Effects Of Integrin-linked Kinase Research Articles

Effects of integrin-linked kinase on protein kinase b, glycogen synthase kinase-3β, and β-catenin molecules in ovarian cancer cells.

Objective(s):This study examines the impact of integrin-linked kinase (ILK), protein kinase B (AKT), glycogen synthase kinase-3β (GSK-3β), and β-catenin signal molecules in SKOV-3 ovarian cancer cells adhered to fibronectin. Materials and Methods:Expression levels of α4, αv, β1, and β6 integrin subunits known as the fibronectin ligand were investigated with the flow cytometry technique. The effects of ILK, AKT, GSK-3β, and β-catenin on the binding of SKOV-3 cells to fibronectin were examined by using the Real-Time Cellular Analysis (RTCA) method. Additionally, the interaction of these proteins was investigated by using Western blot analysis. Results:The results show that the expression levels of integrin subunits were ranked as αv (67.8%), followed by α4 (48.55%), β6 (32.05%), and β1 (31%) on SKOV-3 cells. RTCA results showed that ILK (10 µM Cpd22), GSK-3β (50 μM GSK-3β inhibitor-XI), AKT (35 µM FPA 124), and β-catenin (50 μM cardamonin) inhibitors decreased significantly (P<0.01) binding to fibronectin at 24 hr. Western studies in SKOV-3 cells adhered to fibronectin have shown that in inhibition of ILK, AKT expression was strongly inhibited, whereas, in the inhibition of AKT, ILK expression was strongly inhibited. Furthermore, the expression of β-catenin is partially reduced in inhibition of these two molecules. In β-catenin inhibition, AKT and ILK expressions are also strongly inhibited.Conclusion:ILK, AKT, GSK-3β, and β-catenin were found to be fundamental molecules in binding of SKOV-3 cells to fibronectin. ILK and AKT affect strongly the level of expression of each other, and both also affect the signal path of β-catenin.

Integrin-Linked Kinase (ILK) Regulates Urinary Stem Cells Differentiation into Smooth Muscle via NF-κB Signal Pathway.

Objectives Urinary stem cells (USCs) have the capacity for unlimited growth and are promising tools for the investigations of cell differentiation and urinary regeneration. However, the limited life span significantly restricts their usefulness. This study is aimed at exploring the effect of integrin-linked kinase (ILK) on the smooth muscle cells (SMCs) differentiation of the dog USCs and investigating its molecular mechanism. Methods An immortalized USCs cell line with the molecular markers and biological functions was prepared. After successfully inducing the differentiation of USCs into SMCs, the expression level of the unique key factor and its mechanisms in this process was determined through real-time polymerase chain reaction, Western blot, or Immunofluorescence staining. Results We found that high cell density promoted USCs differentiation SMCs, and ILK was necessary for USCs differentiation into SMCs. Knocking down ILK decreased the expression of SMCs specific-marker, while using a selective ILK agonist increased the expression of SMCs specific-marker. Furthermore, ILK regulated SMCs differentiation in part through the activation of NF-κB pathway in USCs. A NF-κB activity assay showed overexpression of ILK could significantly upregulate NF-κB p50 expression, and NF-κB p50 acts as downstream signal molecular of ILK. Conclusion High cell density induces the differentiation of USCs into SMCs, and ILK is a key regulator of myogenesis. Furthermore, NF-κB signaling pathway might play a crucial role in this process.

Effect of ILK on small-molecule metabolism of human periodontal ligament fibroblasts with mechanical stretching.

Mechanical stimuli can cause periodontal tissue reconstruction. Studies have found that changes in metabolites can be the terminal effect of integrin-mediated mechanical signaling. As a key kinase in integrin regulation, integrin-linked kinase (ILK) mediates mechanical signal transduction, which may contribute to metabolite changes. Defining the components of small-molecule metabolites can optimize mechanical stimuli and periodontal tissue reconstruction. Our purpose is to detect the effect of ILK-mediated mechanical signaling on intracellular small-molecule metabolites (amino acids and organic acids) in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were isolated by enzyme digestion method. Tensile stresses were applied on HPDLFs in vitro using a Flexcell system. ILK gene in HPDLFs was knocked down by RNA interference (RNAi). Twenty common amino acids and seven organic acids in HPDLFs were analyzed by gas chromatography/mass spectrometry technique. Five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) were found to be changed remarkably after mechanical stretching. In addition, baseline levels of four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) were significantly different in ILK knockdown compared with wild-type HPDLFs. This study suggests that five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) may act as cellular mediators for mechanical signals in HPDLFs. Among them, four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) may be closely linked to ILK.

Effect of integrin-linked kinase towards matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 in the process of TGF-β1-induced epithelial-mesenchymal transition of renal tubular cell

Objective To investigate the possible regulating effect of integrin-linked kinase (ILK) towards matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 (MMP-9/TIMP-1) ratio in the process of transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial (HK-2) cells. Methods HK-2 cells were cultured and stimulated with 10 ng/ml TGF-β1. Specific ILK-small interfering RNA (ILK-siRNA) was used to inhibit ILK expression. The characteristic epithelial marker (E-cadherin) and mesenchymal marker (α-SMA) of EMT were examined by Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blot. The expressions of ILK, MMP-9, and TIMP-1 were also examined, to determine the regulating effect of ILK towards MMP-9/TIMP-1 ratio. Results In the HK-2 cells cultured with TGF-β1, the expression of E-cadherin decreased, and α-SMA expression increased; overexpression of ILK and an abnormal changing of MMP-9/TIMP-1 ratio were observed. ILK inhibition by ILK-siRNA could adjust MMP-9/TIMP-1 ratio to near normal. Meanwhile, the overexpressed ILK and α-SMA were decreased. Conclusions Our data indi-cates that ILK-siRNA successfully inhibits ILK expression, which regulates the MMP-9/TIMP-1 ratio in HK-2 cells. The inhibition of ILK expression suppresses TGF-β1-induced EMT partially. Key words: Transforming growth factor beta1; Kidney tubules; Epithelial cells; Biotransformation; Integrins; Matrix metalloproteinase 9; Tissue inhibitor of metalloproteinase-1

Advances in effects of integrin-linked kinase on cutaneous wound healing and the relative mechanism

Previous studies have demonstrated that integrin-linked kinases (ILKs) are abundantly expressed in extracellular matrix (ECM) riche dermis, hair follicles, and basal cells of epidermis. ILKs are not only essential for the maintenance of skin structure, but also play important roles in wound healing. ILKs can promote the formation of granulation tissue by stimulating the proliferation of fibroblasts and secretion of ECM, accelerate wound contraction by inducing the differentiation of fibroblasts to myofibroblasts, and boost reepithelization by promoting proliferation, migration, and differentiation of keratinocytes and follicle epidermal stem cells.

Silencing of ILK attenuates the abnormal proliferation and migration of human Tenon's capsule fibroblasts induced by TGF-β2.

The cytokine, transforming growth factor-β (TGF-β), plays a key role in wound healing and tissue repair. Integrin-linked kinase (ILK) is a downstream factor of the TGF-β signaling pathway. Research on ILK has mainly focused on its role in the invasion and metastasis of cancer cells. However, little has been reported on the effects of ILK in human Tenon's capsule fibroblasts (HTFs). In this study, we investigated the role of ILK in the proliferation and migration of HTFs exposed to TGF-β2. A lentiviral vector targeting ILK was screened from three candidates and the experimental result indicated that RNA interference can be used to inhibit ILK expression at both the mRNA and protein level. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess ILK mRNA expression. Cell proliferation was quantified by MTT assay and cell cycle progression was detected by flow cytometric analysis. Migration was measured by wound healing assay. It was observed that the silencing of ILK suppressed the TGF-β2-induced proliferation of HTFs and led to G1 phase cell cycle arrest and the significant downregulation of cyclin D1 expression. The migration ability of the HTFs decreased following the silencing of ILK, while the downregulation of α-smooth muscle actin expression and the upregulation of E-cadherin expression were observed. The findings of our study indicate that the silencing of ILK attenuates the abnormal proliferation and migration of HTFs induced by TGF-β2, which reveals the therapeutic potential of ILK inhibition in the prevention of scarring following glaucoma filtration surgery.

Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6–Abundant Breast Cancer Cells by Regulating γ-Secretase–Mediated Notch1 Activation in Caveolae

Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6–driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6–driven Notch1 activation by ILK in IL-6–producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7IL-6 cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase–mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6–induced breast CSCs.

Abstract A36: Knockdown of integrin-linked kinase reduces invasive and metastatic potential of renal cell carcinoma

Abstract Introduction: Integrin-linked kinase (ILK) is a serine/threonine kinase implicated in the regulation of cell growth and survival, cell cycle progression, epithelial-mesenchymal transition (EMT), invasion and migration, angiogenesis. However, the role of ILK has not been evaluated in renal cell carcinoma (RCC). We investigated the role of ILK on cancer progression and metastasis and therapeutic potential of ILK inhibition in RCC. Methods: Baseline expression of ILK was evaluated by immunofluorescence and western blotting in non-cancerous renal tubular cells (HK-2) and RCC cells (UMRC-6, UMRC-3 and Caki-1). Molecular EMT markers were examined by Western blotting. RNAi using siRNA was used to knock down ILK in vitro. After transient transfection, crystal violet assay and cell cycle analysis using FACS were performed to check the effect of ILK on tumor growth and cell cycle. Confocal microscopic evaluation was performed to check changes of stress fibers and focal adhesions stained with phalloidin-rhodamine and vinculin antibody. Scratch assay and Matrigel invasion assay were performed to evaluate changes of EMT phenotypes after knockdown of ILK. To evaluate the effects of ILK knockdown in vivo, subcataneuous xenografts and orthotopic renal xenografts were used and tumor growth was traced. Results: ILK is less expressed in normal cells (HK-2) and low stage RCC cells (UMRC-6) but highly expressed in advanced and metastatic RCC cells (UMRC-3 and Caki-1). Caki-1, metastatic RCC cells showed higher expressions of molecular EMT markers including Snail, Zeb1 but decreased activity of GSK3β. Knockdown of ILK using si-ILK inhibited tumor proliferation but the inhibition rate was less than 10% and cell cycle progression was not significantly affected by ILK inhibition. However, ILK knockdown suppressed formation of stress fibers and focal adhesions in UMRC-3 and Caki-1 cells and also effectively impeded phenotypic EMT markers including cell migration and invasion in Caki-1 and UMRC-3 cells. In subcutaneous tumor xenografts with Caki-1-shILK or Caki-1-EV, ILK knockdown inhibited tumor growth and progression. In primery orthotopic animal model, ILK knockdown showed inhibitory effects of invasion and metastasis. Conclusions: ILK is highly expressed in advanced RCC and its high expression is related to EMT-related protein in RCC. Knockdown of ILK inhibited molecular EMT markers and suppressed cell migration and invasion in vitro and metastasis in orthotopic tumor model. These results suggest the therapeutic potential of ILK inhibition on invasion and metastasis in advanced RCC. Citation Format: Han Kyung Seok, Raven Peter, Awrey Shannon, Li Estelle, Fazli Ladan, Gleave Martin, So Alan. Knockdown of integrin-linked kinase reduces invasive and metastatic potential of renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A36.

Effect of integrin-linked kinase on the transforming growth factor-β1/Smad3 signaling pathway in hypertrophic scar

Objective To explore the role of integrin-linked kinase (ILK) in the transforming growth factor-β1 (TGF-β1)/Smad3 signal pathway,and the action mechanism of ILK on the differentiation of fibroblasts in hypertrophic scar.Methods The fibroblasts of human hypertrophic scar were isolated and cultured in vitro.The cells were divided into 4 groups:control group,ILK small interfering RNA (siRNA) group,TGF-β1 group,and ILK siRNA + TGF-β1 group.The ILK expression levels were detected by using immunofiuorescence.The mRNA expression levels of ILK,Smad3 and α-smooth muscle actin (α-SMA) were detected by using real-time fluorescent quantitative polyrnerase chain reaction (FQ-PCR).Results The immunofluorescence showed that ILK expression was decreased after transfection of siRNA,and increased again by stimulation with TGF-β1 (5 μg/L).FQ-PCR revealed that the mRNA expression of Smad3 (0.222 ± 0.089) and α-SMA (0.118 ± 0.080) was decreased with ILK (0.522 ± 0.113) after transfection of ILK siRNA,and then the mRNA expression of Smad3 (2.891 ± 0.475) and α-SMA (2.581 ± 0.826) was decreased with ILK (2.233 ± 0.511) increased significantly after stimulation of TGF-β1 as compared with that in control group and ILK siRNA group (P ＜ 0.05).Conclusion The mRNA level of Smad3 and α-SMA could be up-regulated by ILK,which could be stimulated by TGF-β1.ILK may be an important upstream gene for Smad proteins to affect the differentiation of fibroblasts in hypertrophic scar via TGF-β1/Smad3 signal pathway. Key words: Integrin-linked kinase; Cicatrix; Fibroblast ; Transforming growth factor-β1

Study of the effects of integrin-linked kinase on proliferation and differentiation of fibroblast in hypertrophic scar

To study the role of integrin-linked kinase (ILK) on the proliferation and differentiation of human fibroblast in hypertrophic scar and its effect on the scar formation. The human scar fibroblasts were isolated and cultured in vitro. The cells were divided into 4 groups. (1) control group: only contains DMEM; (2) jetPRIME group: DMEM with 200 microl jetPRIME buffer and 4 microl jetPRIME; (3) ILK siRNA group: DMEM and ILK siRNA; (4) ILK cDNA group: DMEM and ILK cDNA. The cell proliferation was detected by XTT assay and the mRNA and protein expressions of ILK and alpha-SMA were detected by Real-time qPCR and Western blot. (1) XTT results showed that the cellular proliferation level after 48 h in four groups were 0.820 +/- 0.065, 0.873 +/- 0.041, 0.554 +/- 0.013 and 1.296 +/- 0.094, respectively. The cellular proliferation curve showed that the cellular proliferation level was very flat in ILK siRNA group while the cellular proliferation level gradually increased from 12 h. 48 h after transfection, the cellular proliferation level in ILK siRNA group was significant lower than those in other groups (P value were 0.021, 0.034, 0), while the cellular proliferation level in ILK cDNA group was the highest among all 4 groups (P value were 0.017, 0.009, 0). (2) The Real-time qPCR showed that the expressions of ILK mRNA and alpha-SMA mRNA were 0.693 +/- 0.412 and 0.422 +/- 0.037 in control group, were 0.621 +/- 0.183 and 0.388 +/- 0.005 in jetPRIME group, were 0.052 +/- 0.019 and 0.073 +/- 0.023 in ILK siRNA group, were 240.193 +/- 35.170 and 138.056 +/- 24.060 in ILK cDNA group. The expressions of ILK mRNA and alpha-SMA mRNA in ILK siRNA group were significantly lower than those in other three groups (P < 0.05). And the expressions of ILK mRNA and alpha-SMA mRNA in ILK cDNA group were significantly higher than those in other three groups (P < 0.05). (3) The Western blot also showed that the expression of ILK and alpha-SMA proteins were decreased in ILK siRNA group and increased in ILK cDNA group. ILK may promote the proliferation and differentiation of human scar fibroblast. It may play an important role in scar formation and contracture.

Effect of integrin-linked kinase on fibroblast populated collagen lattice contraction

Objective To investigate the influences of integrin-linked kinase (ILK) on fibroblast populated collagen lattice (FPCL) contraction and the mechanism.Methods The human skin fibroblasts from plastic patients were used to construct FPCLs.The effects of ILK-AKT inhibitor LY294002 on FPCL contraction were observed.The expressions of ILK and α-SMA in fibroblasts was detected by using Western blotting after ILK siRNA transfection or LY294002 treatment.Results FPCLs in each group had various extents of contraction,but collagen gels without fibroblasts had no contraction.The contraction rate of FPCLs in LY294002 group at 24 and 48 h [ ( 20.23 ± 9.57 ) ％,(22.47 ± 10.93 ) ％ ] was significantly lower than that in control group [ (48.73 ±6.60)％,(54.33 ± 12.88)％ ] (P ＜0.05).It was also significantly lower than that in control group and DMSO group at 72,96 and 120 h (P ＜0.05).The expression of α-SMA in ILK siRNA transfected group (0.992 ±0.255) and LY294002 group ( 1.225 ±0.323)was significantly lower than other groups (2.009 ± 0.820,2.190 ± 0.577,1.758 ± 0.732 ) ( P ＜ 0.05 ).Conclusion The contraction of FPCL and expression of α-SMA in skin fibroblasts can be inhibited via blocking ILK pathway. Key words: Integrin-linked kinase; Wound; Contraction; α-SMA

Inhibition of high glucose-induced VEGF and ICAM-1 expression in human retinal pigment epithelium cells by targeting ILK with small interference RNA

The aim of this study was to investigate the change of Integrin-linked kinase (ILK) expression of human retinal pigment epithelium (RPE) cells in response to high glucose, and the effect of targeting ILK with small interference RNA (siRNA) on the high glucose-induced expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1). The ILK mRNA and protein expression in human RPE cells were analyzed with RT-PCR and western blot after exposure to 5.5, 30, 40, 50 mM glucose, or 5.5 mM glucose+45.5 mM mannitol for 48 h. The expression of VEGF and ICAM-1 was also determined. Cells were treated with ILK siRNA, to determine the effect of ILK on VEGF and ICAM-1 expression following treatment with high glucose. High concentrations of glucose significantly up-regulated ILK mRNA and protein expression, and the ILK expression increased along with the glucose concentration. The changes of VEGF and ICAM-1 expression were similar to that of ILK expression. Knocking down ILK gene expression with siRNA inhibited the elevation of VEGF and ICAM-1 induced by high glucose treatment. These results suggested that ILK was involved in the response of RPE cells to high glucose and may therefore play a role in the pathogenesis of diabetic ophthalmology.

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.

Effects of Integrin-Linked Kinase (ILK) Inhibitor QLT0267 on Multiple Myeloma Cells and Evaluation of ILK as a Therapeutic Target in Multiple Myeloma.

Multiple myeloma (MM) is a fatal plasma cell tumor that accounts for about 1% of cancers. A hallmark of the disease is its location in the bone marrow where the tumor cells receive prosurvival support from the microenvironment and cause extensive osteolytic damage. Novel drugs are currently being developed into a range of new treatment options. However, because the problems of cancer relapse and eventual selection of therapy-resistant offspring remain, additional therapeutic targets should still be investigated. ILK is a multifunctional protein that, as an adaptor and/or as a kinase, may relay adhesion- and growth factor receptor-mediated signals to downstream signaling cascades that promote growth and survival. We have analysed the expression of ILK in MM cells and have tested the effects of a novel small molecule ILK-inhibitor (QLT0267; QLT Inc., Vancouver, Canada) in MM cell lines, primary MM tumor cells and healthy cells, respectively. ILK expression at either cDNA or protein level was detectable in virtually every MM sample tested. Treatment with QLT0267 for up to 3 days resulted in extensive apoptotic death in MM cell lines (EC50 values below 10 microM in 8/9 MM cell lines tested) and in a majority of primary (anti-CD138-purified) MM samples (EC50 values below 10 microM in 8/14 primary MM samples tested). Drug treatment led to rapid decreases in the levels of phospho-STAT3, phospho-GSK3beta and total Akt protein, whereas levels of ILK and of phospho-ERK were unaffected or, in the latter case, showed a slight increase. Similar to other current pharmacologic approaches, targeting ILK may have several detrimental impacts on the signaling network that sustains MM cells. Such pleiotropic effects could prove valuable for combination treatments. The survival of peripheral blood mononuclear cells and of bone marrow stromal cells (BMSCs) at 20 microM QLT0267 was just slightly affected, indicating that the scope for establishment of a therapeutic window in MM might exist. High (20 microM) concentrations of QLT0267 gradually (and reversibly) promoted detachment of BMSCs from the culture dish, indicating that the drug might be useful to temporarily impair their effectiveness to support myeloma cells. Taken together, these experiments provide a rationale to further explore the utility of ILK-inhibition for the treatment of MM.

Pharmacological inhibition of EGFR tyrosine kinase affects ILK-mediated cellular radiosensitization in vitro

Purpose: Integrin-linked kinase (ILK) mediates signals from β integrins and links integrins to epidermal growth factor receptor (EGFR). Previous studies have identified an antisurvival effect of ILK in irradiated cells. The aim of this study was to evaluate the role of EGFR tyrosine kinase (tk) activity for ILK-mediated radiosensitization.Materials and methods: Human FaDu squamous cell carcinoma (SCC) cells stably transfected with hyperactive ILK (ILK-hk) and ILKfl/fl and ILK−/− mouse fibroblasts were treated with the pharmacological EGFR-tk inhibitor BIBX1382BS without or in combination with single doses of X-rays. Clonogenic radiation survival, protein expression and phosphorylation (EGFR, v-akt murine thymoma viral oncogene homolog 1 (Akt), p42/44 mitogen-activated protein kinase), DNA-double strand break (DSB) repair measured by γH2AX foci, cell morphology and cell cycle distribution were examined.Results: Expression of ILK-hk or ILKfl/fl status resulted in significant radiosensitization relative to vector controls or ILK−/−. Following BIBX1382BS, clonogenic survival of normal fibroblasts and vector controls remained unaffected while ILK-hk-related radiosensitization was significantly diminished. In contrast to BIBX1382BS, which did not affect DNA-DSB repair, ILK-hk-mediated radiosensitization was associated with reduced DNA-DSB repair. At 10 days after BIBX1382BS treatment, FaDu transfectants, in contrast to fibroblasts, showed reduced cell size, accumulation of G1 phase cells and reduced Akt-serine(S)473 phosphorylation.Conclusions: Our findings confirm ILK as a cell type-independent antisurvival factor in irradiated cells, which actions in terms of radiosensitization critically depend on proper EGFR-tk activity.

Overexpression of integrin-linked kinase induces cardiac stem cell expansion

Recent evidence suggests that the adult heart contains stem cells that are capable of self-renewal as well as multilineage differentiation. However, their inherent capacity for self-renewal is limiting to cell replacement applications. Integrin-linked kinase is a multifunctional protein kinase that activates Wnt target genes implicated in the symmetric replication of embryonic stem cells. Primary cultures derived from human fetal cardiac tissue (19-22 weeks' gestation) were grown in serum-free media and evaluated for the presence of cardiac progenitor cells. The effect of integrin-linked kinase was ascertained by adenoviral overexpression. Cultures infected with wild-type integrin-linked kinase yielded a significant (P = .001), approximately 5-fold increase in both the absolute number and the frequency of c-Kit-positive, myosin-negative cells. Cardiospheres, comprised on morphologically homogeneous, anchorage-independent cells, were reproducibly present at days 7 to 10 and formed derivative cardiospheres in multiple passages. Integrin-linked kinase infection of primary cardiac cell cultures resulted in a greater number of primary spheres at each cell density tested, compared with untreated and virus controls (P = .001). Secondary spheres transferred to differentiation medium and 5-aza-deoxycytodine (10 micromol/L) generated cells exhibiting biochemical evidence of differentiation into cardiomyocytes, smooth muscle cells, and endothelial cells. This study demonstrates that self-renewing cardiospheres generated from human fetal cardiac cells are composed of cells exhibiting the properties of stem cells, including the capacity for self-renewal and multilineage differentiation. Our results suggest that integrin-linked kinase promotes stem cell amplification and can be applied therapeutically to overcome a major limitation in the field of cardiac regenerative medicine.

Cell adhesion: Linking Fibronectin Interaction to Transcription

Integrin-mediated cell adhesion is known to regulate gene expression through the activation of the transcription factor AP-1. Troussard et al. show that this event does indeed occur when cells interact with the extracellular matrix component fibronectin (FN). Based on earlier evidence that FN stimulates integrin-linked kinase (ILK) and the known inhibitory effect of ILK on glycogen synthase kinase 3 (GSK-3), the authors determined that in transfected cells, the regulation of AP-1 by FN is mediated through stimulation of ILK and the subsequent inhibition of GSK-3. Although ILK is known to activate protein kinase B (PKB/AKT), this apparently was not involved in the stimulation of AP-1 by FN. The authors propose that inhibition of GSK-3 might prevent phosphorylation of the DNA-binding domain of c-jun, a central component of all AP-1 protein complexes. This would then promote binding to DNA and stimulation of AP-1 activity. Troussard, A.A., Tan, C., Yoganathan, N., and Dedhar, S. (1999) Cell-extracellular matrix interactions stimulate the AP-1 transcription factor in an integrin-linked kinase- and glycogen synthase kinase 3-dependent manner. Mol. Cell. Biol. 19 : 7420-7427. [Online Journal]