Integrin-linked Kinase 1 Research Articles

Eucalyptol ameliorates Snail1/β-catenin-dependent diabetic disjunction of renal tubular epithelial cells and tubulointerstitial fibrosis.

Renal tubulointerstitial fibrosis is an important event in the pathogenesis of diabetic nephropathy. Under pathologic conditions, renal tubular epithelial cells undergo transition characterized by loss of cell-cell adhesion and increased cell migration. This study investigated that eucalyptol inhibited tubular epithelial cell disjunction and tubulointerstitial fibrosis stimulated by glucose. Human renal proximal tubular epithelial cells were incubated for up to 72 h in media containing 27.5 mM mannitol as osmotic controls or 33 mM glucose in the presence of 1-20 μM eucalyptol. Nontoxic eucalyptol inhibited glucose-induced expression of the mesenchymal markers of N-cadherin and α-smooth muscle actin, whereas the induction of E-cadherin was enhanced. Eucalyptol attenuated the induction of connective tissue growth factor and collagen IV by glucose, whereas the membrane type 1-matrix metalloproteinase expression was enhanced with reducing tissue inhibitor of metalloproteinase-2 expression. Oral administration of 10 mg/kg eucalyptol to db/db mice for 8 weeks blunted hyperglycemia and proteinuria. Eucalyptol reversed tissue levels of E-cadherin, N-cadherin and P-cadherin and the collagen fiber deposition in diabetic kidneys. Eucalyptol attenuated the induction of Snail1, β-catenin and integrin-linked kinase 1 (ILK1) in glucose-exposed tubular cells and diabetic kidneys, and the glycogen synthase kinase (GSK)-3β expression was reversely enhanced. Glucose prompted TGF-β1 production in tubular cells, leading to induction of Snail1, β-catenin and ILK1, which was dampened by eucalyptol. Furthermore, the Snail1 gene deletion encumbered the β-catenin induction in glucose/eucalyptol-treated tubular cells accompanying enhanced GSK-3β expression. Therefore, eucalyptol may antagonize hyperglycemia-induced tubular epithelial derangement and tubulointerstitial fibrosis through blocking ILK1-dependent transcriptional interaction of Snail1/β-catenin.

Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease.

Plasma endothelial cell-derived exosomes (EDEs) and platelet-derived exosomes (PDEs) were precipitated and enriched separately by immunospecific absorption procedures for analyses of cargo proteins relevant to atherosclerosis. EDEs had usual exosome size and marker protein content, and significantly higher levels than PDEs of the endothelial proteins vascular cell adhesion molecule-1 (VCAM-1) and endothelial nitric oxide synthase, whereas PDEs had significantly higher levels of platelet glycoprotein VI. EDE levels of VCAM-1, von Willebrand factor, platelet-derived growth factor (PDGF)-BB, angiopoietin-1, and lysyl oxidase-2 and the cerebrovascular-selective proteins glucose transporter 1, permeability-glycoprotein, and large neutral amino acid transporter 1 were significantly higher for 18 patients with cerebrovascular disease (CeVD) than for 18 age- and gender-matched control subjects. PDE levels of PDGF-AA, platelet glycoprotein VI, integrin-linked kinase-1, high mobility group box-1 protein, chemokine CXCL4, and thrombospondin-1 were significantly higher in patients with CeVD than in control subjects, but differences were less with greater overlaps than for EDE proteins. EDE levels of Yes-associated protein (YAP) were higher and of P(S127)-YAP lower in patients with CeVD than in control subjects, consistent with heightened activity of this mechanical force-sensitive system in atherosclerosis. Elevated EDE and PDE levels of atherosclerosis-promoting proteins in CeVD justify clinical studies of their potential value as biomarkers.-Goetzl, E. J., Schwartz, J. B., Mustapic, M., Lobach, I. V., Daneman, R., Abner, E. L., Jicha, G. A. Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease.

HIPPO-Integrin-linked Kinase Cross-Talk Controls Self-Sustaining Proliferation and Survival in Pulmonary Hypertension.

Enhanced proliferation and impaired apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiologic components of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). To determine the role and therapeutic relevance of HIPPO signaling in PAVSMC proliferation/apoptosis imbalance in PAH. Primary distal PAVSMCs, lung tissue sections from unused donor (control) and idiopathic PAH lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. Immunohistochemical and immunoblot analyses demonstrated that the HIPPO central component large tumor suppressor 1 (LATS1) is inactivated in small remodeled pulmonary arteries (PAs) and distal PAVSMCs in idiopathic PAH. Molecular- and pharmacology-based analyses revealed that LATS1 inactivation and consequent up-regulation of its reciprocal effector Yes-associated protein (Yap) were required for activation of mammalian target of rapamycin (mTOR)-Akt, accumulation of HIF1α, Notch3 intracellular domain and β-catenin, deficiency of proapoptotic Bim, increased proliferation, and survival of human PAH PAVSMCs. LATS1 inactivation and up-regulation of Yap increased production and secretion of fibronectin that up-regulated integrin-linked kinase 1 (ILK1). ILK1 supported LATS1 inactivation, and its inhibition reactivated LATS1, down-regulated Yap, suppressed proliferation, and promoted apoptosis in PAH, but not control PAVSMCs. PAVSM in small remodeled PAs from rats and mice with SU5416/hypoxia-induced PH showed down-regulation of LATS1 and overexpression of ILK1. Treatment of mice with selective ILK inhibitor Cpd22 at Days 22-35 of SU5416/hypoxia exposure restored LATS1 signaling and reduced established pulmonary vascular remodeling and PH. These data report inactivation of HIPPO/LATS1, self-supported via Yap-fibronectin-ILK1 signaling loop, as a novel mechanism of self-sustaining proliferation and apoptosis resistance of PAVSMCs in PAH and suggest a new potential target for therapeutic intervention.

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

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 Ca(2+) 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.

Abstract 25: Inducible Depletion of Calpain-2 Attenuates Angiotensin II-induced Cytoskeletal Structural Protein Destruction During Abdominal Aortic Aneurysm Development in Mice

Background and Objective: In abdominal aortic aneurysm (AAA) patients, structural integrity of the aortic wall is disrupted due to the dissociation of cytoskeletal structural proteins that bridge SMC contractile filaments with extracellular matrix by proteases. However, identity of functional proteases which target cytoskeletal structural linker proteins remain unknown. Recently, using the angiotensin II (AngII)-induced AAA model, we demonstrated that AngII significantly increased fragmentation of cytoskeletal structural protein, Filamin A, in AAAs. Further, using a pharmacological inhibitor and genetic deficient mice, we identified that calpain-2 (a class of calcium-activated, intracellular cysteine proteases) plays a critical role in AngII-induced AAA formation in mice. The purpose of this study was to determine the functional contribution of calpain-2 in AngII-induced cytoskeletal structural protein destruction during AAA development. Methods and Results: Calpain-2 floxed mice that were hemizygous for Cre-ERT2 in an LDLr -/- background were produced by breeding male Cre-ERT2 to female calpain-2 floxed mice. At 8 weeks of age, male Calp-2 x Cre-ERT2 (Cre+) and non-Cre littermates (Cre-) mice were injected with tamoxifen (25 mg/kg, i.p.) daily for 5 consecutive days. After 2 weeks, Western blot analyses showed a complete depletion of calpain-2 protein in the aorta from Cre+ mice compared to Cre- littermates. Male Cre+ and Cre- (N=12 each) mice were fed a Western diet and infused with saline or AngII (1,000 ng/kg/min) by osmotic minipumps for 2 weeks. AngII infusion significantly (P<0.05 vs saline) increased c-terminal fragmentation of cytoskeletal structural protein, talin and kinases such as integrin linked kinase-1 (ILK-1), and focal adhesion kinase in addition to filamin A. Inducible depletion of calpain-2 significantly (P<0.05; Cre+ vs Cre-) blunted AngII-induced fragmentation of talin, filamin A and ILK-1. Further siRNA mediated silencing of calpain-2 in aortic SMCs and fibroblasts significantly reduced AngII-induced fragmentation of filamin A and talin. Conclusion: These findings suggest that calpain-2 plays a critical role in AngII-induced cytoskeletal structural protein fragmentation during AAA development in mice.

NOTCH1 intracellular domain negatively regulates PAK1 signaling pathway through direct interaction

p21-Activated kinase 1 (PAK1) is a serine/threonine protein kinase implicated in cytoskeletal remodeling and cell motility. Recent studies have shown that it also promotes cell proliferation, regulates apoptosis, and increases cell transformation and invasion. In this study, we showed that NOTCH1 intracellular domain (NOTCH1-IC) negatively regulated PAK1 signaling pathway. We found a novel interaction between NOTCH1-IC and PAK1. Overexpression of NOTCH1-IC decreased PAK1-induced integrin-linked kinase 1 (ILK1) phosphorylation, whereas inhibition of NOTCH1 signaling increased PAK1-induced ILK1 phosphorylation. Notably, ILK1 phosphorylation was higher in PS1,2−/− cells than in PS1,2+/+ cells. As expected, overexpression of NOTCH1-IC decreased ILK1-induced phosphorylation of glycogen synthase kinase 3 beta (GSK-3beta). Furthermore, NOTCH1-IC disrupted the interaction of PAK1 with ILK1 and altered PAK1 localization by directly interacting with it. This inhibitory effect of NOTCH1-IC on the PAK1 signaling pathway was mediated by the binding of NOTCH1-IC to PAK1 and by the alteration of PAK1 localization. Together, these results suggest that NOTCH1-IC is a new regulator of the PAK1 signaling pathway that directly interacts with PAK1 and regulates its shuttling between the nucleus and the cytoplasm.

Periostin Enhances Migration, Invasion, and Adhesion of Human Endometrial Stromal Cells Through Integrin-Linked Kinase 1/Akt Signaling Pathway.

Although our previous study confirmed that periostin (PN) was overexpressed in the eutopic and ectopic endometrial stroma of women with endometriosis by immunohistochemitry, the role of PN in the pathophysiology of endometriosis remains unknown. Thus, we aimed to investigate the effects of PN on endometrial stromal cells (ESCs) migration, invasion, adhesion, and proliferation and to further study the mechanism under this process. Eutopic (EuSCs), ectopic (EcSCs), and normal ESCs (NSCs) were isolated and cultured. We evaluated the above-mentioned biology behaviors and the expression of PN, integrin-linked kinase 1 (ILK1), and phospho-Akt (p-Akt) in NSCs, EuSCs as well as EcSCs before and after receiving PN small-interfering RNA (siRNA). The protein and messenger RNA (mRNA) levels of PN were upregulated in EuSCs (P < .05; P = .2261 in proliferative phase and P = .3385 in secretory phase) and EcSCs (P < .001; P < .001 in proliferative phase and P < .05 in secretory phase) compared with NSCs, although there was no significant difference in PN mRNA between EuSCs and NSCs. In EcSCs, abilities of migration, invasion, and adhesion and the expressions of ILK1 and p-Akt were enhanced; and all of those were downregulated after PN siRNA interference. Thus, PN enhanced ESCs migration, invasion, and adhesion due to the ILK1/Akt signal pathway. As an agonist in the development and progression of endometriosis, PN may be a new clinical treatment target of endometriosis.

Prolactin-induced protein in breast cancer.

Prolactin-induced protein (PIP) is a 17-kDa single polypeptide chain that is secreted by a number of normal apocrine cells, such as milk, saliva, and seminal fluid. PIP is widely expressed in breast cancer and is commonly used as a diagnostic biomarker for the histopathological diagnosis of this disease. Expression of PIP in breast cancer is regulated by androgen and prolactin through a number of transcription factors and signaling cross-talks, including STAT5, Runx2, and CREB1. Notably, PIP is induced by a positive feedback loop between androgen receptor (AR) and extracellular signal-regulated kinase (ERK). The available data indicate that PIP has an aspartyl protease activity that can degrade fibronectin. Importantly, PIP is necessary for outside-in activation of integrin-β1 signaling pathway and regulation of key downstream signaling targets of this pathway such as interaction of integrin-β1 with integrin-linked kinase 1 (ILK1) and ErbB2. Furthermore, the importance of PIP in cell proliferation has been demonstrated by the fact that purified PIP promotes growth of breast cancer cells and PIP expression is necessary for the proliferation of T-47D and MDA-MB-453 cell lines. In addition to cell proliferation, PIP mediates invasion of breast cancer cells in a process that partially depends on the degradation of fibronectin by this protein. Therefore, PIP is a breast cancer-related protein that is expressed in a majority of breast tumors and has a significant function in the biology of this disease.

Overexpression of ILK1 in breast cancer associates with poor prognosis

Integrin-linked kinase 1 (ILK1), a member of the serine/threonine kinases, has been demonstrated to be associated with numerous biological and pathological processes. However, the role of ILK1 in breast cancer has not been thoroughly elucidated. The purpose of this study was to assess ILK1 expression and to explore its contribution to breast cancer. The ILK1 mRNA expression was measured by real-time quantitative reverse transcriptase-polymerase chain reaction. In addition, ILK1 expression was analyzed by immunohistochemistry in 163 clinicopathologically characterized breast cancer cases. The relationship between ILK1 expression and clinicopathological features was analyzed by appropriate statistics. Kaplan-Meier analysis and Cox proportional hazard regression models were used to investigate the correlation between ILK1 expression and prognosis of breast cancer patients. The relative mRNA expression of ILK1 was significantly higher in breast cancer tissues than in adjacent noncancerous tissues (P < 0.001). In addition, ILK1 expression was significantly correlated with tumor size (P = 0.016), grade (P = 0.024), stage (P = 0.029), lymph node metastases (P = 0.007), and estrogen receptor status (P = 0.002). Kaplan-Meier analysis indicated that patients with high ILK1 expression had poor overall survival (P < 0.001). Multivariate analysis showed that high ILK1 expression was an independent predictor of overall survival. In conclusion, our data suggest for the first time that the increased expression of ILK1 in breast cancer is associated significantly with aggressive progression and poor prognosis. ILK1 may be an important molecular marker for predicting the carcinogenesis, progression, and prognosis of breast cancer.

Abstract 2445: Cucurbitacin B suppresses breast cancer growth by inhibiting TGFβ-mediated integrin-HER2 signaling.

Abstract Integrins have recently been shown to play an important role in promotion of cell motility and survival in various cancers through cooperative signaling with EGFRs. Integrins transmit cell survival signals through ILK-Paxillin complex and further play role in activating AKT signaling. Curcubitacin B (CuB) is a steroidal class of chemical present in the plants belonging to cucurbitaceae family. CuB has shown cytotoxic potential in several cancers by causing G2/M cell cycle arrest and inhibition of JAK/STAT3 pathway. However, the exact mechanism of CuB and its role in metastasis and cell survival is not known. Our results show that CuB significantly suppressed the growth of MDA-MB-231, SKBR3, MCF-7, MDA-MB-231 (HER2) and MCF-7 (HER2), five different breast cancer cells in a concentration and time-dependent manner. CuB also induced apoptosis and down-regulated Integrin linked kinase 1(ILK1) expression. In addition, CuB down-regulated Integrin β4 (ITGB4) and Integrin α 6 (ITGA6) protein expression and suppressed the phosphorylation of AKT at Ser473, paxillin at Tyr118. Previous studies have shown that Transforming growth factor β (TGFβ) mediates crosstalk between ITGB4 and HER2, through Src kinase to enhance cell survival and motility. Interestingly we observed significant downregulation of Src, EGFR and HER2 in various breast cancer cells, suggesting that CuB suppresses cooperative signaling of Integrins and HER2. We also observed that TGFβ pretreatment imparted protection against CuB induced cytotoxic effects, indicating that CuB modulates TGFβ mediated Integrin signaling. Taken together our results indicate the anti-cancer effects of CuB in breast cancer cells by targeting ITGB4/ITGA6, Src, ILK and TGFβ. Further detailed mechanistic work is in progress. [Supported in part by R01 grants CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Parul Gupta, Sanjay K. Srivastava. Cucurbitacin B suppresses breast cancer growth by inhibiting TGFβ-mediated integrin-HER2 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2445. doi:10.1158/1538-7445.AM2013-2445

Characterization of Nuclear Localization Signal in the N Terminus of Integrin-linked Kinase-associated Phosphatase (ILKAP) and Its Essential Role in the Down-regulation of RSK2 Protein Signaling

Integrin-linked kinase-associated phosphatase (ILKAP) is a serine/threonine (S/T) phosphatase that belongs to the protein phosphatase 2C (PP2C) family. Many previous studies have demonstrated that ILKAP plays key roles in the regulation of cell survival and apoptosis. Researchers have thus far considered ILKAP a cytoplasmic protein that negatively regulates integrin signaling by interacting with and phosphorylating integrin-linked kinase 1 (ILK1). In this study, we found that both endogenous and tagged ILKAP mainly localize to the nucleus and that the nuclear transport of ILKAP is nuclear localization signal (NLS) importin-mediated. The ILKAP protein interacts directly with importin α1, α3, and α5. The NLS in ILKAP is located in the N-terminal region between amino acids 71 and 86, and the NLS-deleted ILKAP protein was distributed in the cytoplasm. In addition, we show that Lys-78 and Arg-79 are critical for the binding of ILKAP to importin α. We also found that nuclear ILKAP interacts with ribosomal protein S6 kinase-2 (RSK2) and induces apoptosis by inhibiting RSK2 activity and down-regulating the expression level of the RSK2 downstream substrate cyclin D1. These results indicate that ILKAP is a nuclear protein that regulates cell survival and apoptosis through the regulation of RSK2 signaling.

Abstract 2567: DIM regulates anoikis through sonic hedgehog in ovarian cancer cells

Abstract Ovarian cancer is the second leading gynecological cancer with poor prognosis. By the time of detection, it usually metastasizes to peritoneum or organs such as stomach and liver. Detachment of a cell from extracellular matrix (ECM) allows the cells to travel and lodge at metastatic site. Anoikis is an emerging form of cell death that is associated with the apoptosis of cells detached from ECM. Normal or immortal epithelial cells are sensitive to anoikis. However, carcinomas are generally associated with resistance to anoikis. Hence, induction of anoikis is an important step in preventing metastasis. We have recently shown that diindolylmethane (DIM), a compound present in cruciferous vegetables reduces the survival of ovarian cancer cells by inducing apoptosis. Here, we evaluated the effect of DIM in inducing anoikis in ovarian cancer cells. A2780, SKOV-3 and OVCAR-429 cells were grown on low attachment plates and treated with various concentrations of DIM for 24 hours and analyzed. Our results show that DIM treatment resulted in the concentration-dependent anoikis in all the cell lines. For example, anoikis induction was approximately 50-70% in all the cell lines treated with 75µM DIM. Furthermore, Integrin Linked Kinase 1 (ILK-1), an important regulator of anoikis was significantly down-regulated by DIM treatment. Western blotting revealed cleavage of caspase-3 and PARP by DIM treatment, confirming anoikis induction in our model. Few recent studies showed that Sonic Hedgehog (Shh) pathway is activated in ovarian tumors. Shh pathway is also associated with metastasis of various cancers. We therefore next determined whether Shh pathway was involved in anoikis induction by DIM. Our results demonstrated that DIM treatment reduced the expression of Shh and Gli1 in a concentration-dependent manner. Moreover, Shh secretion as well was reduced by DIM treatment in ovarian cancer cells. Importantly, Shh pretreatment significantly inhibited DIM-induced anoikis in A2780 ovarian cancer cells. Treatment of cells with Shh also reduced the cleavage of caspase-3 or PARP induced by DIM treatment indicating the involvement of Shh pathway in anoikis. Taken together, these results suggest that DIM induces anoikis in ovarian cancer cells by regulating sonic hedgehog signaling. Further in-depth mechanistic studies are in progress to establish anoikis induction by DIM and to validate it in an in vivo model. [Supported in part by R01 grants CA 1065953 and CA 129038 awarded to S.K.S by National Cancer Institute, NIH] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2567. doi:1538-7445.AM2012-2567

Abstract 3874: Galectin-3 Enhances Post Ischemic Angiogenesis Via Modulation Of Integrin And Akt Pathways.

Introduction: As restoration of blood flow is critical for improved functional recovery following ischemic stroke, enhancing cerebral angiogenesis might be immensely helpful. Galectin-3 (gal-3) is a β-galactoside-binding lectin that plays key roles in inflammation, survival, and angiogenesis. Methods: We used transient middle cerebral artery occlusion (MCAO), oxygen-glucose deprivation (OGD) model, western blot analysis, RT-PCR, Immunofluorescence staining. Results: We currently report that intra-cerebroventricular infusion of recombinant gal-3 in rats subjected to transient MCAO increases the post-ischemic functional recovery. We further show that gal-3 mRNA and protein expression is up-regulated in vitro when activated microglial BV2 cells were subjected to either oxygen-glucose deprivation (OGD) or LPS (lipo-polysaccharide) stimulation. Immunofluorescence staining showed increased cytoplasmic gal-3 that is known to have pro-survival function. This increase in gal-3 is associated with increased number of pro-angiogenic structures in a 3D human vein umbilical cord endothelial (HUVEC) and microglia co-culture model. The pro-angiogenic effect of gal-3 was suppressed by treatment with gal-3 siRNA indicating the specific role of gal-3 in promoting angiogenesis. Exogenous gal-3 treatment further augmented the angiogenic potential of BV2 microglia cells. In addition, gal-3 increased survival of microglial BV2 cells, HUVEC, and neuro-progenitor cells. Gal-3 levels were also directly correlated with increased levels of Integrin-linked kinase 1(ILK1), pro-survival factor AKT and their down stream effector pro-angiogenic factors bFGF and MMP2. Conclusion: Taken together, our studies emphasize the importance of gal-3 in enhancing angiogenesis that is critical for neuron survival and neurogenesis in the post-stroke brain.

BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

We present a novel cell-signaling paradigm in which bone morphogenetic protein 2 (BMP-2) consecutively and interdependently activates the wingless (Wnt)-β-catenin (βC) and Wnt-planar cell polarity (PCP) signaling pathways to facilitate vascular smooth muscle motility while simultaneously suppressing growth. We show that BMP-2, in a phospho-Akt-dependent manner, induces βC transcriptional activity to produce fibronectin, which then activates integrin-linked kinase 1 (ILK-1) via α4-integrins. ILK-1 then induces the Wnt-PCP pathway by binding a proline-rich motif in disheveled (Dvl) and consequently activating RhoA-Rac1-mediated motility. Transfection of a Dvl mutant that binds βC without activating RhoA-Rac1 not only prevents BMP-2-mediated vascular smooth muscle cell motility but promotes proliferation in association with persistent βC activity. Interfering with the Dvl-dependent Wnt-PCP activation in a murine stented aortic graft injury model promotes extensive neointima formation, as shown by optical coherence tomography and histopathology. We speculate that, in response to injury, factors that subvert BMP-2-mediated tandem activation of Wnt-βC and Wnt-PCP pathways contribute to obliterative vascular disease in both the systemic and pulmonary circulations.

Integrin-linked kinase 1 role in hormonal cancer progression

Integrin-linked kinase 1 (ILK1) is a serine/threonine kinase that plays important roles in a variety of cellular functions including cell survival, migration and angiogenesis. ILK1 is normally expressed in numerous tissues and activated by growth factors, cytokines and hormones. Dysregulation of ILK1 expression or function is found in several hormonal tumors including breast, ovary and prostate. Emerging evidence suggests that ILK overexpression promotes cellular transformation, cell survival, epithelial mesenchymal transition (EMT), and metastasis of hormonal cancer cells while inhibition of ILK1 reduces tumor growth and progression. The recent development of ILK1 inhibitors has provided novel mechanisms for blocking ILK1 signaling to curb metastasis and therapy resistance of hormonal tumors. This review will focus on recent advances made towards understanding the role of ILK signaling axis in progression of hormonal cancer.

Anti-Angiogenic Therapy Induces Integrin-Linked Kinase 1 Up-Regulation in a Mouse Model of Glioblastoma

BackgroundIn order to improve our understanding of the molecular pathways that mediate tumor proliferation and angiogenesis, and to evaluate the biological response to anti-angiogenic therapy, we analyzed the changes in the protein profile of glioblastoma in response to treatment with recombinant human Platelet Factor 4-DLR mutated protein (PF4-DLR), an inhibitor of angiogenesis.Methodology/Principal FindingsU87-derived experimental glioblastomas were grown in the brain of xenografted nude mice, treated with PF4-DLR, and processed for proteomic analysis. More than fifty proteins were differentially expressed in response to PF4-DLR treatment. Among them, integrin-linked kinase 1 (ILK1) signaling pathway was first down-regulated but then up-regulated after treatment for prolonged period. The activity of PF4-DLR can be increased by simultaneously treating mice orthotopically implanted with glioblastomas, with ILK1-specific siRNA. As ILK1 is related to malignant progression and a poor prognosis in various types of tumors, we measured ILK1 expression in human glioblatomas, astrocytomas and oligodendrogliomas, and found that it varied widely; however, a high level of ILK1 expression was correlated to a poor prognosis.Conclusions/SignificanceOur results suggest that identifying the molecular pathways induced by anti-angiogenic therapies may help the development of combinaatorial treatment strategies that increase the therapeutic efficacy of angiogenesis inhibitors by association with specific agents that disrupt signaling in tumor cells.

Prognostic significance of integrin‐linked kinase1 overexpression in astrocytoma

Integrin-linked kinase 1 (ILK1), a member of the serine/threonine kinases, has been demonstrated to be associated with numerous biological and pathological processes. However, the clinical and functional significance of ILK1 expression has not been characterized previously in human astrocytoma. In this study, we found that ILK1 was overexpressed, at both mRNA and protein levels, in astrocytoma cell lines as compared with normal human astrocytes. The ILK1 mRNA and protein were significantly increased up to 5.6-fold and 10.1-fold, respectively, in primary astrocytoma in comparison with the paired adjacent noncancerous brain tissues obtained from the same patient. Furthermore, immunohistochemical analysis revealed that ILK1 protein was positive in 208 of 228 (91.2%) paraffin-embedded archival astrocytoma specimens. Statistical analysis suggested that the upregulation of ILK1 was significantly correlated with the histological grading of astrocytoma (p = 0.000), and that patients with high ILK1 level exhibited shorter survival time (p < 0.001). Multivariate analysis revealed that ILK1 upregulation might be an independent prognostic indicator for the survival of patients with astrocytoma. Taken together, our results suggest that ILK1 might represent a novel and useful prognostic marker for astrocytoma and play a role during the development and progression of the disease.

Inositol Hexaphosphate Suppresses Growth and Induces Apoptosis in Prostate Carcinoma Cells in Culture and Nude Mouse Xenograft: PI3K-Akt Pathway as Potential Target

Constitutive activation of phosphoinositide 3-kinase (PI3K)-Akt pathway transmits growth-regulatory signals that play a central role in promoting survival, proliferation, and angiogenesis in human prostate cancer cells. Here, we assessed the efficacy of inositol hexaphosphate (IP6) against invasive human prostate cancer PC-3 and C4-2B cells and regulation of PI3K-Akt pathway. IP6 treatment of cells suppressed proliferation, induced apoptosis along with caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, and inhibited constitutive activation of Akt and its upstream regulators PI3K, phosphoinositide-dependent kinase-1 and integrin-linked kinase-1 (ILK1). Downstream of Akt, IP6 inhibited the phosphorylation of glycogen synthase kinase-3alpha/beta at Ser(21/9) and consequently reduced cyclin D1 expression. Efficacy studies employing PC-3 tumor xenograft growth in nude mice showed that 2% (w/v) IP6 feeding in drinking water inhibits tumor growth and weight by 52% to 59% (P < 0.001). Immunohistochemical analysis of xenografts showed that IP6 significantly reduces the expression of molecules associated with cell survival/proliferation (ILK1, phosphorylated Akt, cyclin D1, and proliferating cell nuclear antigen) and angiogenesis (platelet endothelial cell adhesion molecule-1 or CD31, vascular endothelial growth factor, endothelial nitric oxide synthase, and hypoxia-inducible factor-1alpha) together with an increase in apoptotic markers (cleaved caspase-3 and PARP). These findings suggest that, by targeting the PI3K-ILK1-Akt pathway, IP6 suppresses cell survival, proliferation, and angiogenesis but induces death in prostate cancer cells, which might have translational potential in preventing and controlling the growth of advanced and aggressive prostate cancer for which conventional chemotherapy is not effective.

A New PIXel in the Puzzle

Hypertension is a clinical situation associated with endothelial dysfunction and increased vascular reactive oxygen species (ROS) formation. The observations that antioxidants, such as Tempol or apocynin, lower the blood pressure in spontaneously hypertensive rats gave rise to the concept that ROS essentially contribute to hypertension. Recent work, however, has sharpened this view, and it is now clear that the underlying mechanism of ROS-induced hypertension may not be endothelial dysfunction but rather an ROS-dependent modulation of the sympathetic drive,1 the kidney, and potentially alterations in the immune system.2 Despite these findings, hypertension has been consistently linked to increased ROS formation in the vascular system. It is, therefore, plausible that ROS are not necessarily the cause but potentially the consequence of hypertension. This notion certainly does not exclude that ROS are involved in the pathogenesis of specific aspects of hypertension, such as the development of fibrosis. In keeping with the concept of hypertension-induced ROS formation, it was noted previously that distension of a vessel with an oversized balloon acutely increases ROS formation.3 Moreover, acute elevation of pressure from 80 to 160 mm Hg enhances the ROS formation and induces an ROS-dependent attenuation of the endothelium-dependent relaxation in isolated rat femoral arteries.4 Moreover, even in chronic models of isolated hypertension, such as aortic banding, the ROS formation was consistently higher in the hypertensive rather than in the normotensive part. There is, however, uncertainty regarding the enzymatic sources of ROS, and a contribution of mitochondria, endothelial NO …

Phosphorylation of ser246residue in integrin‐linked kinase 1 by serum‐ and glucocorticoid‐induced kinase 1 is required to form a protein‐protein complex with 14–3–3

Abstract Integrin‐linked kinase 1 (ILK1) regulates several protein kinases, including PKB/Akt kinase and glycogen synthase kinase β. ILK1 is also involved distinctively in the cell morphological and structural functions by interacting with the components of the extracellular matrix or integrin. According to the information of serum‐ and glucocorticoid‐induced kinase 1 (SGK1) substrate specificity (R‐X‐R‐X‐X‐(S/T)‐ϕ; ϕ indicates a hydrophobic amino acid), two putative phosphorylation sites, Thr181 and Ser246, were found in ILK1. We showed that ILK1 fusion protein and two fluorescein‐labeled ILK1 peptides, FITC‐174RTRPRNGTLN183 and FITC‐239CPRLRIFSHP248, were phosphorylated by SGK1 in vitro. We also identified that 14–3–3 θ, ϵ and ζ among several 14–3–3 isotypes (β, γ, ϵ, η, σ, θ, τ and ζ) formed protein complex with ILK1 in COS‐1 cells. Furthermore, the phosphorylation of Ser246 by SGK1 induced the binding with 14–3–3. It was also demonstrated that 14–3–3‐bound ILK1 has reduced kinase activity. Thus, these...