemic brain. We currently show that Gal-3 significantly increases the viability of microglia BV2 cells subjected to oxygen glucose deprivation (OGD) and re-oxygenation. Exogenous Gal-3 promoted the formation of pro-angiogenic structures in an in vitro human umbilical vein endothelial (HUVEC) and BV2 cell co-culture model. Gal-3 induced angiogenesis was associated with increased expression of vascular endothelial growth factor. The conditioned medium of BV2 cells exposed to OGD contained increased Gal-3 levels, and promoted the formation of pro-angiogenic structures in an in vitro HUVEC culture model. Gal-3 also augmented the in vitro migratory potential of BV2 microglia. Gal-3 mediated functions were associated with increased levels of integrin-linked kinase (ILK) signaling as demonstrated by the impaired angiogenesis and migration of BV2 cells following targeted silencing of ILK expression by SiRNA. Furthermore, we show that ILK levels correlate with the levels of phos-AKT and ERK1/2 that are downstream effectors of ILK pathway. Taken together, our studies indicate that Gal-3 contributes to angiogenesis and microglia migration that may have implications in post stroke repair. PMID:23246924

Epidermal growth factor induction of front–rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

PubMed Central

Ho, Ernest; Dagnino, Lina

2012-01-01

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front–rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front–rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front–rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front–rear polarity and forward movement. PMID:22160594

Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2.

PubMed

Ho, Ernest; Dagnino, Lina

2012-02-01

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.

Subchronic exposure to arsenic through drinking water alters expression of cancer-related genes in rat liver.

PubMed

Cui, Xing; Li, Song; Shraim, Amjad; Kobayashi, Yayoi; Hayakawa, Toru; Kanno, Sanae; Yamamoto, Megumi; Hirano, Seishiro

2004-01-01

Although arsenic exposure causes liver disease and/or hepatoma, little is known about molecular mechanisms of arsenic-induced liver toxicity or carcinogenesis. We investigated the effects of arsenic on expression of cancer-related genes in a rat liver following subchronic exposure to sodium arsenate (1, 10, 100 ppm in drinking water), by using real-time quantitative RT-PCR and immunohistochemical analyses. Arsenic accumulated in the rat liver dose-dependently and caused hepatic histopathological changes, such as disruption of hepatic cords, sinusoidal dilation, and fatty infiltration. A 1-month exposure to arsenic significantly increased hepatic mRNA levels of cyclin D1 (10 ppm), ILK (1 ppm), and p27(Kip1) (10 ppm), whereas it reduced mRNA levels of PTEN (1 ppm) and beta-catenin (100 ppm). In contrast, a 4-month arsenic exposure showed increased mRNA expression of cyclin D1 (100 ppm), ILK (1 ppm), and p27(Kip1) (1 and 10 ppm), and decreased expression of both PTEN and beta-catenin at all 3 doses. An immunohistochemical study revealed that each protein expression accords closely with each gene expression of mRNA level. In conclusion, subchronic exposure to inorganic arsenate caused pathological changes and altered expression of cyclin D1, p27(Kip1), ILK, PTEN, and beta-catenin in the liver. This implies that arsenic liver toxicity involves disturbances of some cancer-related molecules.

An oyster species-specific miRNA scaffold42648_5080 modulates haemocyte migration by targeting integrin pathway.

PubMed

Chen, Hao; Wang, Hao; Jiang, Shuai; Xu, Jiachao; Wang, Lingling; Qiu, Limei; Song, Linsheng

2016-10-01

miRNAs are important gene regulators at post-transcriptional level and can modulate diverse biological processes, including immune response. Dozens of species-specific miRNAs have been identified in oyster Crassostrea gigas while their functions remain largely unknown. In the present study, an oyster species-specific miRNA scaffold42648_5080 was found responsive to LPS stimulation and might target a total of 31 oyster genes possibly involved in cell communication, cellular localization and cellular response to stimulus. Besides, in gain-of-function assay of scaffold42648_5080 in vivo, the phagocytosis (30.90% in miRNA group verse 23.20% in miRNA control group), apoptosis (3.10% in miRNA group verse 5.30% in miRNA control group) and migration rate (13.88% in miRNA group verse 21.03% in miRNA control group) of oyster haemocytes were found significantly altered after the injection of scaffold42648_5080 mimics. Among the target genes, integrin-linked kinase (CgILK) was considered crucial in cell migration and its interaction with scaffold42648_5080 was then verified both in vitro and in vivo. Consequently, a significant decrease of relative luciferase ratio was observed in CgILK 3'-UTR luciferase reporter assay after transfection of scaffold42648_5080 mimics (0.70-fold of that in blank group, p < 0.01). Meanwhile, when scaffold42648_5080 was overexpressed in vivo (5.41-fold of miRNA control group, p < 0.01), the expression of CgILK declined significantly to 0.25-fold of miRNA control group (p < 0.01). Comparatively, a significant decrease of the haemocyte migration rate (19.76% verse 34.82% in siEGFP control group, p < 0.01) was observed after knock-down of CgILK in vivo. The present study, as far as we know, for the first time revealed the immunomodulation role of an oyster species-specific miRNA, which might provide new insights into miRNA-mediated adaptation mechanism of oysters. Copyright © 2016 Elsevier Ltd. All rights reserved.

Galectin-3 Mediates Tumor Cell-Stroma Interactions by Activating Pancreatic Stellate Cells to Produce Cytokines via Integrin Signaling.

PubMed

Zhao, Wei; Ajani, Jaffer A; Sushovan, Guha; Ochi, Nobuo; Hwang, Rosa; Hafley, Margarete; Johnson, Randy L; Bresalier, Robert S; Logsdon, Craig D; Zhang, Zhiqian; Song, Shumei

2018-04-01

Pancreatic ductal adenocarcinoma (PDAC) is characterized by activated pancreatic stellate cells (PSCs), abundance of extracellular matrix (ECM), and production of cytokines and chemokines. Galectin 3 (GAL3), a β-galactoside-specific lectin, contributes to PDAC development but its effects on the stroma and cytokine production are unclear. The effect of recombinant human GAL3 (rGAL3) on activation of PSCs, production of cytokines, and ECM proteins was determined by proliferation, invasion, cytokine array, and quantitative polymerase chain reaction. We assessed co-cultures of PDAC cells with GAL3 genetic alterations with PSCs. Production of interleukin 8 (IL8) and activities of nuclear factor (NF)-κB were determined by enzyme-linked immunosorbent assay and luciferase reporter analyses. We studied the effects of inhibitors of NF-κB and integrin-linked kinase (ILK) on pathways activated by rGAL3. In analyses of the Gene Expression Omnibus database and our dataset, we observed higher levels of GAL3, IL8, and other cytokines in PDAC than in nontumor tissues. Production of IL8, granulocyte-macrophage colony-stimulating factor, chemokine ligand 1, and C-C motif chemokine ligand 2 increased in PSCs exposed to rGAL3 compared with controls. Culture of PSCs with PDAC cells that express different levels of GAL3 resulted in proliferation and invasion of PSCs that increased with level of GAL3. GAL3 stimulated transcription of IL8 through integrin subunit beta 1 (ITGB1) on PSCs, which activates NF-κB through ILK. Inhibitors of ILK or NF-κB or a neutralizing antibody against ITGB1 blocked transcription and production of IL8 from PSCs induced by rGAL3. The GAL3 inhibitor significantly reduced growth and metastases of orthotopic tumors that formed from PDAC and PSC cells co-implanted in mice. GAL3 activates PSC cells to produce inflammatory cytokines via ITGB1signaling to ILK and activation of NF-κB. Inhibition of this pathway reduced growth and metastases of pancreatic

Determining the Number of Component Clusters in the Standard Multivariate Normal Mixture Model Using Model-Selection Criteria.

DTIC Science & Technology

1983-06-16

has been advocated by Gnanadesikan and 𔃾ilk (1969), and others in the literature. This suggests that, if we use the formal signficance test type...American Statistical Asso., 62, 1159-1178. Gnanadesikan , R., and Wilk, M..B. (1969). Data Analytic Methods in Multi- variate Statistical Analysis. In

GPU-enabled molecular dynamics simulations of ankyrin kinase complex

NASA Astrophysics Data System (ADS)

Gautam, Vertika; Chong, Wei Lim; Wisitponchai, Tanchanok; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abd.; Tayapiwatana, Chatchai; Lee, Vannajan Sanghiran

2014-10-01

The ankyrin repeat (AR) protein can be used as a versatile scaffold for protein-protein interactions. It has been found that the heterotrimeric complex between integrin-linked kinase (ILK), PINCH, and parvin is an essential signaling platform, serving as a convergence point for integrin and growth-factor signaling and regulating cell adhesion, spreading, and migration. Using ILK-AR with high affinity for the PINCH1 as our model system, we explored a structure-based computational protocol to probe and characterize binding affinity hot spots at protein-protein interfaces. In this study, the long time scale dynamics simulations with GPU accelerated molecular dynamics (MD) simulations in AMBER12 have been performed to locate the hot spots of protein-protein interaction by the analysis of the Molecular Mechanics-Poisson-Boltzmann Surface Area/Generalized Born Solvent Area (MM-PBSA/GBSA) of the MD trajectories. Our calculations suggest good binding affinity of the complex and also the residues critical in the binding.

A Signaling Network Induced by β2 Integrin Controls the Polarization of Lytic Granulesin Cytotoxic Cells

PubMed Central

Zhang, Minggang; March, Michael E.; Lane, William S.; Long, Eric O.

2014-01-01

Cytotoxic lymphocyte skill target cells by polarized release of the content of perforin-containing granules. In natural killer cells, the binding of β2 integrin to its ligand ICAM-1 is sufficient to promote not only adhesion but also lytic granule polarization. This provided a unique opportunity to study polarization in the absence of degranulation, and β2 integrin signaling independently of inside-out signals from other receptors. Using an unbiased proteomics approach we identified a signaling network centered on an integrin-linked kinase (ILK)–Pyk2–Paxillin core that was required for granule polarization. Downstream of ILK, the highly conserved Cdc42–Par6 signaling pathway that controls cell polarity was activated and required for granule polarization. These results delineate two connected signaling networks induced upon β2 integrin engagement alone, which are integrated to control polarization of the microtubule organizing center and associated lytic granules toward the site of contact with target cells during cellular cytotoxicity. PMID:25292215

Role of the Integrin-Linked Kinase, ILK, in Mammary Carcinogensis

DTIC Science & Technology

2000-08-01

have been implicated in environmental stress clonei 6-10 responses in yeasts, plants and mammals, as well as regulating abscisic acid signal transduction...phosphatase 2C involved in abscisic acid signal transduction in higher plants. Proc. Natl Acad. Sci. USA, 95, 975-980. Strovel,E.T., Wu,D. and Sussman,D.J...contain a 450bp open reading frame, coding for 149 amino acids and a poly A tail 245bp downstream of the stop codon, although no polyadenylation site

The Democratic-Republican Societies: An Educational Dream Deferred

ERIC Educational Resources Information Center

Dotts, Brian W.

2010-01-01

Historians often identify Horace Mann and other middle-class reformers of the mid-nineteenth century as the catalysts of the Common School Movement, the forerunner of today's public school systems. Despite education historians' focus on Mann and his ilk as the original advocates of common schooling, the notion of free universal public education…

Advanced Ultra-Violet (UV) Aircraft Fire Detection System. Volume 3. Ground Support Equipment (GSE) for System Check-Out

DTIC Science & Technology

1982-08-01

linsutaled * ifr rnif OC used nstead of ins resistors). 76 _ - Thil positions are as follows. 76 .APPENDIX A-2 3.2 PAper Loading TI’a6414ý-;jies lphnumeic ji...34i I I S.... . . I. ..... . .... •..................... ..- 4 . . o .. o . . .... . 149 :. ; , e~Vh -rom PPE ~t~ilk A-9 e" OSE S ~1: el/THIGH Is

USAF/SCEEE Summer Faculty Research Program (1979). Volume 2

DTIC Science & Technology

1979-12-01

Summer Faculty Research Program participants. The program designed to stimulate ’Ilk scientific and engineering interaction between university faculty...Prog., Dept. of Industrial Engineering Facility design and location theory University of Oklahoma and routing and distribution systems 202 W. Boyd...Theory & Assistant Professor of Management Adninistration, 1975 University of Akron S.ec aIty: Organization Design Akron, OH 44325 Assigned: AFBRMC

Organizational Environment and Preferences for Leadership and Power in the Officer Corps

DTIC Science & Technology

1977-06-10

satisfaction and performance or output may be higher in open organizational environments. In the work by Schwartz, et al, mentioned above. Ilk... satisfaction , anxiety/stress, and performance . Administrative Science Quarterly. 1975, 20(2), 272-280. Koontz, H. The management theory jungle...CowsertTjGarrett T. ,:IVIa$^SyCCH^o - ^t- , a ^ a) O- O 9. PERFORMING ORGANIZATION NAME AND ADDRESS Student at the U.S. Army Command and General

[ICD-10 diagnosis and quality of life. A pilot study of quality of life of children and adolescents with psychiatric disorders].

PubMed

Schubert, M T; Herle, M; Wurst, E

2003-11-01

The present study examined whether the quality of life in children and adolescents with psychological disorders, as judged by the patients themselves and their mothers, differed according to the various ICD-10 diagnoses or the number of axes involved. 151 children/adolescents and 125 mothers, referred consecutively to the clinic, completed the Inventory for Evaluation of Quality of Life in Children and Adolescents (Inventar zur Erfassung der Lebensqualität bei Kindern und Jugendlichen; ILK) by Mattejat et al. ICD-10 diagnoses were grouped for evaluation. No significant interaction between the five diagnostic axes and the several domains of quality of life was found. However, mothers of children and adolescents with attention deficit/hyperactivity disorders and/or conduct disorder more often tended to judge their children's quality of life as unsatisfactory in all domains, while the patients themselves did not. Thus, rather than the children themselves it seems to be the children's environment which considers "external disorders" to be distressing. The authors conclude that the quality of life as measured by the ILK cannot be captured by ICD-10 criteria. Apparently it is not so much the diagnosis itself but its subjective meaning that has the most essential impact on an individual's assessment of quality of life.

Applied Physics Research for Innovation in Pulsed Power

DTIC Science & Technology

1994-09-30

yet li.... hilh I"*~ OWLC Figeue of (r-esoh gad) worik So e 0o0 baeer i*s es..esem or": Lane. bndgs, low N bu ilk ,, ly e w a, ver h voka" Oiemoed...4)~~ ~ 2WV"tW ~ electron drift mobility C) 2 V 3 FurLow forward voltage drop and 4) CO V OA. PEfT fast switching speed In majority carriler devices t

The Toxicology, Environmental Fate, and Human Risk of Herbicide Orange and Its Associated Dioxin

DTIC Science & Technology

1978-10-01

CONTENTS Pao@ PREFACE v CHAPTER I THE USE OF HERBICIDES IN SOUTH VIETNAM I. INTRODUCTION I-1 II. THE...SUMMARY V-32 LITERATURE CITED V-33 LIST OF TABLES 1. Total United States production and use of 2,4,5-T herbicide for the period 1961 through 1969. V-3...reduction. Techniques to recover a useful product included use , return to manufacturers, fractionation and chlorinolysis. LI -A,7 4~ilk Of these

U. S. Naval Forces, Vietnam Monthly Historical Summary for May 1970

DTIC Science & Technology

1970-07-15

of 18 of the vessels, which were loaded with clothing, utensils, food , and weapons. They received automatic weapons fire from the ground but completed...Investigation revealed a camouflaged food cache about three days old containing 2400 pounds of rice and 150 pounds of salt. Crew members destroyed the... food with 46 white phosphorous grenades. 17 On ICU q UNp Ilk,-- - - W T , - - - W - - - CONFIDENTIAL Riverine Strike Group ", During May, all RAC

Evidence for Integrin - Venus Kinase Receptor 1 Alliance in the Ovary of Schistosoma mansoni Females Controlling Cell Survival.

PubMed

Gelmedin, Verena; Morel, Marion; Hahnel, Steffen; Cailliau, Katia; Dissous, Colette; Grevelding, Christoph G

2017-01-01

In metazoan integrin signaling is an important process of mediating extracellular and intracellular communication processes. This can be achieved by cooperation of integrins with growth factor receptors (GFRs). Schistosoma mansoni is a helminth parasite inducing schistosomiasis, an infectious disease of worldwide significance for humans and animals. First studies on schistosome integrins revealed their role in reproductive processes, being involved in spermatogenesis and oogenesis. With respect to the roles of eggs for maintaining the parasite´s life cycle and for inducing the pathology of schistosomiasis, elucidating reproductive processes is of high importance. Here we studied the interaction of the integrin receptor Smβ-Int1 with the venus kinase receptor SmVKR1 in S. mansoni. To this end we cloned and characterized SmILK, SmPINCH, and SmNck2, three putative bridging molecules for their role in mediating Smβ-Int1/SmVKR1 cooperation. Phylogenetic analyses showed that these molecules form clusters that are specific for parasitic platyhelminths as it was shown for integrins before. Transcripts of all genes colocalized in the ovary. In Xenopus oocytes germinal vesicle breakdown (GVBD) was only induced if all members were simultaneously expressed. Coimmunoprecipitation results suggest that a Smβ-Int1-SmILK-SmPINCH-SmNck2-SmVKR1 complex can be formed leading to the phosphorylation and activation of SmVKR1. These results indicate that SmVKR1 can be activated in a ligand-independent manner by receptor-complex interaction. RNAi and inhibitor studies to knock-down SmILK as a representative complex member concurrently revealed effects on the extracellular matrix surrounding the ovary and oocyte localization within the ovary, oocyte survival, and egg production. By TUNEL assays, confocal laser scanning microscopy (CLSM), Caspase-3 assay, and transcript profiling of the pro-apoptotic BCL-2 family members BAK/BAX we obtained first evidence for roles of this signaling

Evaluating the Significance of CDK2-PELP1 Axis in Tumorigenesis and Hormone Therapy Resistance

DTIC Science & Technology

2011-02-01

reagents an breast cancer cells MCF7, ZR75, IMR-90, NIH3T3, 93T were obtained from the American Type Culture tion. All stable cell lines were generated...galactosidase activity or tal protein concentration. ime PCR and cell cycle microarray s were harvested with Trizol Reagent (Invitrogen), and NA was isolated...Margue, F Huselstein, G, Grignard , W Dippel, M, Nathan, S Giacchi, R Scheiden. ILK as a potential marker gene to ascertain specific adenocarcinoma

Translations from Red Flag No. 8, 1 August 1978

DTIC Science & Technology

1978-09-29

marketing and credit cooperatives should call regular meetings of repre- sentatives of the poor and lower-middle peasants, at least once a year. Urban ... urban and rural areas at present have more than 3 billion yuan worth of commodities in transit, tying up large quan- tities of goods and funds. The...will not change its cannibal nature. Why was Hitler able to initiate a war? Wasn’t it because of Chamberlain, Daladier and their ilk that he became

A Comparison of the Usefulness of the Facilities Management Program in the Graduate School of Systems and Logistics and Similar Programs in Civilian Institutions as Perceived by Former Students

DTIC Science & Technology

1980-06-01

8217 \\ PhlipM./Ray ap ta in, usA~Yj AUG 5 1980 IlkJ D DETTBIBUTION STATE?4ENT A Apprtvncl foT public TOIOO.B0 e e i hi U == I The contents off the...education in the context of course content and their programs are useful; the current FM program is current and relevant, however, it does need to be...dedi- cate this thesis to Judy and Becky. TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .". . ..... ......... ......... ......... iii. LIST OF TABLES

XLMS: A Linguistic Memory System,

DTIC Science & Technology

1980-09-01

summum- genus as the root. The node tree is used by XLMS for accessing concepts via their ilks (or nodes via their genuses ) and as the basis for the...2 stereotype 6 *p 6 subclassp 17 #P 9 subcouceptp 17 parenthesized pair 5 subtree extent pointer 35 partitive 6 summum- genus 1, 17.19,.32F path...type of object, the node. A node has two essential components, known as the genus and specializer, which are its immediate constituents. The genus of a

A Deductive Approach to Computer Programming.

DTIC Science & Technology

1986-01-01

82] K. L. (’lark and S.-A. Thrnlund (editors). Logic Programming, Academic Press (1982). A.R.(’. Studies in Data Processing No. 16. : .(;Goguen and...Tiii Siiillii’>>- oftlie t ralnSforunatloll rukisi 5 (v ielt Since e’ach prodite’ ani (’xjpl’ssiill equliv- * ~ Llil i t’qi ii (ilk t it(’ theo’try...S. Boyer and J S. Moore, A Computational Logic, Academic Press, New York, N.Y., 1979. Brand [751 D. Brand, Proving theorems with the modification

Development of a Facility Management and Improvement Manual for Army Service Schools.

DTIC Science & Technology

1983-03-01

Ehhhhomhmhhhhlo EEEEEEEohhhhhI L-Ii Ilk . ~MICROCOPY RESOLUTION TEST CHART NATIONAL BURESU Of STA OSN-T63-A NA01BRA FSADRS16- construction m*nilhlli 8mbtg 10b...U.S. Army Construction Engineering Research Lab. P.O. Box 4005 • 4A762731AT41-A-001 Champaign, IL 61820 _ It. CONTROLLING OFFICtNAME AND ADDRESS 37...developing a data collection and analysis method. The prototype method was discussed in CERL Interim Report P-ill. 3 This prototype method was tested by

Zeolites: Exploring Molecular Channels

ScienceCinema

Arslan, Ilke; Derewinski, Mirek

2018-05-16

Synthetic zeolites contain microscopic channels, sort of like a sponge. They have many uses, such as helping laundry detergent lather, absorbing liquid in kitty litter, and as catalysts to produce fuel. Of the hundreds of types of zeolites, only about 15 are used for catalysis. PNNL catalysis scientists Ilke Arslan and Mirek Derewinksi are studying these zeolites to understand what make them special. By exploring the mystery of these microscopic channels, their fundamental findings will help design better catalysts for applications such as biofuel production.

Zeolites: Exploring Molecular Channels

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

Arslan, Ilke; Derewinski, Mirek

2015-05-22

Synthetic zeolites contain microscopic channels, sort of like a sponge. They have many uses, such as helping laundry detergent lather, absorbing liquid in kitty litter, and as catalysts to produce fuel. Of the hundreds of types of zeolites, only about 15 are used for catalysis. PNNL catalysis scientists Ilke Arslan and Mirek Derewinksi are studying these zeolites to understand what make them special. By exploring the mystery of these microscopic channels, their fundamental findings will help design better catalysts for applications such as biofuel production.

The Snail-Induced Sulfonation Pathway in Breast Cancer Metastasis

DTIC Science & Technology

2014-09-01

of the SNAIL protein with DNA The model of SNAIL, containing 4 Zn fingers bound to DNA, was created using PDB structures 1tf3 (TFIIIA protein, for...AutoDOCK (17) analysis of fragmented LIMD2 structure against that of the pdb struc- ture 3kmw (ILK/a-Parvin), rethreading the LIMD2 structure through the top...Fig. 5E). We assessed the structural similarity between LIMD2 and other reported LIM structures present in the PDB . The superposition of LIMD2 onto the

Wing Axial Loads and Stresses

DTIC Science & Technology

1950-10-04

Appendix "A" A-l to 2 4 7 8 8 15 16 17 19 22 22 26 33 36 44 45 56 56 57 58 61 100 101 102 104 105 107 110 13 A-3 14 A-4...RrpoatNo 70I7C-I •• j-’J"- - .-•*• ’ Ilk I 4 -.- / ’• X .6 *B*t£Sb FAS Ö-1Q-50® rHb <A1QIRrPÖFT fO inr. MODEL y^i^ C

Defense Science Study Group IV: Study Reports 1994-1995. Volume I

DTIC Science & Technology

1996-02-01

100_<- 9 800 70 _ 60- > ~50- P CD 20- 10 450 500 550 600 650 700 750 800 850 900 WAVELENGTH NANOMETEtS 01 GEN II +- EARLY GEN III 0 TYPICAL GEN III...lva•) 6.5 Somlval.ief1 Cd : 3.25 "sle Cd :. 1 Cd : 60 TI:3.25 .pglAdscm[ Pbt:I0 Pb:100 Cd.Pb40 Pb:is Low Volotie ",metas 0 (Am. Be. Cr. ON (Ilk As. Cr...mixed radioactive-hazardous wastes include steam reforming, wet air oxidation, and high pressure hydrothermal processing[11]. High pressure hydrothermal

Fire Support Requirements Methodology Study, Phase 2 Proceedings of the Fire Support Methodology Workshop

DTIC Science & Technology

1975-12-18

It was not immediatei- clear that the -approach- would- succeed in overcoming the deficiencies of present fire support methodologies which demand- an...support require analysis up to Level 6. They also felt that deficiencies in f technique were most serious at Levels 3, 4 and 5. It was accepted that...defined as: Tk2 = _Tkl ilk2 kl (2) Tkt = Tk,t-l - ’lMktMk,t-l + 𔃼kt ,t-2 I t > (3. where Mt refers to the-number of type k targets killed in time

iTRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

PubMed

Geng, Xiaofang; Wang, Gaiping; Qin, Yanli; Zang, Xiayan; Li, Pengfei; Geng, Zhi; Xue, Deming; Dong, Zimei; Ma, Kexue; Chen, Guangwen; Xu, Cunshuan

2015-01-01

The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR) remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

Stress History Simulation. Volume 2. A User’s Manual for a Computer Program to Modify Stress History Simulations

DTIC Science & Technology

1977-03-01

4444444.000000I00071 7 Z 7 7 > > > > 𔄁 > > 27 Z 2 *.JUJUJ30T. 4444.4 .4.4 .4 .4 . . . . .4 . . Utiulti iLK) LMUJL) it)LJit)Li it)Lit)LULNJt)Lit .31)0 MiLK ...M4-cUro r4-4-t m4~qoIN 0--r..44-m.O40iinn’O4-in.0C’JNO.4tDMCU~r-- (uor-r-04-* V44 1 \\J.-4’ 1 C’) -PM.-4. .- 4 11-4 W4 I4 .4-* fl44C .tl. I4 MUCU 3 I

Command History. 1966. Sanitized

DTIC Science & Technology

1966-01-01

ticaps th vftit~d at I ilk afil th 3vt. :; La Mh I . I .0.. 11t% 0 Ř. ’t1m L~-twi Order .. I A ’Ali~ COMMAND HISTORY Appowed tmot uiyiec reas hltl...8217 " A A.: S’.•, 1•• choosing. The buildup in the nunber of battalions, and particularly the infiltration of la -ger NVA units, would be done covert2...autd attachen inoreasod, both la Quang Tin and. Quang Nga± Provinces., with the. latter reasiving the greater number. Throughout the MyZ the VC retained

Reviews Equipment: Chameleon Nano Flakes Book: Requiem for a Species Equipment: Laser Sound System Equipment: EasySense VISION Equipment: UV Flash Kit Book: The Demon-Haunted World Book: Nonsense on Stilts Book: How to Think about Weird Things Web Watch

NASA Astrophysics Data System (ADS)

2011-03-01

WE RECOMMEND Requiem for a Species This book delivers a sober message about climate change Laser Sound System Sound kit is useful for laser demonstrations EasySense VISION Data Harvest produces another easy-to-use data logger UV Flash Kit Useful equipment captures shadows on film The Demon-Haunted World World-famous astronomer attacks pseudoscience in this book Nonsense on Stilts A thought-provoking analysis of hard and soft sciences How to Think about Weird Things This book explores the credibility of astrologers and their ilk WORTH A LOOK Chameleon Nano Flakes Product lacks good instructions and guidelines WEB WATCH Amateur scientists help out researchers with a variety of online projects

Burning Velocities of a Solid Propellant, via a Microwave Technique, at Elevated Pressures and Pressurization Rates.

DTIC Science & Technology

1979-10-01

b 0 FRQUNC (I/SE Fiue4.Acmltv Eeg pcrmfrte noddMaue hs Anl n h ’ Sed-taeTs 109 M 0Jc M a-cv- 0E 0: -3.- C3 c o:-- I- 0 - . 0 40 0 BO.0 200 000 200 24...aeo irto et1V tm 141 0 Ci. C,’ 0 I- O- r 0.00 3.00 6 .00 9.00 lk.Q0 15S.00 ih-oo 2,1.01 2 4.00 TIMlE ( MSEC) Figure 80. Change in Relative Amplitude...equation (E-3) gives the desired result: A iiA(E-6) A- Cos (Om a) -Asin tm ac in C ilk 210 APPENDIX F EQUIPMENT LIST 1. 211 EQUIPMENT LIST In the

HIF-2α-ILK Is Involved in Mesenchymal Stromal Cell Angiogenesis in Multiple Myeloma Under Hypoxic Conditions

PubMed Central

Zhang, Xiaoying; Xu, Yinhui; Liu, Hongbo; Zhao, Pan; Chen, Yafang; Yue, Zhijie; Zhang, Zhiqing; Wang, Xiaofang

2018-01-01

Mesenchymal stromal cells are proven to be likely induce the angiogenic response in multiple myeloma and thus represent an enticing target for antiangiogenesis therapies for multiple myeloma. Substantial evidence indicates that angiogenesis in multiple myeloma is complex and involves direct production of angiogenic cytokines by abnormal plasma cells and these B-cell neoplasia generated pathophysiology change within the microenvironment. In this study, we demonstrated that mesenchymal stromal cells cultured with U266/Lp-1 under hypoxic conditions resulted in an increased α-smooth muscle actin expression and high productive levels of both hypoxia-inducible factor-2α and integrin-linked kinase proteins. Moreover, inhibition of hypoxia-inducible factor-2α by Small interfering RNA (siRNA) in mesenchymal stromal cells decreased the protein levels of both α-smooth muscle actin and integrin-linked kinase after mesenchymal stromal cells cultured with U266 under hypoxic conditions. We further demonstrated that transfection of integrin-linked kinase-siRNA reduced the protein level of α-smooth muscle actin and attenuated angiogenesis in vitro by decreasing the attachment of Q-dot labeled cells and secretion of angiogenic factors. In conclusion, our research showed that mesenchymal stromal cells cultured with myeloma cells under hypoxia participated in the angiogenesis of multiple myeloma, which is regulated by the hypoxia-inducible factor-2α-integrin-linked kinase pathway. Thus, targeting integrin-linked kinase may represent an effective strategy to block hypoxia-inducible factor-2α-induced angiogenesis in the treatment of multiple myeloma. PMID:29656700

24-Methylenecycloartanyl ferulate, a major compound of γ-oryzanol, promotes parvin-beta expression through an interaction with peroxisome proliferator-activated receptor-gamma 2 in human breast cancer cells.

PubMed

Kim, Heon Woong; Lim, Eun Joung; Jang, Hwan Hee; Cui, XueLei; Kang, Da Rae; Lee, Sung Hyen; Kim, Haeng Ran; Choe, Jeong Sook; Yang, Young Mok; Kim, Jung Bong; Park, Jong Hwan

2015-12-25

Parvin-β is an adaptor protein that binds to integrin-linked kinase (ILK) and is significantly downregulated in breast tumors and breast cancer cell lines. We treated the breast cancer cell line MCF7 with 24-methylenecycloartanyl ferulate (24-MCF), a γ-oryzanol compound. We observed upregulation of parvin-β (GenBank Accession No. AF237769) and peroxisome proliferator-activated receptor (PPAR)-γ2 (GenBank Accession No. NM_015869). Among γ-oryzanol compounds, only treatment with 24-MCF led to the formation of reverse transcription-PCR products of parvin-β (650 and 500 bp) and PPAR-γ2 (580 bp) in MCF7 cells, but not in T47D, SK-BR-3, or MDA-MB-231 cells. 24-MCF treatment increased the mRNA and protein levels of parvin-β in MCF7 cells in a dose-dependent manner. We hypothesized that there is a correlation between parvin-β expression and induction of PPAR-γ2. This hypothesis was investigated by using a promoter-reporter assay, chromatin immunoprecipitation, and an electrophoretic mobility shift assay. 24-MCF treatment induced binding of PPAR-γ2 to a peroxisome proliferator response element-like cis-element (ACTAGGACAAAGGACA) in the parvin-β promoter in MCF7 cells in a dose-dependent manner. 24-MCF treatment significantly decreased anchorage-independent growth and inhibited cell movement in comparison to control treatment with dimethyl sulfoxide. 24-MCF treatment reduced the levels of GTP-bound Rac1 and Cdc42. Evaluation of Akt1 inhibition by 24-MCF revealed that the half maximal effective concentration was 33.3 μM. Docking evaluations revealed that 24-MCF binds to the ATP-binding site of Akt1(PDB ID: 3OCB) and the compound binding energy is -8.870 kcal/mol. Taken together, our results indicate that 24-MCF treatment increases parvin-β expression, which may inhibit ILK downstream signaling. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

An analysis of gene expression data involving examination of signaling pathways activation reveals new insights into the mechanism of action of minoxidil topical foam in men with androgenetic alopecia

PubMed Central

Wu, Jeff; Pappas, Apostolos; Mirmirani, Paradi; McCormick, Thomas S.; Cooper, Kevin D.; Schastnaya, Jane; Ozerov, Ivan V.; Aliper, Alexander; Zhavoronkov, Alex

2017-01-01

ABSTRACT Androgenetic alopecia is the most common form of hair loss. Minoxidil has been approved for the treatment of hair loss, however its mechanism of action is still not fully clarified. In this study, we aimed to elucidate the effects of 5% minoxidil topical foam on gene expression and activation of signaling pathways in vertex and frontal scalp of men with androgenetic alopecia. We identified regional variations in gene expression and perturbed signaling pathways using in silico Pathway Activation Network Decomposition Analysis (iPANDA) before and after treatment with minoxidil. Vertex and frontal scalp of patients showed a generally similar response to minoxidil. Both scalp regions showed upregulation of genes that encode keratin associated proteins and downregulation of ILK, Akt, and MAPK signaling pathways after minoxidil treatment. Our results provide new insights into the mechanism of action of minoxidil topical foam in men with androgenetic alopecia. PMID:28594262

An analysis of gene expression data involving examination of signaling pathways activation reveals new insights into the mechanism of action of minoxidil topical foam in men with androgenetic alopecia.

PubMed

Stamatas, Georgios N; Wu, Jeff; Pappas, Apostolos; Mirmirani, Paradi; McCormick, Thomas S; Cooper, Kevin D; Consolo, Mary; Schastnaya, Jane; Ozerov, Ivan V; Aliper, Alexander; Zhavoronkov, Alex

2017-01-01

Androgenetic alopecia is the most common form of hair loss. Minoxidil has been approved for the treatment of hair loss, however its mechanism of action is still not fully clarified. In this study, we aimed to elucidate the effects of 5% minoxidil topical foam on gene expression and activation of signaling pathways in vertex and frontal scalp of men with androgenetic alopecia. We identified regional variations in gene expression and perturbed signaling pathways using in silico Pathway Activation Network Decomposition Analysis (iPANDA) before and after treatment with minoxidil. Vertex and frontal scalp of patients showed a generally similar response to minoxidil. Both scalp regions showed upregulation of genes that encode keratin associated proteins and downregulation of ILK, Akt, and MAPK signaling pathways after minoxidil treatment. Our results provide new insights into the mechanism of action of minoxidil topical foam in men with androgenetic alopecia.

Chemical Composition and antiproliferative activity of essential oil from the leaves of a medicinal herb, Levisticum officinale, against UMSCC1 head and neck squamous carcinoma cells.

PubMed

Sertel, Serkan; Eichhorn, Tolga; Plinkert, Peter K; Efferth, Thomas

2011-01-01

Oral squamous cell carcinoma (OSCC) is a challenging disease with a high mortality rate. Natural products represent a valuable source for the development of novel anticancer drugs. We investigated the cytotoxic potential of essential oil from the leaves of a medicinal plant, Levisticum officinale (lovage) on head and neck squamous carcinoma cells (HNSCC). Cytotoxicity of lovage essential oil was investigated on the HNSCC cell line, UMSCC1. Additionally, we performed pharmacogenomics analyses. Lovage essential oil extract had an IC₅₀ value of 292.6 μg/ml. Genes involved in apoptosis, cancer, cellular growth and cell cycle regulation were the most prominently affected in microarray analyses. The three pathways to be most significantly regulated were extracellular signal-regulated kinase 5 (ERK5) signaling, integrin-linked kinase (ILK) signaling, virus entry via endocytic pathways and p53 signaling. Levisticum officinale essential oil inhibits human HNSCC cell growth.

24-Methylenecycloartanyl ferulate, a major compound of γ-oryzanol, promotes parvin-beta expression through an interaction with peroxisome proliferator-activated receptor-gamma 2 in human breast cancer cells

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

Kim, Heon Woong; Lim, Eun Joung; Jang, Hwan Hee

2015-12-25

Parvin-β is an adaptor protein that binds to integrin-linked kinase (ILK) and is significantly downregulated in breast tumors and breast cancer cell lines. We treated the breast cancer cell line MCF7 with 24-methylenecycloartanyl ferulate (24-MCF), a γ-oryzanol compound. We observed upregulation of parvin-β (GenBank Accession No. (AF237769)) and peroxisome proliferator-activated receptor (PPAR)-γ2 (GenBank Accession No. (NM-015869)). Among γ-oryzanol compounds, only treatment with 24-MCF led to the formation of reverse transcription-PCR products of parvin-β (650 and 500 bp) and PPAR-γ2 (580 bp) in MCF7 cells, but not in T47D, SK-BR-3, or MDA-MB-231 cells. 24-MCF treatment increased the mRNA and protein levels of parvin-β inmore » MCF7 cells in a dose-dependent manner. We hypothesized that there is a correlation between parvin-β expression and induction of PPAR-γ2. This hypothesis was investigated by using a promoter-reporter assay, chromatin immunoprecipitation, and an electrophoretic mobility shift assay. 24-MCF treatment induced binding of PPAR-γ2 to a peroxisome proliferator response element-like cis-element (ACTAGGACAAAGGACA) in the parvin-β promoter in MCF7 cells in a dose-dependent manner. 24-MCF treatment significantly decreased anchorage-independent growth and inhibited cell movement in comparison to control treatment with dimethyl sulfoxide. 24-MCF treatment reduced the levels of GTP-bound Rac1 and Cdc42. Evaluation of Akt1 inhibition by 24-MCF revealed that the half maximal effective concentration was 33.3 μM. Docking evaluations revealed that 24-MCF binds to the ATP-binding site of Akt1(PDB ID: (3OCB)) and the compound binding energy is -8.870 kcal/mol. Taken together, our results indicate that 24-MCF treatment increases parvin-β expression, which may inhibit ILK downstream signaling. - Highlights: • Treatment with 24-MCF increases gene expression of parvin-β and PPAR-ϒ2 in MCF7 cells. • PPAR-ϒ2 interacts with the parvin

Investigation of Primary School Teachers' Attitudes towards Gifted Education=Ilkögretim Ögretmenlerinin Üstün Yeteneklilerin Egitimine Iliskin Tutumlarinin Incelenmesi

ERIC Educational Resources Information Center

Tortop, Hasan Said; Kunt, Kürsat

2013-01-01

Gifted education is very important issue for the development of societies. This importance has been increased with opening of the Center for Science and Art in Turkey. These institutions provide with formal educational institutions coordination for development of gifted students. In this respect, primary school teachers' attitude towards gifted…

The raf-like kinase ILK1 and the high affinity K+ transporter HAK5 are required for innate immunity and abiotic stress response

USDA-ARS?s Scientific Manuscript database

Plants combat bacterial infection by detecting conserved molecular signatures called pathogen-associated molecular patterns (PAMPs) and producing defensive compounds to restrict pathogen entry and reproduction. Numerous ion fluxes are activated within minutes of PAMP perception, including Ca2+ influ...

A plea for virtue in ethics.

PubMed

Sisti, Dominic A; Baum-Baicker, Cynthia

2012-01-01

Comments on the original article, "Nonrational processes in ethical decision making" by M. D. Rogerson et al (see record 2011-19198-001). The current authors suggest that Rogerson, Gottlieb, Handelsman, Knapp, and Younggren (October 2011) presumed that the only ethical theories available for grounding decision-making models are of the rational, neoliberal variety. Rogerson et al stated, "Contextual, interpersonal, and intuitive factors are inextricably linked and inexorably influential in the process of ethical decision making. Ethical theory would benefit from encompassing these subtle yet powerful forces" (Rogerson et al., 2011, p. 616). They sought to augment these models with a cluster of contextual considerations, appending to them accounts of emotion, context, and intuition. First, notwithstanding the theories attributed to (the caricature of) Kant and his ilk, there are several ethical theories that include an account of what Rogerson et al. (2011) consider to be "nonrational" processes. From feminist theories to narrative ethics, sophisticated contextual theories have been developed and are readily available. Second, we question whether thick contextual considerations can simply be tacked on to extant models of decision making originally built upon a philosophical foundation that assumes a rational, autonomous agent who deliberates independently and logically.

Aloe-emodin inhibits HER-2 expression through the downregulation of Y-box binding protein-1 in HER-2-overexpressing human breast cancer cells.

PubMed

Ma, Jui-Wen; Hung, Chao-Ming; Lin, Ying-Chao; Ho, Chi-Tang; Kao, Jung-Yie; Way, Tzong-Der

2016-09-13

Human epidermal growth factor receptor-2 (HER-2)-positive breast cancer tends to be aggressive, highly metastatic, and drug resistant and spreads rapidly. Studies have indicated that emodin inhibits HER-2 expression. This study compared the HER-2-inhibitory effects of two compounds extracted from rhubarb roots: aloe-emodin (AE) and rhein. Our results indicated that AE exerted the most potent inhibitory effect on HER-2 expression. Treatment of HER-2-overexpressing breast cancer cells with AE reduced tumor initiation, cell migration, and cell invasion. AE was able to suppress YB-1 expression, further suppressing downstream HER-2 expression. AE suppressed YB-1 expression through the inhibition of Twist in HER-2-overexpressing breast cancer cells. Our data also found that AE inhibited cancer metastasis and cancer stem cells through the inhibition of EMT. Interestingly, AE suppressed YB-1 expression through the downregulation of the intracellular integrin-linked kinase (ILK)/protein kinase B (Akt)/mTOR signaling pathway in HER-2-overexpressing breast cancer cells. In vivo study showed the positive result of antitumor activity of AE in nude mice injected with human HER-2-overexpressing breast cancer cells. These findings suggest the possible application of AE in the treatment of HER-2-positive breast cancer.

Assessment of the cerebellar neurotoxic effects of nicotine in prenatal alcohol exposure in rats.

PubMed

Bhattacharya, Dwipayan; Majrashi, Mohammed; Ramesh, Sindhu; Govindarajulu, Manoj; Bloemer, Jenna; Fujihashi, Ayaka; Crump, Bailee-Ryan; Hightower, Harrison; Bhattacharya, Subhrajit; Moore, Timothy; Suppiramaniam, Vishnu; Dhanasekaran, Muralikrishnan

2018-02-01

The adverse effects of prenatal nicotine and alcohol exposure on human reproductive outcomes are a major scientific and public health concern. In the United States, substantial percentage of women (20-25%) of childbearing age currently smoke cigarettes and consume alcohol, and only a small percentage of these individuals quit after learning of their pregnancy. However, there are very few scientific reports on the effect of nicotine in prenatal alcohol exposure on the cerebellum of the offspring. Therefore, this study was conducted to investigate the cerebellar neurotoxic effects of nicotine in a rodent model of Fetal Alcohol Spectrum Disorder (FASD). In this study, we evaluated the behavioral changes, biochemical markers of oxidative stress and apoptosis, mitochondrial functions and the molecular mechanisms associated with nicotine in prenatal alcohol exposure on the cerebellum. Prenatal nicotine and alcohol exposure induced oxidative stress, did not affect the mitochondrial functions, increased the monoamine oxidase activity, increased caspase expression and decreased ILK, PSD-95 and GLUR1 expression without affecting the GSK-3β. Thus, our current study of prenatal alcohol and nicotine exposure on cerebellar neurotoxicity may lead to new scientific perceptions and novel and suitable therapeutic actions in the future. Copyright © 2017. Published by Elsevier Inc.

Genistein Promotes Endothelial Colony-Forming Cell (ECFC) Bioactivities and Cardiac Regeneration in Myocardial Infarction

PubMed Central

Lee, Sang Hun; Lee, Jun Hee; Asahara, Takayuki; Kim, Yong Sook; Jeong, Hae Chang; Ahn, Youngkeun; Jung, Jin Sup; Kwon, Sang-Mo

2014-01-01

Although stem cell-mediated treatment of ischemic diseases offers significant therapeutic promise, the limitation in the therapeutic efficacy of transplanted stem cells in vivo because of poor engraftment remains a challenge. Several strategies aimed at improving survival and engraftment of stem cells in the ischemic myocardium have been developed, such as cell transplantation in combination with growth factor delivery, genetic modification of stem cells, and/or cell therapy using scaffolds. To improve therapeutic efficacy, we investigated the effects of genistein on the engraftment of transplanted ECFCs in an acute myocardial ischemia model. Results: We found that genistein treatment enhanced ECFCs' migration and proliferation, which was accompanied by increases in the expression of ILK, α-parvin, F-actin, and phospholylation of ERK 1/2 signaling. Transplantation of genistein-stimulates ECFCs (GS-ECFCs) into myocardial ischemic sites in vivo induced cellular proliferation and secretion of angiogenic cytokines at the ischemic sites and thereby enhanced neovascularization and decreased myocardial fibrosis as well as improved cardiac function, as shown by echocardiography. Taken together, these data suggest that pretreatment of ECFCs with genistein prior to transplantation can improve the regenerative potential in ischemic tissues, providing a novel strategy in adult stem cell therapy for ischemic diseases. PMID:24830850

Assessing quality of life: mother-child agreement in depressed and non-depressed Hungarian.

PubMed

Kiss, Eniko; Kapornai, Krisztina; Baji, Ildikó; Mayer, László; Vetró, Agnes

2009-05-01

An important question in child psychiatry is the agreement between parents and children. We studied mother-child concordance about the quality of life of children (QoL). We hypothesized that mothers of depressed children rate lower QoL than children for themselves while mothers of non-depressed children rate better QoL; that inter-informant agreement is higher in the non-depressed sample; and finally that agreement increases with age of the child. QoL of depressed children (N = 248, mean age 11.45 years, SD 2.02) were compared to that of non-depressed children (N = 1695, mean age 10.34 years, SD 2.19). QoL was examined by a 7 item questionnaire (ILK). Mothers of depressed children rated lower QoL than their children while mothers of nondepressed children rated higher QoL than their children. Agreement was low in both samples but higher in the controls. Inter-informant agreement was only influenced by depression. Our results show that mothers relate more serious negative effects to childhood depression than their children and rate less problems for their non-depressed children compared to self-reports. Mother-child agreement is negatively influenced by depression which further stresses the importance of obtaining reports from the child and at least one parent in order to understand the subjective experiences caused by the illness.

Gestational Diabetes Alters Offspring DNA Methylation Profiles in Human and Rat: Identification of Key Pathways Involved in Endocrine System Disorders, Insulin Signaling, Diabetes Signaling, and ILK Signaling.

PubMed

Petropoulos, Sophie; Guillemin, Claire; Ergaz, Zivanit; Dimov, Sergiy; Suderman, Matthew; Weinstein-Fudim, Liza; Ornoy, Asher; Szyf, Moshe

2015-06-01

Gestational diabetes is associated with risk for metabolic disease later in life. Using a cross-species approach in rat and humans, we examined the hypothesis that gestational diabetes during pregnancy triggers changes in the methylome of the offspring that might be mediating these risks. We show in a gestation diabetes rat model, the Cohen diabetic rat, that gestational diabetes triggers wide alterations in DNA methylation in the placenta in both candidate diabetes genes and genome-wide promoters, thus providing evidence for a causal relationship between diabetes during pregnancy and DNA methylation alterations. There is a significant overlap between differentially methylated genes in the placenta and the liver of the rat offspring. Several genes differentially methylated in rat placenta exposed to maternal diabetes are also differentially methylated in the human placenta of offspring exposed to gestational diabetes in utero. DNA methylation changes inversely correlate with changes in expression. The changes in DNA methylation affect known functional gene pathways involved in endocrine function, metabolism, and insulin responses. These data provide support to the hypothesis that early-life exposures and their effects on metabolic disease are mediated by DNA methylation changes. This has important diagnostic and therapeutic implications.

[Prediction of School Dropout among Turkish High School Students: A Model Testing with Moderator and Mediator Variables.

PubMed

Ozer, Arif; Gençtanirim, Dilek; Ergene, Tuncay

2011-01-01

Bu araştırmada ilk olarak, dürtüsel davranma ile okulu terk etme riski arasındaki ilişkiye disiplin cezası almanın, antisosyal davranışların ve sigara-alkol kullanımının aracılık edip etmediği incelenmiştir. İkinci olarak, öğretmen desteği ve antisosyal davranış etkileşiminin okulu terk etme riski üzerindeki etkisi test edilmiştir. Araştırma grubunu 2009-2010 yılında Ankara İlinde genel liselere devam eden 478 öğrenci oluşturmuştur. Sonuçlar okulu terk etme riskini aile ve arkadaş desteğinin azalttığını, dürtüsel davranmanın ise artırdığını göstermiştir. Ayrıca disiplin cezası, alkol-sigara kullanma ve antisosyal davranışlar okulu terk etme riskini artıran aracı değişkenlerdir. Antisosyal davranışlarla okulu terk etme arasındaki ilişki öğretmen desteğine bağlı olarak değişmektedir. Öğrencilerin cinsiyet ve başarıları ile okulu terk etme riskleri arasında anlamlı bir ilişki bulunmamaktadır.

Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors.

PubMed

Sander, Suzanne; Arora, Neha; Smith, Emily A

2012-06-01

Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10(-9) cm(2)/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein's expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand-integrin-cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties.

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome.

PubMed

Alachkar, Houda; Santhanam, Ramasamy; Maharry, Kati; Metzeler, Klaus H; Huang, Xiaomeng; Kohlschmidt, Jessica; Mendler, Jason H; Benito, Juliana M; Hickey, Christopher; Neviani, Paolo; Dorrance, Adrienne M; Anghelina, Mirela; Khalife, Jihane; Tarighat, Somayeh S; Volinia, Stefano; Whitman, Susan P; Paschka, Peter; Hoellerbauer, Pia; Wu, Yue-Zhong; Han, Lina; Bolon, Brad N; Blum, William; Mrózek, Krzysztof; Carroll, Andrew J; Perrotti, Danilo; Andreeff, Michael; Caligiuri, Michael A; Konopleva, Marina; Garzon, Ramiro; Bloomfield, Clara D; Marcucci, Guido

2014-04-01

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

Natural and molecular history of prolactinoma: insights from a Prlr-/- mouse model.

PubMed

Bernard, Valérie; Villa, Chiara; Auguste, Aurélie; Lamothe, Sophie; Guillou, Anne; Martin, Agnès; Caburet, Sandrine; Young, Jacques; Veitia, Reiner A; Binart, Nadine

2018-01-19

Lactotroph adenoma, also called prolactinoma, is the most common pituitary tumor but little is known about its pathogenesis. Mouse models of prolactinoma can be useful to better understand molecular mechanisms involved in abnormal lactotroph cell proliferation and secretion. We have previously developed a prolactin receptor deficient ( Prlr -/- ) mouse, which develops prolactinoma. The present study aims to explore the natural history of prolactinoma formation in Prlr -/- mice, using hormonal, radiological, histological and molecular analyses to uncover mechanisms involved in lactotroph adenoma development. Prlr -/- females develop large secreting prolactinomas from 12 months of age, with a penetrance of 100%, mimicking human aggressive densely granulated macroprolactinoma, which is a highly secreting subtype. Mean blood PRL measurements reach 14 902 ng/mL at 24 months in Prlr -/- females while PRL levels were below 15 ng/mL in control mice ( p < 0.01). By comparing pituitary microarray data of Prlr -/- mice and an estrogen-induced prolactinoma model in ACI rats, we pinpointed 218 concordantly differentially expressed (DE) genes involved in cell cycle, mitosis, cell adhesion molecules, dopaminergic synapse and estrogen signaling. Pathway/gene-set enrichment analyses suggest that the transcriptomic dysregulation in both models of prolactinoma might be mediated by a limited set of transcription factors (i.e., STAT5, STAT3, AhR, ESR1, BRD4, CEBPD, YAP, FOXO1) and kinases (i.e., JAK2, AKT1, BRAF, BMPR1A, CDK8, HUNK, ALK, FGFR1, ILK). Our experimental results and their bioinformatic analysis provide insights into early genomic changes in murine models of the most frequent human pituitary tumor.

Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells.

PubMed

Shkumatov, Artem; Thompson, Michael; Choi, Kyoung M; Sicard, Delphine; Baek, Kwanghyun; Kim, Dong Hyun; Tschumperlin, Daniel J; Prakash, Y S; Kong, Hyunjoon

2015-06-01

Multiple pulmonary conditions are characterized by an abnormal misbalance between various tissue components, for example, an increase in the fibrous connective tissue and loss/increase in extracellular matrix proteins (ECM). Such tissue remodeling may adversely impact physiological function of airway smooth muscle cells (ASMCs) responsible for contraction of airways and release of a variety of bioactive molecules. However, few efforts have been made to understand the potentially significant impact of tissue remodeling on ASMCs. Therefore, this study reports how ASMCs respond to a change in mechanical stiffness of a matrix, to which ASMCs adhere because mechanical stiffness of the remodeled airways is often different from the physiological stiffness. Accordingly, using atomic force microscopy (AFM) measurements, we found that the elastic modulus of the mouse bronchus has an arithmetic mean of 23.1 ± 14 kPa (SD) (median 18.6 kPa). By culturing ASMCs on collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we found that gels designed to be softer than average airway tissue significantly increased cellular secretion of vascular endothelial growth factor (VEGF). Conversely, gels stiffer than average airways stimulated cell proliferation, while reducing VEGF secretion and agonist-induced calcium responses of ASMCs. These dependencies of cellular activities on elastic modulus of the gel were correlated with changes in the expression of integrin-β1 and integrin-linked kinase (ILK). Overall, the results of this study demonstrate that changes in matrix mechanics alter cell proliferation, calcium signaling, and proangiogenic functions in ASMCs. Copyright © 2015 the American Physiological Society.

Türk Lise Öğrencilerinde Okul Terkinin Yordanması: Aracı ve Etkileşim Değişkenleri ile Bir Model Testi

PubMed Central

Özer, Arif; Gençtanirim, Dilek; Ergene, Tuncay

2011-01-01

Bu araştırmada ilk olarak, dürtüsel davranma ile okulu terk etme riski arasındaki ilişkiye disiplin cezası almanın, antisosyal davranışların ve sigara-alkol kullanımının aracılık edip etmediği incelenmiştir. İkinci olarak, öğretmen desteği ve antisosyal davranış etkileşiminin okulu terk etme riski üzerindeki etkisi test edilmiştir. Araştırma grubunu 2009-2010 yılında Ankara İlinde genel liselere devam eden 478 öğrenci oluşturmuştur. Sonuçlar okulu terk etme riskini aile ve arkadaş desteğinin azalttığını, dürtüsel davranmanın ise artırdığını göstermiştir. Ayrıca disiplin cezası, alkol-sigara kullanma ve antisosyal davranışlar okulu terk etme riskini artıran aracı değişkenlerdir. Antisosyal davranışlarla okulu terk etme arasındaki ilişki öğretmen desteğine bağlı olarak değişmektedir. Öğrencilerin cinsiyet ve başarıları ile okulu terk etme riskleri arasında anlamlı bir ilişki bulunmamaktadır. PMID:22003257

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome

PubMed Central

Alachkar, Houda; Santhanam, Ramasamy; Maharry, Kati; Metzeler, Klaus H.; Huang, Xiaomeng; Kohlschmidt, Jessica; Mendler, Jason H.; Benito, Juliana M.; Hickey, Christopher; Neviani, Paolo; Dorrance, Adrienne M.; Anghelina, Mirela; Khalife, Jihane; Tarighat, Somayeh S.; Volinia, Stefano; Whitman, Susan P.; Paschka, Peter; Hoellerbauer, Pia; Wu, Yue-Zhong; Han, Lina; Bolon, Brad N.; Blum, William; Mrózek, Krzysztof; Carroll, Andrew J.; Perrotti, Danilo; Andreeff, Michael; Caligiuri, Michael A.; Konopleva, Marina; Garzon, Ramiro; Bloomfield, Clara D.; Marcucci, Guido

2014-01-01

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML. PMID:24590286

Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors.

PubMed

Chen, Xiaofei; Liu, Liang; Mims, Jade; Punska, Elizabeth C; Williams, Kristin E; Zhao, Weiling; Arcaro, Kathleen F; Tsang, Allen W; Zhou, Xiaobo; Furdui, Cristina M

2015-01-01

Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.

Distinctive gene expression profiles characterize donor biopsies from HCV-positive kidney donors.

PubMed

Mas, Valeria R; Archer, Kellie J; Suh, Lacey; Scian, Mariano; Posner, Marc P; Maluf, Daniel G

2010-12-15

Because of the shortage of organs for transplantation, procurement of kidneys from extended criteria donors is inevitable. Frequently, donors infected with hepatitis C virus (HCV) are used. To elucidate an initial compromise of molecular pathways in HCV graft, gene expression profiles were evaluated. Twenty-four donor allograft biopsies (n=12 HCV positive (+) and n=12 HCV negative (-)) were collected at preimplantation time and profiled using microarrays. Donors were age, race, gender, and cold and warm ischemia time matched between groups. Probe level data were read into the R programming environment using the affy Bioconductor package, and the robust multiarray average method was used to obtain probe set expression summaries. To identify probe sets exhibiting differential expression, a two sample t test was performed. Molecular and biologic functions were analyzed using Interaction Networks and Functional Analysis. Fifty-eight probe sets were differentially expressed between HCV (+) versus HCV (-) donors (P<0.001). The molecular functions associated with the two top scored networks from the analysis of the differentially expressed genes were connective tissue development and function and tissue morphology (score 34), cell death, cell signaling, cellular assembly, and organization (score 32). Among the differentially affected top canonical pathways, we found the role of RIG1-like receptors in antiviral innate immunity (P<0.001), natural killer cell signaling (P=0.007), interleukin-8 signaling (P=0.048), interferon signaling (P=0.0 11; INFA21, INFGR1, and MED14), ILK signaling (P=0.001), and apoptosis signaling. A unique gene expression pattern was identified in HCV (+) kidney grafts. Innate immune system and inflammatory pathways were the most affected.

Breast Cancer Clinical Trial of Chemotherapy and Trastuzumab: Potential Tool to Identify Cardiac Modifying Variants of Dilated Cardiomyopathy

PubMed Central

Serie, Daniel J.; Crook, Julia E.; Necela, Brian M.; Axenfeld, Bianca C.; Dockter, Travis J.; Colon-Otero, Gerardo; Perez, Edith A.; Thompson, E. Aubrey; Norton, Nadine

2017-01-01

Doxorubicin and the ERBB2 targeted therapy, trastuzumab, are routinely used in the treatment of HER2+ breast cancer. In mouse models, doxorubicin is known to cause cardiomyopathy and conditional cardiac knock out of Erbb2 results in dilated cardiomyopathy and increased sensitivity to doxorubicin-induced cell death. In humans, these drugs also result in cardiac phenotypes, but severity and reversibility is highly variable. We examined the association of decline in left ventricular ejection fraction (LVEF) at 15,204 single nucleotide polymorphisms (SNPs) spanning 72 cardiomyopathy genes, in 800 breast cancer patients who received doxorubicin and trastuzumab. For 7033 common SNPs (minor allele frequency (MAF) > 0.01) we performed single marker linear regression. For all SNPs, we performed gene-based testing with SNP-set (Sequence) Kernel Association Tests: SKAT, SKAT-O and SKAT-common/rare under rare variant non-burden; rare variant optimized burden and non-burden tests; and a combination of rare and common variants respectively. Single marker analyses identified seven missense variants in OBSCN (p = 0.0045–0.0009, MAF = 0.18–0.50) and two in TTN (both p = 0.04, MAF = 0.22). Gene-based rare variant analyses, SKAT and SKAT-O, performed very similarly (ILK, TCAP, DSC2, VCL, FXN, DSP and KCNQ1, p = 0.042–0.006). Gene-based tests of rare/common variants were significant at the nominal 5% level for OBSCN as well as TCAP, DSC2, VCL, NEXN, KCNJ2 and DMD (p = 0.044–0.008). Our results suggest that rare and common variants in OBSCN, as well as in other genes, could have modifying effects in cardiomyopathy. PMID:29367538

Exosomes Secreted under Hypoxia Enhance Invasiveness and Stemness of Prostate Cancer Cells by Targeting Adherens Junction Molecules

PubMed Central

Ramteke, Anand; Ting, Harold; Agarwal, Chapla; Mateen, Samiha; Somasagara, Ranganathan; Hussain, Anowar; Graner, Michael; Frederick, Barbara; Agarwal, Rajesh; Deep, Gagan

2015-01-01

Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2) or normoxic (21% O2) conditions, and exosomes secreted under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that ExoHypoxic have smaller average size as compared to ExoNormoxic. Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70) and Annexin II in ExoHypoxic compared to ExoNormoxic. Co-culturing with ExoHypoxic increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. ExoHypoxic also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to ExoNormoxic, ExoHypoxic showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in ExoHypoxic (160 proteins) compared to ExoNormoxic (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, ExoHypoxic targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that ExoHypoxic are loaded with unique proteins that could enhance invasiveness, stemness and induce microenvironment changes; thereby, promoting PCA aggressiveness. PMID:24347249

Tectonic Interpretation of CHAMP Geopotential Data over the Northern Adriatic Sea.

NASA Astrophysics Data System (ADS)

Taylor, P. T.; Kim, H. R.; Mayer-Gürr, T.

2006-05-01

Recent aeromagnetic anomaly compilations (Chiappini et al., 2000 and Tontini et al., 2004) show a large positive (>700 nT) northwest-southeast trending magnetic anomaly off the Dalmatian coast. Unfortunately these aeromagnetic data cover only a part of this anomaly. We wanted to investigate if this large magnetic anomaly could be detected at satellite altitude and what is the extent and source of this feature. Therefore, magnetic and gravity anomaly maps were made from the CHAMP geopotential data, measured at the current low altitude of 345-350 km over the northern Adriatic Sea. We made the magnetic anomaly map over this relatively small region using 36 descending and 85 ascending orbits screened to be at the lowest altitude and the most magnetically quietest data. We removed the main field component (i.e., IGRF-10 up to degree and order 13) and then demeaned individual tracks and subtracted a second order polynomial to remove regional and/or un-modeled external field features. The resulting map from these well-correlated anomalies revealed a positive magnetic anomaly (>2 nT). Reduction-to-the pole brought these CHAMP anomaly features into coincidence with the aeromagnetic data. Previously Cantini et al. (1999) compared the surface magnetic data with MAGSAT by continuing upward the former and downwards the latter to 100 km and found a good correlation for wavelengths of 300-500 km. We also investigated the CHAMP gravity data. They were reduced using the kinematic short-arc integration method (Ilk et al., 2005 and Mayer Gürr et al., 2005). However, no corresponding short-wavelength gravity anomaly was observed in our study area. This tectonically complex region is under horizontal stress and the source of the large magnetic anomaly can be modelled by an associated ophiolite melange.

HANAC Syndrome Col4a1 Mutation Causes Neonate Glomerular Hyperpermeability and Adult Glomerulocystic Kidney Disease

PubMed Central

Chen, Zhiyong; Migeon, Tiffany; Verpont, Marie-Christine; Zaidan, Mohamad; Sado, Yoshikazu; Kerjaschki, Dontscho; Ronco, Pierre

2016-01-01

Hereditary angiopathy, nephropathy, aneurysms, and muscle cramps (HANAC) syndrome is an autosomal dominant syndrome caused by mutations in COL4A1 that encodes the α1 chain of collagen IV, a major component of basement membranes. Patients present with cerebral small vessel disease, retinal tortuosity, muscle cramps, and kidney disease consisting of multiple renal cysts, chronic kidney failure, and sometimes hematuria. Mutations producing HANAC syndrome localize within the integrin binding site containing CB3[IV] fragment of the COL4A1 protein. To investigate the pathophysiology of HANAC syndrome, we generated mice harboring the Col4a1 p.Gly498Val mutation identified in a family with the syndrome. Col4a1 G498V mutation resulted in delayed glomerulogenesis and podocyte differentiation without reduction of nephron number, causing albuminuria and hematuria in newborns. The glomerular defects resolved within the first month, but glomerular cysts developed in 3-month-old mutant mice. Abnormal structure of Bowman’s capsule was associated with metalloproteinase induction and activation of the glomerular parietal epithelial cells that abnormally expressed CD44, α-SMA, ILK, and DDR1. Inflammatory infiltrates were observed around glomeruli and arterioles. Homozygous Col4a1 G498V mutant mice additionally showed dysmorphic papillae and urinary concentration defects. These results reveal a developmental role for the α1α1α2 collagen IV molecule in the embryonic glomerular basement membrane, affecting podocyte differentiation. The observed association between molecular alteration of the collagenous network in Bowman’s capsule of the mature kidney and activation of parietal epithelial cells, matrix remodeling, and inflammation may account for glomerular cyst development and CKD in patients with COL4A1-related disorders. PMID:26260163

HANAC Syndrome Col4a1 Mutation Causes Neonate Glomerular Hyperpermeability and Adult Glomerulocystic Kidney Disease.

PubMed

Chen, Zhiyong; Migeon, Tiffany; Verpont, Marie-Christine; Zaidan, Mohamad; Sado, Yoshikazu; Kerjaschki, Dontscho; Ronco, Pierre; Plaisier, Emmanuelle

2016-04-01

Hereditary angiopathy, nephropathy, aneurysms, and muscle cramps (HANAC) syndrome is an autosomal dominant syndrome caused by mutations in COL4A1 that encodes the α1 chain of collagen IV, a major component of basement membranes. Patients present with cerebral small vessel disease, retinal tortuosity, muscle cramps, and kidney disease consisting of multiple renal cysts, chronic kidney failure, and sometimes hematuria. Mutations producing HANAC syndrome localize within the integrin binding site containing CB3[IV] fragment of the COL4A1 protein. To investigate the pathophysiology of HANAC syndrome, we generated mice harboring the Col4a1 p.Gly498Val mutation identified in a family with the syndrome. Col4a1 G498V mutation resulted in delayed glomerulogenesis and podocyte differentiation without reduction of nephron number, causing albuminuria and hematuria in newborns. The glomerular defects resolved within the first month, but glomerular cysts developed in 3-month-old mutant mice. Abnormal structure of Bowman's capsule was associated with metalloproteinase induction and activation of the glomerular parietal epithelial cells that abnormally expressed CD44,α-SMA, ILK, and DDR1. Inflammatory infiltrates were observed around glomeruli and arterioles. Homozygous Col4a1 G498V mutant mice additionally showed dysmorphic papillae and urinary concentration defects. These results reveal a developmental role for the α1α1α2 collagen IV molecule in the embryonic glomerular basement membrane, affecting podocyte differentiation. The observed association between molecular alteration of the collagenous network in Bowman's capsule of the mature kidney and activation of parietal epithelial cells, matrix remodeling, and inflammation may account for glomerular cyst development and CKD in patients with COL4A1-related disorders. Copyright © 2016 by the American Society of Nephrology.

Mesenchymal change and drug resistance in neuroblastoma.

PubMed

Naiditch, Jessica A; Jie, Chunfa; Lautz, Timothy B; Yu, Songtao; Clark, Sandra; Voronov, Dimitry; Chu, Fei; Madonna, Mary Beth

2015-01-01

Metastatic initiation has many phenotypic similarities to epithelial-to-mesenchymal transition, including loss of cell-cell adhesion, increased invasiveness, and increased cell mobility. We have previously demonstrated that drug resistance is associated with a metastatic phenotype in neuroblastoma (NB). The purpose of this project was to determine if the development of doxorubicin resistance is associated with characteristics of mesenchymal change in human NB cells. Total RNA was isolated from wild type (WT) and doxorubicin-resistant (DoxR) human NB cell lines (SK-N-SH and SK-N-BE(2)C) and analyzed using the Illumina Human HT-12 version 4 Expression BeadChip. Differentially expressed genes (DEGs) were identified. Volcano plots and heat maps were generated. Genes of interest with a fold change in expression >1.5 and an adjusted P < 0.1 were analyzed. Immunofluorescence (IF) and Western blot analysis confirmed microarray results of interest. Matrigel invasion assay and migration wounding assays were performed. Volcano plots and heat maps visually demonstrated a similar pattern of DEGs in the SK-N-SH and SK-N-BE(2)C DoxR cell lines relative to their parental WT lines. Venn diagramming revealed 1594 DEGs common to both DoxR cell lines relative to their parental cell lines. Network analysis pointed to several significantly upregulated epithelial-to-mesenchymal transition pathways, through TGF-beta pathways via RhoA, PI3K, and ILK and via SMADs, as well as via notch signaling pathways. DoxR cell lines displayed a more invasive phenotype than respective WT cell lines. Human SK-N-SH and SK-N-BE(2)C NB cells display characteristics of mesenchymal change via multiple pathways in the transition to a drug-resistant state. Copyright © 2015 Elsevier Inc. All rights reserved.

Expansion Formulation of General Relativity: the Gauge Functions for Energy-Momentum Tensor

NASA Astrophysics Data System (ADS)

Beloushko, Konstantin; Karbanovski, Valeri

At present the one of the GR (General Relativity) basic problem remains a definition of the gravitation field (GF) energy. We shall analyze this content. As well known, the energy-momentum ``tensor'' (EMT) of GF was introduced by Einstein [1] with purpose of the SRT (Special Relativity Theory) generalization. It supposed also, that EMT of matter satisfy to the condition begin{equation} ⪉bel{GrindEQ__1_1_} T^{ik} _{;i} =0 (a semicolon denotes a covariant differentiation with respect to coordinates). In absence of GF the equation (ref{GrindEQ__1_1_}) reduces to a corresponding SRT expression begin{equation} ⪉bel{GrindEQ__1_2_} T^{ik} _{,i} =0 (a comma denotes a differentiation with respect to coordinates of space-time). Obviously, the ``conservation law'' (ref{GrindEQ__1_2_}) is not broken by transformation begin{equation} ⪉bel{GrindEQ__1_3_} T^{ik} to tilde{T}^{ik} =T^{ik} +h^{ikl} _{,l} , where for h(ikl) takes place a constrain begin{equation} ⪉bel{GrindEQ__1_4_} h^{ikl} =-h^{ilk} Later the given property has been used for a construction ``pseudo-tensor'' tau (ik) of ``pure'' GF [2, S 96] begin{equation} ⪉bel{GrindEQ__1_5_} -gleft(frac{c^{4} }{8pi G} left(R^{ik} -frac{1}{2} g^{ik} Rright)+tau ^{ik} right)=h^{ikl} _{,l} However such definition was a consequence of non-covariant transition from a reference system with condition g(ik) _{,l} =0 to an arbitrary frame. Therefore the Landau-Lifshitz pseudo-tensor has no physical contents and considered problem remains actual. ``The non-covariant character'' of GF energy was the reason for criticism of GR as Einstein's contemporaries [3, 4], as and during the subsequent period (see, for example, [5]). In [6] were analyzed the grounds of given problem, which are connected with a formulation indefiniteness of ``the conservation law'' in curved space-time. In [7] contends, that the gravitational energy in EMT can be separated only ``artificially'' by a choice of the certain coordinate system. In [8] is concluded

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

PubMed

Sawitzky, Mandy; Zeissler, Anja; Langhammer, Martina; Bielohuby, Maximilian; Stock, Peggy; Hammon, Harald M; Görs, Solvig; Metges, Cornelia C; Stoehr, Barbara J M; Bidlingmaier, Martin; Fromm-Dornieden, Carolin; Baumgartner, Bernhard G; Christ, Bruno; Brenig, Bertram; Binder, Gerhard; Metzger, Friedrich; Renne, Ulla; Hoeflich, Andreas

2012-01-01

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

Multifaced Roles of the αvβ3 Integrin in Ehlers–Danlos and Arterial Tortuosity Syndromes’ Dermal Fibroblasts

PubMed Central

Zoppi, Nicoletta; Chiarelli, Nicola; Ritelli, Marco; Colombi, Marina

2018-01-01

The αvβ3 integrin, an endothelial cells’ receptor-binding fibronectin (FN) in the extracellular matrix (ECM) of blood vessels, regulates ECM remodeling during migration, invasion, angiogenesis, wound healing and inflammation, and is also involved in the epithelial mesenchymal transition. In vitro-grown human control fibroblasts organize a fibrillar network of FN, which is preferentially bound on the entire cell surface to its canonical α5β1 integrin receptor, whereas the αvβ3 integrin is present only in rare patches in focal contacts. We report on the preferential recruitment of the αvβ3 integrin, due to the lack of FN–ECM and its canonical integrin receptor, in dermal fibroblasts from Ehlers–Danlos syndromes (EDS) and arterial tortuosity syndrome (ATS), which are rare multisystem connective tissue disorders. We review our previous findings that unraveled different biological mechanisms elicited by the αvβ3 integrin in fibroblasts derived from patients affected with classical (cEDS), vascular (vEDS), hypermobile EDS (hEDS), hypermobility spectrum disorders (HSD), and ATS. In cEDS and vEDS, respectively, due to defective type V and type III collagens, αvβ3 rescues patients’ fibroblasts from anoikis through a paxillin-p60Src-mediated cross-talk with the EGF receptor. In hEDS and HSD, without a defined molecular basis, the αvβ3 integrin transduces to the ILK-Snail1-axis inducing a fibroblast-to-myofibroblast-transition. In ATS cells, the deficiency of the dehydroascorbic acid transporter GLUT10 leads to redox imbalance, ECM disarray together with the activation of a non-canonical αvβ3 integrin-TGFBRII signaling, involving p125FAK/p60Src/p38MAPK. The characterization of these different biological functions triggered by αvβ3 provides insights into the multifaced nature of this integrin, at least in cultured dermal fibroblasts, offering future perspectives for research in this field. PMID:29587413

Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice

PubMed Central

Sawitzky, Mandy; Zeissler, Anja; Langhammer, Martina; Bielohuby, Maximilian; Stock, Peggy; Hammon, Harald M.; Görs, Solvig; Metges, Cornelia C.; Stoehr, Barbara J. M.; Bidlingmaier, Martin; Fromm-Dornieden, Carolin; Baumgartner, Bernhard G.; Christ, Bruno; Brenig, Bertram; Binder, Gerhard; Metzger, Friedrich; Renne, Ulla; Hoeflich, Andreas

2012-01-01

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice. PMID:22768110

MO-G-18C-03: Evaluation of Deformable Image Registration for Lung Motion Estimation Using Hyperpolarized Gas Tagging MRI

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

Huang, Q; Zhang, Y; Liu, Y

2014-06-15

Purpose: Hyperpolarized gas (HP) tagging MRI is a novel imaging technique for direct measurement of lung motion during breathing. This study aims to quantitatively evaluate the accuracy of deformable image registration (DIR) in lung motion estimation using HP tagging MRI as references. Methods: Three healthy subjects were imaged using the HP MR tagging, as well as a high-resolution 3D proton MR sequence (TrueFISP) at the end-of-inhalation (EOI) and the end-of-exhalation (EOE). Ground truth of lung motion and corresponding displacement vector field (tDVF) was derived from HP tagging MRI by manually tracking the displacement of tagging grids between EOI and EOE.more » Seven different DIR methods were applied to the high-resolution TrueFISP MR images (EOI and EOE) to generate the DIR-based DVFs (dDVF). The DIR methods include Velocity (VEL), MIM, Mirada, multi-grid B-spline from Elastix (MGB) and 3 other algorithms from DIRART toolbox (Double Force Demons (DFD), Improved Lucas-Kanade (ILK), and Iterative Optical Flow (IOF)). All registrations were performed by independent experts. Target registration error (TRE) was calculated as tDVF – dDVF. Analysis was performed for the entire lungs, and separately for the upper and lower lungs. Results: Significant differences between tDVF and dDVF were observed. Besides the DFD and IOF algorithms, all other dDVFs showed similarity in deformation magnitude distribution but away from the ground truth. The average TRE for entire lung ranged 2.5−23.7mm (mean=8.8mm), depending on the DIR method and subject's breathing amplitude. Larger TRE (13.3–23.7mm) was found in subject with larger breathing amplitude of 45.6mm. TRE was greater in lower lung (2.5−33.9 mm, mean=12.4mm) than that in upper lung (2.5−11.9 mm, mean=5.8mm). Conclusion: Significant differences were observed in lung motion estimation between the HP gas tagging MRI method and the DIR methods, especially when lung motion is large. Large variation among different

A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway.

PubMed

Salašová, Alena; Yokota, Chika; Potěšil, David; Zdráhal, Zbyněk; Bryja, Vítězslav; Arenas, Ernest

2017-07-11

Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Our study shows for the first time that LRRK2 activates the WNT

Validating the use of a luciferase labeled breast cancer cell line, MDA435LCC6, as a means to monitor tumor progression and to assess the therapeutic activity of an established anticancer drug, docetaxel (Dt) alone or in combination with the ILK inhibitor, QLT0267

PubMed Central

Anantha, Malathi; Warburton, Corinna; Waterhouse, Dawn; Yan, Hong; Yang, Young-joo; Strut, Dita; Osooly, Maryam; Masin, Dana; Bally, Marcel B

2011-01-01

A significant issue in drug efficacy studies is animal study design. Here we hypothesize that when evaluating new or existing therapeutics for the treatment of cancer, the location of disease burden will influence drug efficacy. To study this, female NCr nude mice were inoculated with luciferase-positive human breast cancer cells (LCC6WT-luc) orthotopically (o.t.), intraperitoneally (i.p.) or intracardiacly (i.c.) to create localized, ascites or disseminated disease, respectively. Tumor development was monitored using bioluminescence imaging. Docetaxel (Dt) pharmacokinetics and distribution to sites of tumor growth were determined. Disease progression was followed in animals treated with Dt alone and in combination with QLT0267, an integrin linked kinase inhibitor. Tumor related morbidity was most rapid when cells were inoculated i.c., where disease progression was observed in brain, ovaries, adrenal glands and lungs. Dt pharmacokinetics were comparable regardless of the model used (mean plasma AUC0–24 hrs 482.6 ng/ml*hr), however, Dt levels were lowest in those tissues developing disease following i.c. cell injection. Treatment with low dose Dt (5 mg/kg) increased overall survival and reduced tumor cell growth in all three models but the activity was greatest in mice with orthotopic tumors. Higher doses of Dt (15 mg/kg) was able to prolong survival in animals bearing i.p. tumors but not i.c. tumors. Addition of QLT0267 provided no added benefit above Dt alone in the disseminated model. These studies highlight a need for more comprehensive in vivo efficacy studies designed to assess multiple disease models and multiple endpoints, focusing analysis of drug parameters on the most chemoresistant disease. PMID:21358264

The prevention of benign prostatic hyperplasia (bph).

PubMed

Roper, W G

2017-03-01

Barry Marshall and Robin Warren were the first to show that the chronic diseases (gastric ulcer and chronic gastritis) were caused by an infection (Helicobacter pylori). The chronic disease benign prostatic hyperplasia belongs to the same ilk, except that the infection process is much more subtle and complex. The enzyme Phospholipase D (PLD) which is attached to the outer membrane of Escherichia coli (E. coli) has now been almost completely proven to be the basic cause of BPH. The evidence for this process is now extremely strong and compelling. PLD obtained from the organism Streptomyces chromofuscus has been used in past research because of its PLD content. It is commercially available. In vitro, on a culture of prostatic smooth muscle, PLD stimulated the production of lysophosphatidic acid (LPA) which acted on and caused substantial growth of that muscle in accordance with the quantity of PLD/ LPA generated. It has been asserted that repeated colonization by E. coli of the transitional zone of the prostate gland and the release of PLD following repeated destruction of these colonized bacteria, is the basic cause of BPH. The evidence for colonizing and re-colonizing infection is now overwhelming. PLD is a simple lipid consisting of a phosphate, glycerol and a fatty acid. After absorption into the prostatic stroma (which consists of connective tissue and of smooth muscle), it stimulates the production of LPA which, in turn, apart from directly stimulating prostatic smooth muscle, also acts on the connective tissue in the prostate and induces a complex mixture of growth regulatory proteins, which include members of the fibroblast, insulin-like and growth transforming factor families and implicates autocrine hormones acting on the stroma and paracrine hormones acting on epithelium. Also involved, are a variety of interleukins and other inflammatory cell cytokines, secreted by the stroma, which may further promote autocrine/paracrine proliferation of BPH cells

Revised Stratigraphy of The Nallıhan-Dudaş (Beypazarı) Area and Significance of the Campanian-Maastrichtian Reef Occurrences Based on the Foraminiferal and Rudist Data

NASA Astrophysics Data System (ADS)

Görmüş, Muhittin; Sami Us, Muhammed; Özer, Sacit; Tekin, Erdoǧan; Akpınar, Serap; Kabakcı, Büşra

2016-04-01

Transgressive to regressive succession of the Cretaceous Period and Cretaceous Paleogene boundary from the Nallıhan-Beypazarı area have significant data to interpret the past geological history of northwestern Turkey. In the literature, main scientific differences are seen on the formation/lithodem names, their ages, contact relations and environmental interpretations. In the study, a revision has been made for a proper stratigraphy of the area. For the revised stratigraphy, the obtained results from our field and laboratory works and the literature information were used. The following stratigraphy were established from basement to top: the Permo-Triassic aged Sekli metamorphics, Jurassic to lower Cretaceous Soǧukçam formation, Campanian to Maastrichtian aged Dereköy Group-Haremiköy conglomerates, Çeǧiköy reefs, Nardin formation (Seben formation), Taraklı formation; the Paleogene aged Kızılçay group including Kızılbayır formation, Karaköy volcanoclastics, Selvipınar limestone, the Miocene-Pliocene terrestrial sediments, Çoraklar formation, Hırka formation, Akpınar formation, Çayırhan formation, Teke volkanics, Bozbelen formation, Kirmir formation. The main unconformities are between Jura and Campanian, Maastrichtian and Paleogene, Eocene and Miocene times. Among the geological units, the Çeǧiköy reefs having rich rudist fauna overlie the Haremiköy conglomerates in both sides at the north-Yeşilyurt village and at the south-Gökçeöz village. Another outcrop, Emincik is between two mentioned villages. Biohermal reefs mainly includes very rich rudists up to 40 centimetres in size around the Yeşilköy such as: Pironaea polystyla, Vaccinites loftusi, Hippurites sublaevis. Larger foraminifera Orbitoides medius, O. apiculatus, Siderolites calcitrapoides, Pseodosiderolites vidali are also common in the fore reef areas. Around the Gökçeöz at the south part, the identified rudists are as follows: Vaccinites sp., Hippurites aff. sublaevis

Geology and mineral resources of central Antioquia Department (Zone IIA), Colombia

USGS Publications Warehouse

Hall, R.B.; Alvarez A., Jairo; Rico H., Hector

1973-01-01

This report summarizes the geology of an area of some 6000 square kilometers in the northern part of the Central Cordillera of the Colombian Andes. The area, in north-central Department of Antioquia, was mapped between 1964 and 1968 as part of the Inventario Minero Nacional (IMN) project. Mineral resources are summarized within a larger area, designated as subzone ILK of IMN Zone If, which comprises almost 22,000 sq. kin, including the area mapped geologically by IMN and additional areas mapped by other agencies. The oldest formation is a micaceous paragneiss of early Paleozoic or possibly late Precambrian age. A thick geosynclinal sedimentary series accumulated during the Paleozoic Era and became regionally metamorphosed to greenschist (locally amphibolite) facies during the Permian or early Triassic; these schists and gneisses are designated collectively as the Valdivia Group. The Permian(?) orogenic episode included intrusion of concordant syntectonic plutons, mostly of tonalitic composition. Rocks of unequivocal Triassic or Jurassic age are not recognized. The Cretaceous is well represented by both igneous and sedimentary assemblages. Eugeosynclinal alpine ophiolites comprising submarine basalt flows and numerous intrusions of gabbro and serpentinite are prominent in the Lower Cretaceous, together with flysch composed of marine shale and lesser sandstone and conglomerate. The Upper Cretaceous is represented along the west border of the mapped area by submarine basalt flows and pyroclastic rocks, locally Interbedded with fine-grained clastic sedimentary beds, and lenses of dark laminated chert, at least part of which is radiolarian. The Late Cretaceous was marked by an orogenic event that profoundly folded and faulted all rocks and in the Central Cordillera caused low-grade metamorphism, the overprint of which is hardly observable in pre-Cretaceous rocks elsewhere. The Late Cretaceous orogeny culminated with discordant intrusion of the epizonal tonalitic

Re-arrangements of Global Plate Motion: Role of True Polar Wander (TPW)

NASA Astrophysics Data System (ADS)

Bostrom, R. C.

2004-12-01

may now be significant that with increased understanding of the paleomagnetic record (M. Prevot et al. 2000; J. Besse and V. Courtillot 2002), shift of Earth's pole of rotation (TPW) has become a well demonstrated occurrence, although agreement is far from universal as to its modus operandi. Prior to -130 Ma interpretation still is subject to much uncertainty, the major subsequent event having been the end of the -130 to -60 Ma period of relatively fast polar wander. Standstill may have set in as from about -50 Ma. An earlier period of standstill, currently estimated to extend from -200Ma to about -150 Ma, was co-extensive with the Jurassic, being replaced by the highly TPW-active Cretaceous. Agreement has not been reached as to the rapidity of a 20° ultra-fast episode or `jump' culminating at around -115 Ma, at the time of most active break-up of Gondwana into its constituent continents and formation of such ranges as the Rockies and European Alps. If the Cadek/Ricard summary of plate motion or one similar is sound, TPW entails equivalent change in the direction 'west'. The liberty is now taken to suggest that rather than being subsidiary to unexplained reorganizations of global tectonics, TPW is causative and primary. Thermodynamics requires that under TPW, junctures must occur at which Earth's heat efflux is effected most efficiently via convection compliant with a greatly different Pole location, compelling the occurrence of a fast episode or `jump' of the ilk reported by Prevot et al. Within the grossly inhomogeneous, convective Earth, the operation of the conjugate factors NLR and TPW seems basic; of which, the latter has constituted the primary geotectonic determinant.