Overexpression Of Integrin-linked Kinase Research Articles

The oncogenic and growth-suppressive functions of the integrin-linked kinase are distinguished by JNK1 expression in human cancer cells

While most reports detail an oncogenic function for the integrin-linked kinase (ILK) in human cancer, few describe a contradictory growth-suppressive function. We previously reported that ILK functions as either a tumor suppressor or an oncogene in rhabdomyosarcoma (RMS), in a manner linked to expression of the c-jun amino terminal kinase-1 (JNK1). However, studies in other tumors are lacking. With the advent of bioavailable small molecule inhibitors of ILK, defining both the function of ILK and biomarkers to predict its behaviour are of critical importance. Here, we studied the role of ILK in a panel of tumor cell lines. We demonstrate that ILK functions as either a growth-promoter or suppressor in numerous tumor cell lines. Further, cell lines in which ILK functioned as a growth suppressor displayed elevated JNK1 expression relative to cells in which ILK functioned as an oncogene. Comparison of endogenous JNK1 and JNK1β isoform expression levels to the cellular response to ILK overexpression demonstrated that JNK1β isoforms represent biomarkers differentiating the two functions of ILK. Moreover, RNAi and overexpression-based alteration of JNK1 expression levels was sufficient to switch the function of ILK in both transformed and untransformed cells. These results indicate widespread oncogenic and growth-suppressive functions for ILK in multiple human malignancies and suggest that JNK1 isoforms represent biomarkers for ILK neoplastic activity. These results provide a rationale for stratifying patients to receive ILK kinase inhibitors based on individualized tumor-specific ILK function.

ILK expression in human basal cell carcinoma correlates with epithelial–mesenchymal transition markers and tumour invasion

Epithelial-mesenchymal transition (EMT) has been known to play a significant role in tumour progression. Integrin-linked kinase (ILK) has been recently added to the growing list of EMT regulators that control some aspect of carcinogenesis. The aim was to study ILK expression and its relevance to EMT markers in human basal cell carcinoma (BCC). Paraffin-embedded tissue sections from 100 human BCC cases were processed by immunohistochemistry for the expression of ILK, E-cadherin, Snail, beta-catenin and alpha-smooth muscle actin (alpha-SMA). ILK overexpression was observed in 100% of cases and strongly correlated with tumour invasion and infiltrative BCC. Loss of membranous E-cadherin was found in 71% of cases while nuclear immunoreactivity for E-cadherin was also observed in 90% of the tumours. Snail, nuclear beta-catenin and alpha-SMA expression was detected in 100%, 99% and 97% of tumours, respectively. Aberrant expression of E-cadherin, nuclear beta-catenin and alpha-SMA correlated with BCC tumour invasion. Interestingly, there was a significant correlation between ILK expression and all the EMT markers examined. ILK overexpression in BCC is implicated in tumour progression probably through the induction of an EMT-related molecular profile. Nuclear localization of E-cadherin in BCC is also associated with aggressive tumour features.

Abstract 2374: Integrin-linked kinase (ILK) promotes melanoma angiogenesis by activating IL-6 signaling pathway

Abstract Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase involved in cell-ECM interactions, cytoskeletal organization, and cell signaling. Overexpression of ILK in epithelial cells leads to anchorage-independent growth with increased cell cycle progression. Previously, we have shown that ILK upregulation strongly correlates with melanoma progression, invasion and inversely correlates with 5-year survival of melanoma patients. We also found that ILK knockdown impeded melanoma cell migration, invasion and impaired the growth of melanoma xenografts in severe combined immunodeficient (SCID) mice. However, the molecular mechanism by which ILK enhances melanoma angiogenesis is currently unknown. In the present study, we found that pro-angiogenic molecule interleukin-6 (IL-6) is the downstream transcriptional target of ILK in melanoma cells. ILK overexpression increased IL-6 whereas shRNA mediated silencing of ILK suppressed IL-6 expression both transcriptionally and translationally. By electromobility shift assays, we found that ILK enhanced the binding of transcription factor (NF-κB) to IL-6 promoter and this binding was reduced in ILK knockdown cells. Furthermore, Conditioned media (CM) from ILK- overexpressing cells enhanced the tube forming ability of HUVEC cells in vitro. This tube formation by HUVEC cells was IL-6 dependent as CM from IL6-siRNA treated ILK-overexpressing melanoma cells inhibited the tube formation of HUVEC cells. In order to delineate the mechanism by which ILK-upregulated IL-6 can enhance the growth of endothelial cells, further analysis of the downstream targets of IL-6 signaling showed an increase in activity and nuclear translocation of STAT3 in ILK-overexpressing cells. As STAT3 binds to VEGF promoter, we found that VEGF levels were elevated in ILK-overexpressing cells and declined in ILK knockdown cells, suggesting that ILK may regulate VEGF expression through IL-6 pathway by upregulating and activating STAT3. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2374.

There's something about ILK

Purpose: Integrin-Linked Kinase (ILK) is associated with integrin and growth factor receptor signalling. As both signalling pathways contribute to cancer cell resistance, ILK seems well suited as a promising tumour target.Material and methods: Data were obtained by performing a PubMed database search and summarised with a focus on the function of ILK in cancer biology.Results: The findings on the catalytic function of ILK, on the putative substrates of ILK and on the expression of ILK in tumour and normal tissues are heterogeneous. In the context of cancer, two of these issues might be of importance. First, a variety of reports indicate a lack of ILK overexpression in tumours. Second, wild-type or overexpression of ILK has been found to considerably sensitise tumour cells to ionising irradiation as compared to ILK knockout or ILK knockdown conditions. In contrast, wild-type or overexpression of ILK has been shown to protect tumour cells from chemotherapy-induced cell death.Conclusions: Due to these conflicting data, it is difficult to evaluate if therapeutic targeting of ILK is a reasonable strategy in cancer therapy. A more comprehensive understanding of the molecular mechanisms controlled by ILK may help to answer this question.

Clinical Summaries

Clinical Summaries

Increased Expression of Integrin-Linked Kinase Attenuates Left Ventricular Remodeling and Improves Cardiac Function After Myocardial Infarction

Left ventricular (LV) remodeling is associated with the development of heart failure after myocardial infarction. Here we investigated whether integrin-linked kinase (ILK) may regulate LV remodeling and function after myocardial infarction. Adenoviral vector expressing ILK (n=25) or empty adeno-null (n=25) was injected into rat peri-infarct myocardium after left anterior descending coronary artery ligation. ILK expression was confirmed by Western blotting and immunofluorescence. Echocardiographic and hemodynamic analyses demonstrated relatively preserved cardiac function in adeno-ILK animals. ILK treatment was associated with reduced infarct scar size, increased scar thinning ratio, and preserved LV diameter, wall thickness, cardiomyocyte size, and myofilament density. Enhanced angiogenesis and reduced fibrosis were observed in the adeno-ILK group, along with reduced apoptosis as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis. Moreover, increased cardiomyocyte proliferation was found in adeno-ILK animals, as measured by proliferating cell nuclear antigen, Ki-67, and phosphohistone-H3 staining. At long-term follow-up, most indices of cardiac function and hemodynamics showed no difference between adeno-ILK and control animals by 9 weeks, although LV end-systolic diameter and infarct scar size were reduced in the adeno-ILK group at this time point. Additionally, ILK overexpression was found to exert a rescue effect on remodeling when administered in a delayed fashion 1 week after coronary artery ligation. ILK gene therapy improves cardiac remodeling and function in rats after myocardial infarction and is associated with increased angiogenesis, reduced apoptosis, and increased cardiomyocyte proliferation. This may represent a new approach to the treatment of postinfarct remodeling and subsequent heart failure.

Integrin-linked kinase associated with integrin activation

AbstractPlatelet integrin αIIbβ3 activation is tightly controlled by intracellular signaling pathways, and several molecules, including talin, have been identified as critical for αIIbβ3 activation. However, the whole pathway associated with αIIbβ3 activation remains to be determined. To address this issue, we established a Chinese hamster ovary cell line (parental cells) that expresses constitutively activated chimeric integrin αIIbα6Bβ3, and then obtained mutant cells expressing inactivated αIIbα6Bβ3 by genome-wide mutagenesis. We have performed expression cloning to isolate signaling molecules responsible for integrin activation in the mutant cells. We show that integrin-linked kinase (ILK) complements defective integrin activation in the mutant cells. ILK mRNAs in the mutant cells contained 2 nonsense mutations, R317X and W383X, in a compound heterozygous state, resulting in a complete loss of ILK expression. Moreover, the mutant cells showed partially impaired activation of endogenous β1 integrins. Knockdown of ILK in parental cells significantly suppressed the activated state of αIIbα6Bβ3. However, ILK overexpression did not rescue the impaired integrin activation in talin knocked-down parental cells, whereas overexpression of talin-F3, a subdomain of the talin head domain, restored the function. Our present data suggest that ILK contributes to inside-out integrin activation.

Integrin-linked kinase – essential roles in physiology and cancer biology

Integrin-linked kinase (ILK) is a multifunctional intracellular effector of cell-matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The use of recently developed Cre-lox-driven recombination and RNA-interference technologies has enabled the evaluation of the physiological roles of ILK in several major organ systems. Significant developmental and tissue-homeostasis defects occur when the gene that encodes ILK is deleted, whereas the expression of ILK is often elevated in human malignancies. Although the cause(s) of ILK overexpression remain to be fully elucidated, accumulating evidence suggests that its oncogenic capacity derives from its regulation of several downstream targets that provide cells with signals that promote proliferation, survival and migration, supporting the concept that ILK is a relevant therapeutic target in human cancer. Furthermore, a global analysis of the ILK 'interactome' has yielded several novel interactions, and has revealed exciting and unexpected cellular functions of ILK that might have important implications for the development of effective therapeutic agents.

Integrin-linked kinase regulates smooth muscle differentiation marker gene expression in airway tissue

Phenotypic changes in airway smooth muscle occur with airway inflammation and asthma. These changes may be induced by alterations in the extracellular matrix that initiate signaling pathways mediated by integrin receptors. We hypothesized that integrin-linked kinase (ILK), a multidomain protein kinase that binds to the cytoplasmic tail of beta-integrins, may be an important mediator of signaling pathways that regulate the growth and differentiation state of airway smooth muscle. We disrupted signaling pathways mediated by ILK in intact differentiated tracheal muscle tissues by depleting ILK protein using ILK antisense. The depletion of ILK protein increased the expression of the smooth muscle differentiation marker genes myosin heavy chain (SmMHC), SM22alpha, and calponin and increased the expression of SmMHC protein. Conversely, the overexpression of ILK protein reduced the mRNA levels of SmMHC, SM22alpha, and calponin and SmMHC protein. Analysis by chromatin immunoprecipitation showed that the binding of the transcriptional regulator serum response factor (SRF) to the promoters of SmMHC, SM22alpha, and calponin genes was increased in ILK-depleted tissues and decreased in tissues overexpressing ILK. ILK depletion also increased the amount of SRF that localized within the nucleus. ILK depletion and overexpression, respectively, decreased and increased the activation of its downstream substrate protein kinase B (PKB/Akt). The pharmacological inhibition of Akt activity also increased SRF binding to the promoters of smooth muscle-specific genes and increased expression of smooth muscle proteins, suggesting that ILK may exert its effects by regulating the activity of Akt. We conclude that ILK is a critical regulator of airway smooth muscle differentiation. ILK may mediate signals from integrin receptors that control airway smooth muscle differentiation in response to alterations in the extracellular matrix.

Integrin‐linked kinase regulates E‐cadherin expression through PARP‐1

Repression of E-cadherin expression by the transcription factor, Snail, is implicated in epithelial to mesenchymal transition and cancer progression. We show here that Integrin-Linked Kinase (ILK) regulates E-cadherin expression through Poly(ADP-ribose) polymerase-1 (PARP-1). ILK overexpression in Scp2 cells resulted in stimulation of Snail expression and loss of E-cadherin expression. Silencing of ILK, Akt or Snail resulted in re-expression of E-cadherin in PC3 cells. To elucidate the signaling pathway downstream of ILK, we identified candidate Snail promoter ILK Responsive Element (SIRE) binding proteins. PARP-1 was identified as a SIRE-binding protein. ILK silencing inhibited binding of PARP-1 to SIRE. PARP-1 silencing resulted in inhibition of Snail and ZEB1, leading to up-regulation of E-cadherin. We suggest a model in which ILK represses E-cadherin expression by regulating PARP-1, leading to the binding of PARP-1 to SIRE and modulation of Snail expression.

OPPOSING FUNCTIONS FOR A PROTEIN KINASE: A JNK1 DEPENDENT SWITCH DETERMINES THE ONCOGENIC OR TUMOR SUPPRESSIVE ACTIVITY OF ILK INRHABDOMYOSARCOMA

Background:The integrin-linked kinase (ILK) is a protein kinase involved in the regulation of pathogenic cancer cell behaviours, such as proliferation, survival and invasion. ILK appears to be pro-oncogenic in vitro and in vivo models of tumorigenesis. Rhabdomyosarcoma (RMS) is a primitive mesenchyme-derived tumor and is subclassified into primarily embryonal (ERMS) and alveolar (ARMS) variants. Patients who present with metastatic RMS tumors have a less than 20% chance of cure, suggesting a need to define novel targets for chemotherapeutic intervention. Methods: We used cell culture, murine xenografts and primary human tumors to examine ILK expression and functionality. RNAi and adenoviruses were used to knock down or over expressproteins, and SP600125 was used to inhibit JNK kinase activity. ERMS cells stablye xpressing PAX3-FOXO1A we regenerated using pcDNA3.1 with the full length PAX3-FOXO1A cDNA insert. Results: RNAi-mediated ablation of ILK induced stimulation of ERMS and inhibition of ARMS cell growth in vitro and in vivo. Overexpression of ILK, but not the ILK-R211A mutant reversed these effects. High-throughput screening of multiple tumor cell lines and mesenchymal progenitor cells demonstrated similar ILK anti-growth effects. Consistent with these results, clinical correlations made between ILK immunohistochemical staining intensity and patterns on an ERMS tumor tissue microarray revealed downregulation of ILK in stage III/IV primary tumors. Mechanistically, ILK silencing induced selective phosphorylation of the c-jun amino terminal kinase (JNK) and its target c-Jun in ERMS cells with attenuated phosphorylation in ARMS cells. ERMS cells express higher levels of JNK1 isoforms than ARMS cells. Introduction of the ARMS-associated PAX3-FOXO1A fusion gene into ERMS cells restored the oncogenic function of ILK and downregulated of JNK1. Coupling ILK siRNA with inhibition of the JNK-c-Jun signaling pathway in ERMS cells resulted in growth reductions and apoptotic induction. In contrast, coupling ILK knockdown with overexpression of JNK1 in ARMS cells resulted in growth and c-jun phosphorylation. Conclusion: In summary, these data suggest a model whereby the effect of ILK as an oncogene or tumor suppressor is determined by JNK1. Finally, this data suggests that ILK kinase inhibition may be warranted in ARMS tumors, and may be contraindicated in ERMS.

Astragaloside IV improves high glucose-induced podocyte adhesion dysfunction via α 3β 1 integrin upregulation and integrin-linked kinase inhibition

Impaired podocyte adhesion to glomerular basement membrane (GBM) may contribute to podocyte detachment from GBM, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we examined the effects of Astragaloside IV (AS-IV), a saponin purified from Astragalus membranaceus (Fisch) Bge, on high glucose-induced cell adhesion dysfunction in cultured mouse podocytes. Cells were seeded into 96-well plates coated with basement membrane protein complex (BMC). The cells were incubated for 12 h in media containing 30 mM glucose (HG) with 10, 50 and 100 μg/ml of AS-IV. The cells were also exposed to HG media with 100 μg/ml of AS-IV for 3, 6, 12 and 24 h. Cell adhesion assays were performed by fluorescence and centrifugation methods, respectively. Levels of mRNA were determined by quantitative reverse transcriptase real-time PCR and protein expression was analyzed by immunoblotting. HG strongly inhibited adhesion of podocytes to BMC, accompanied by reduction in α 3β 1 integrin mRNA and protein expression, as well as increase in integrin-linked kinase (ILK) activity and expression. When podocytes under HG stimulation were treated with AS-IV, a dose- and time-dependent increase in cell–matrix adhesion was observed, which was significant from 10 μg/ml of AS-IV and from 6 h of incubation of AS-IV with 100 μg/ml. This was accompanied by significant increases in α 3β 1 integrin mRNA and protein expression, as well as inhibition of ILK activation and overexpression. These results suggest that AS-IV improve HG-induced podocyte adhesion dysfunction, which is partly attributed to α 3β 1 integrin upregulation and ILK inhibition.

Cell Adhesion-dependent Cofilin Serine 3 Phosphorylation by the Integrin-linked Kinase·c-Src Complex

Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.

Regulation of cell–matrix contacts and β-catenin signaling in VSMC by integrin-linked kinase: implications for intimal thickening

Vascular smooth muscle cell (VSMC) proliferation and migration is responsible for intimal thickening that occurs in restenosis and atherosclerosis. Integrin-linked kinase (ILK) is a serine/threonine protein kinase implicated in signaling pathways involved in cell proliferation and migration. We studied the involvement of ILK in intimal thickening. ILK expression was significantly increased in two models of intimal thickening: balloon-injured rat carotid arteries and human saphenous vein organ cultures. Over-expression of a dominant negative ILK (DN-ILK) significantly reduced intimal thickening by approximately 50% in human saphenous vein organ cultures, demonstrating an important role in intimal thickening. ILK protein and activity was reduced on laminin and up-regulated on fibronectin, indicating ILK protein expression is modulated by extracellular matrix composition. Inhibition of ILK by siRNA knockdown and DN-ILK significantly decreased VSMC proliferation and migration while wild type ILK significantly increased proliferation and migration on laminin, confirming an essential role of ILK in both processes. Localization of paxillin and vinculin and protein levels of FAK and phospho-FAK indicated that inhibition of ILK reduced focal adhesion formation. Additionally, inhibition of ILK significantly attenuated the presence of the cell-cell complex proteins N-cadherin and beta-catenin, and beta-catenin signaling. We therefore suggest ILK modulates VSMC proliferation and migration at least in part by acting as a molecular scaffold in focal adhesions as well as modulating the stability of cell-cell contact proteins and beta-catenin signaling. In summary, ILK plays an important role in intimal thickening by modulating VSMC proliferation and migration via regulation of cell-matrix and cell-cell contacts and beta-catenin signaling.

Overexpression of integrin-linked kinase in bovine retinal microvascular pericytes induced by glucose

目的 研究高糖培养的牛视网膜微血管周细胞的凋亡和整合蛋白连接酶(ILK)表达的变化及二者的关系.方法 原代培养周细胞,用流式细胞Annexin V检测周细胞的凋亡,用免疫细胞化学和Western blot检测牛视网膜微血管周细胞ILK的表达.结果 用30 mmol/L D-葡萄糖培养24、48、72 h后,牛视网膜微血管周细胞凋亡显著增加,分别为47.6%、58.4%和68.7%且具有时间依赖性.用5 mmol/L D-葡萄糖培养72 h后未见周细胞ILK表达.在30 mmol/L D-葡萄糖或5 μmol/L H2O2条件下培养72 h后,周细胞形态发生改变,ILK表达显著增强,主要分布在细胞质;Western blot检测发现30 mmol/L D-葡萄糖培养的条件下周细胞ILK蛋白水平随时间延长显著增加.结论 高糖可以诱导视网膜微血管周细胞凋亡,高糖条件下周细胞ILK过表达可能参与周细胞的损伤。

A JNK-dependent switch determines the oncogenic or tumor suppressor activity of ILKs

Background: Integrin-linked kinase (ILK) is a potent intracellular kinase involved in the regulation of multiple proliferation and survival kinases, including protein kinase B/Akt, glycogen synthase kinase-3β (GSK3β) and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Evidence suggests ILK is overexpressed and acts oncogenically in a wide variety of primary tumors and genetic models, resulting in induction of tumor cell proliferation, migration, adhesion and angiogenic behaviours. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Histological subclassification of RMS yields two major variants, embryonal (ERMS) and alveolar (ARMS). ERMS, as well as multiple other tumors including breast and lung carcinoma, are commonly associated with loss of heterozygosity at 11p15.5, a region harboring the ILK locus. 
 Methods: We utilized cell culture and primary tumors to examine the expression and function of ILK. 
 Results: We demonstrate differential expression of ILK between ERMS and ARMS tumors in vitro and in vivo. RNAi to ILK induces potent gene silencing, with decreases in the phosphorylation of multiple ILK targets. ILK silencing induced inhibition of ARMS, and a contrasting potent stimulation of ERMS cell growth in survival and proliferation assays. These effects were reversed by adenoviral overexpression of ILK in multiple RMS and other tumor cell lines. Moreover, ILK silencing has multiple signaling effects, including induction of cell cycle progression through bmi-1and p16INK4a in ERMS with repression in ARMS, and phosphorylation of c-Jun NH2 terminal kinase (JNK) and c-Jun in ERMS and repression in ARMS cells. Coupling of JNK inhibition with ILK knockdown in ERMS cells inverted the ILK knockdown phenotype, resulting in a significant loss of cell proliferation. 
 Conclusions: Together, these data confirm the oncogenic role of ILK in alveolar rhabdomyosarcoma, and suggest a novel tumor-suppressive role for ILK signaling in embryonal rhabdomyosarcoma, mediated through the novel ILK target axis, JNK-c-Jun.

Tubular Epithelial Cell Dedifferentiation Is Driven by the Helix-Loop-Helix Transcriptional Inhibitor Id1

In the fibrotic kidney, tubular cells undergo epithelial-to-mesenchymal transition (EMT), a phenotypic conversion that is characterized by sequential loss of epithelial markers and gain of mesenchymal features. For understanding of the molecular mechanism that governs this process, a high-throughput gene expression microarray analysis was used to identify the critical genes in the initial phase of the TGF-beta1-mediated EMT. Inhibitor of differentiation-1 (Id1), a dominant negative antagonist of the basic helix-loop-helix transcription factors, was found to be induced rapidly in human proximal tubular epithelial cells after TGF-beta1 treatment. This induction of Id1 depended on intracellular Smad signaling. Ectopic expression of Id1 suppressed epithelial E-cadherin and zonula occludens-1 expression. Id1 physically formed complex with basic helix-loop-helix transcription factor HEB (Hela E-box binding factor), sequestered its ability to bind to E-box, and repressed the trans-activation of E-cadherin promoter. However, overexpression of Id1 failed to induce alpha-smooth muscle actin, matrix metalloproteinase-2, fibronectin, and integrin-linked kinase (ILK), indicating its inability to confer a complete EMT. Overexpression of ILK or inhibition of ILK activity had no effect on Id1 induction by TGF-beta1, suggesting that Id1 and ILK have independent roles in epithelial dedifferentiation and EMT. In vivo, Id1 was induced exclusively in the degenerated, dilated renal tubular epithelium after unilateral ureteral obstruction. These studies identify Id1 transcriptional inhibitor as a crucial player in mediating cell dedifferentiation of renal tubular epithelium and suggest that EMT is a multistep process in which loss of epithelial adhesion does not necessarily lead to an autonomous mesenchymal transition.

Loss of integrin linked kinase from mouse hepatocytesin vitro andin vivo results in apoptosis and hepatitis

Extracellular matrix (ECM) is fundamental for the survival of cells within a tissue. Loss of contact with the surrounding ECM often causes altered cell differentiation or cell death. Hepatocytes cultured without matrix lose patterns of hepatocyte-specific gene expression and characteristic cellular micro-architecture. However, differentiation is restored after the addition of hydrated matrix preparations to dedifferentiated hepatocytes. Integrin-linked kinase (ILK) is an important component of cell-ECM adhesions transmitting integrin signaling to the interior of the cell. ILK has been implicated in many fundamental cellular processes such as differentiation, proliferation, and survival. In this study, we investigated the role of ILK in mouse hepatocytes in vitro as well as in vivo. Depletion of ILK from primary mouse hepatocytes resulted in enhanced apoptosis. This was accompanied by increased caspase 3 activity and a significant decrease in expression of PINCH and alpha-parvin, which, along with ILK, form a stable well-characterized ternary complex at cell-ECM adhesions. The induction of apoptosis caused by ILK depletion could be substantially reversed by simultaneous overexpression of ILK, indicating that apoptosis is indeed a consequence of ILK removal. These results were further corroborated via in vivo data showing that adenoviral delivery of Cre-recombinase in ILK-floxed animals by tail vein injection resulted in acute hepatitis, with a variety of pathological findings including inflammation, fatty change, and apoptosis, abnormal mitoses, hydropic degeneration, and necrosis. Our results demonstrate the importance of ILK and integrin signaling for the survival of hepatocytes and the maintenance of normal liver function.

Integrin-Linked Kinase Induces Both Senescence-Associated Alterations and Extracellular Fibronectin Assembly in Aging Cardiac Fibroblasts

Integrin-linked kinase (ILK) is an integrin-binding cytoplasmic protein that is involved in regulating numerous cellular processes and extracellular matrix accumulation. We reported that ILK may be involved in cellular senescence, but whether ILK is the cause of senescence or an accompanying phenomenon still remains to be explored. Here, RNA interference and gene transfer techniques were used to knock down and overexpress ILK in 3-month-old and 28-month-old rat primary cardiac fibroblasts. The results show that, in younger cells, ILK overexpression induces larger cell shapes, lower proliferation capacity, and higher levels of enzymatic beta-galactosidase activity, and increases basal p53 and p21 protein levels, whereas knock-down of ILK prevents phenotypic changes typical of senescence in aging cells. In addition, ILK could induce the cytoskeleton proteins to organize into dense, thick bundles of filaments, which contribute to cellular enlargement and extracellular fibronectin assembly. The results indicate that ILK can accelerate the process of cellular senescence.

Integrin-Linked Kinase Inhibitor KP-392 Demonstrates Clinical Benefitsin an Orthotopic Human Non-small Cell Lung Cancer Model

The overexpression of integrin-linked kinase (ILK) has been implicated in the promotion of tumor invasion and metastasis. We studied the anticancer effects of KP-392, a potent selective inhibitor of ILK in the NCI-H460 cell line. In vitro, KP-392 inhibited ILK activity of H460 cells. In vivo, the effect of KP-392 was investigated in a metastatic H460 orthotopic lung cancer model. Intraperitoneal KP-392 (5 mg/day per animal) was administered both alone and in combination with cisplatin (5 mg/kg per week for 3 weeks). In group I, all treated animals were followed until death to assess therapeutic effect on survival. In group II, tumor growth and metastasis were evaluated by sacrificing one animal from each treatment when a control animal died. Both cisplatin and KP-392 significantly enhanced survival (37.8 +/- 3.7 and 34.9 +/- 5.2 days) compared with the control (30.2 +/- 3.6 days, p < 0.0001 and p = 0.0418, respectively), and the survival benefit from combination treatment was greater than that of either agent alone (45.8 +/- 3.9 days, p < 0.0001). Although KP-392 alone did not impact the incidence of metastasis, in combination with cisplatin a consistent trend of inhibition was seen for metastases in the kidney, bone, and the contralateral lung. KP-392 was well tolerated throughout the study. KP-392 demonstrated increased tumor necrosis and decreased nuclear phospho-protein kinase/Akt but did not change the levels of phospho-extracellular signal-regulated kinase 1/2. ILK inhibitor does not enhance the toxicity of standard chemotherapy and may have a beneficial therapeutic effect in lung cancer.