Integrin-linked kinase links dynactin-1/dynactin-2 with cortical integrin receptors to orient the mitotic spindle relative to the substratum.

Cells must divide strictly along a plane to form an epithelial layer parallel to the basal lamina. The axis of cell division is primarily governed by the orientation of the mitotic spindle and spindle misorientation pathways have been implicated in cancer initiation. While ß1-Integrin and the Dynein/Dynactin complex are known to be involved, the pathways linking these complexes in positioning mitotic spindles relative to the basal cortex and extracellular matrix remain to be elucidated. Here, we show that Integrin-Linked Kinase (ILK) and α-Parvin regulate mitotic spindle orientation by linking Dynactin-1 and Dynactin-2 subunits of the Dynein/Dynactin complex to Integrin receptors at the basal cortex of mitotic cells. ILK and α-Parvin are required for spindle orientation. ILK interacts with Dynactin-1 and Dynactin-2 and ILK siRNA attenuates Dynactin-2 localization to the basal cortex. Furthermore we show that Dynactin-2 can no longer colocalize or interact with Integrins when ILK is absent, suggesting mechanistically that ILK is acting as a linking protein. Finally we demonstrate that spindle orientation and cell proliferation are disrupted in intestinal epithelial cells in vivo using tissue-specific ILK knockout mice. These data demonstrate that ILK is a linker between Integrin receptors and the Dynactin complex to regulate mitotic spindle orientation.

Regulation of mitotic cytoskeleton dynamics and cytokinesis by integrin-linked kinase in retinoblastoma cells.

During cell division integrin-linked kinase (ILK) has been shown to regulate microtubule dynamics and centrosome clustering, processes involved in cell cycle progression, and malignant transformation. In this study, we examine the effects of downregulating ILK on mitotic function in human retinoblastoma cell lines. These retinal cancer cells, caused by the loss of function of two gene alleles (Rb1) that encode the retinoblastoma tumour suppressor, have elevated expression of ILK. Here we show that inhibition of ILK activity results in a concentration-dependent increase in nuclear area and multinucleated cells. Moreover, inhibition of ILK activity and expression increased the accumulation of multinucleated cells over time. In these cells, aberrant cytokinesis and karyokinesis correlate with altered mitotic spindle organization, decreased levels of cortical F-actin and centrosome de-clustering. Centrosome de-clustering, induced by ILK siRNA, was rescued in FLAG-ILK expressing Y79 cells as compared to those expressing FLAG-tag alone. Inhibition of ILK increased the proportion of cells exhibiting mitotic spindles and caused a significant G2/M arrest as early as 24 hours after exposure to QLT-0267. Live cell analysis indicate ILK downregulation causes an increase in multipolar anaphases and failed cytokinesis (bipolar and multipolar) of viable cells. These studies extend those indicating a critical function for ILK in mitotic cytoskeletal organization and describe a novel role for ILK in cytokinesis of Rb deficient cells.

Requirement of epithelial integrin-linked kinase for facilitation of Citrobacter rodentium-induced colitis.

BACKGROUND: Integrin-linked kinase (ILK) is a serine-threonine kinase that transduces extracellular matrix-related cues into intracellular signals, with fundamental roles in cell motility, development and cancer. Recently ILK been shown to have an important role in bacterial epithelial cell attachment, through ILK-bacterial OspE binding. Here we report on the role of epithelial derived ILK in response to Citrobacter rodentium infection. METHODS: C. rodentium was administered to both control and intestinal epithelial cell ILK knockout mice. Histological inflammatory scores were assessed, and cytokines measured by ELISA as well as RT-PCR, in mouse colons. Bacterial colonization was determined by plating homogenates onto MacConkey agar, and immunofluorescence microscopy performed using anti-LPS and anti-Tir antibodies. RESULTS: ILK-ko mice exhibited reduced weight loss at 15 days post-infection (p < 0.01) and demonstrated reduced histological inflammatory scores (p < 0.01), reduced CCL2 and pro-inflammatory cytokines. This was not due to reduced colonization, but was associated with an altered pattern of C. rodentium bacterial migration. Attenuated fibronectin expression was found in the ILK-ko mice. C. rodentium exposure was shown to increase ILK expression in cell lines, and in murine epithelium in vivo. In ILK-ko mice reduced activation of ser473Akt and reduced crypt proliferation, together with reduced cyclin D1 expression were observed. CONCLUSIONS: ILK influences the host response to C. rodentium -induced infection, independently of reduced colonization in the ILK knockout mice. The reduced inflammation and dramatically attenuated hyperplastic cryptal response to infection in this group, are at least in part the result of, the reduction in CCL2 and cyclin D1 expression respectively.

The importance of integrin-linked kinase in the regulation of bladder cancer invasion.

It is important to understand the molecular mechanisms of bladder cancer progression not only to prevent cancer progression but also to detect new therapeutic targets against advanced bladder cancer. The integrin-linked kinase (ILK) is a major signaling integrator in mammalian cells and plays an important role in epithelial-mesenchymal transition (EMT) of human cancers, but its mechanisms are not completely understood. In this study, we investigated the importance and mechanisms of ILK in bladder cancer progression. When the expression of ILK in bladder cancer cell lines and N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced murine bladder cancer was evaluated, ILK has a tendency to be overexpressed in invasive cell lines and invasive BBN-induced murine bladder cancer. Overexpression of ILK in 253J bladder cancer cells suppressed E-cadherin expression, resulting in the promotion of cell invasion. Conversely, ILK knockdown by siRNA suppresses cell invasion in invasive bladder cancer cells through the regulation of E-cadherin or matrix metalloprotease 9 (MMP-9). To regulate E-cadherin expression, our results showed that the glycogen synthase kinase 3ß (GSK3ß)-Zeb1 pathway may play an important role downstream of ILK. Finally, the results of a human bladder tissue microarray (TMA) showed that ILK expression correlates with the invasiveness of human bladder cancer. Our study suggests that ILK is overexpressed in invasive bladder cancer and plays an important role in the EMT of bladder cancer via the control of E-cadherin and MMP-9 expression. ILK may be a new molecular target to suppress tumor progression in advanced and high-risk bladder cancer patients.

Role of epithelial integrin-linked kinase in promoting intestinal inflammation: effects on CCL2, fibronectin and the T cell repertoire.

BACKGROUND: The role of integrin signaling in mucosal inflammation is presently unknown. Hence, we aimed to investigate the role of epithelial-derived integrin-linked kinase (ILK), a critical integrin signaling intermediary molecule, in colonic inflammation. METHODS: Conditional intestinal epithelial cell ILK knockout mice were used for assessment of acute and chronic dextran sodium sulfate (DSS)-induced colitis. Disease activity was scored using standard histological scoring, mucosal cytokines were measured using ELISA, chemokines were determined using reverse-transcription polymerase chain reaction, as well as Q-PCR, and intracellular cytokine staining performed using FACS analysis. RESULTS: In both acute and chronic DSS-induced colitis, compared to wild-type mice, ILK-ko mice exhibit less weight loss, and have reduced inflammatory scores. In an in vitro model system using HCT116 cells, we demonstrate that si-RNA mediated down-regulation of ILK results in a reduction in monocyte chemoattractant protein 1 (MCP1, CCL2) chemokine expression. A reduction in CCL2 levels is also observed in the tissue lysates of chronically inflamed colons from ILK-ko mice. Examination of mesenteric lymph node lymphocytes from ILK-ko mice reveals that there is a reduction in the levels of IFN gamma using intracellular staining, together with an increase in Foxp3+ T regulatory cells. Immunohistochemistry demonstrates that reduced fibronectin expression characterizes the inflammatory lesions within the colons of ILK-ko mice. Intriguingly, we demonstrate that fibronectin is directly capable of downregulating T regulatory cell development. CONCLUSIONS: Collectively, the data indicate for the first time that ILK plays a pro-inflammatory role in intestinal inflammation, through effects on chemokine expression, the extracellular matrix and immune tolerance.

Cutting edge: PHLPP regulates the development, function, and molecular signaling pathways of regulatory T cells.

Regulatory T cells (Tregs) have a reduced capacity to activate the PI3K/Akt pathway downstream of the TCR, and the resulting low activity of Akt is necessary for their development and function. The molecular basis for the failure of Tregs to activate Akt efficiently, however, remains unknown. We show that PH-domain leucine-rich-repeat protein phosphatase (PHLPP), which dephosphorylates Akt, is upregulated in Tregs, thus suppressing Akt activation. Tregs expressed higher levels of PHLPP than those of conventional T cells, and knockdown of PHLPP1 restored TCR-mediated activation of Akt in Tregs. Consistent with their high Akt activity, the suppressive capacity of Tregs from PHLPP1(-/-) mice was significantly reduced. Moreover, the development of induced Tregs was impaired in PHLPP1(-/-) mice. The increased level of Akt's negative regulator, PHLPP, provides a novel mechanism used by T cells to control the Akt pathway and the first evidence, to our knowledge, for a molecular mechanism underlying the functionally essential reduction of Akt activity in Tregs.

The single IgG IL-1-related receptor controls TLR responses in differentiated human intestinal epithelial cells.

Intestinal epithelial cells (IECs) are constantly exposed to enteric microbes. Although IECs express TLRs that recognize bacterial products, the activation of these TLRs is strictly controlled through poorly understood mechanisms, producing a state of hyporesponsiveness and preventing unwanted inflammation. The single IgG IL-1-related receptor (Sigirr) is a negative regulator of TLRs that is expressed by IECs and was recently shown to inhibit experimental colitis. However, the importance of Sigirr in IEC hyporesponsiveness and its distribution within the human colon is unknown. In this study, we investigated the role of Sigirr in regulating epithelial-specific TLR responses and characterized its expression in colonic biopsy specimens. Transformed and nontransformed human IECs were cultured as monolayers. Transient gene silencing and stable overexpression of Sigirr was performed to assess innate IEC responses. Sigirr expression in human colonic biopsy specimens was examined by immunohistochemistry. Bacterial infection of IECs and exposure to flagellin transiently decreased Sigirr protein expression, concurrent with secretion of the neutrophil chemokine IL-8. Sigirr gene silencing augmented chemokine responses to bacterial flagellin, Pam3Cys, and the cytokine IL-1beta. Conversely, stable overexpression of Sigirr diminished NF-kappaB-mediated IL-8 responses to TLR ligands. We also found that Sigirr expression increased as IECs differentiated in culture. This observation was confirmed in biopsy sections, in which Sigirr expression within colonic crypts was prominent in IECs at the apex and diminished at the base. Our findings show that Sigirr broadly regulates innate responses in differentiated human IECs; therefore, it may modulate epithelial involvement in infectious and inflammatory bowel diseases.

Tumor expression of integrin-linked kinase (ILK) correlates with the expression of the E-cadherin repressor snail: an immunohistochemical study in ductal pancreatic adenocarcinoma.

Integrin-linked kinase (ILK) is a key molecule involved in mediating several biological functions including cell-matrix interactions, angiogenesis, and invasion, as well as playing a role in epithelial to mesenchymal transition (EMT) in cancer cells. In ductal pancreatic adenocarcinoma, increased expression of ILK has been linked to tumor prognosis and correlated with increased chemoresistance to drugs, such as gemcitabine. However, the precise relationship between ILK, Snail, E-cadherin, and N-cadherin expression on the stepwise development of pancreatic cancer is unknown. Hence, the purpose of this work was to investigate levels of expression of ILK, Snail, and the cadherins in pancreatic intraepithelial neoplasia (PanIN), and cancer. Resection specimens of 25 randomly selected patients, who underwent a pyloric preserving pancreatoduodenectomy for ductal pancreatic adenocarcinoma, were utilized for this study. Formalin-fixed paraffin embedded pancreatic tissue was immunostained for ILK, E-cadherin, N-cadherin, and Snail by standard techniques. The extent of staining positivity was scored and the results correlated with clinicopathological parameters. In 23 of 25 cases, ILK expression showed extensive positivity (>50%), while two cases did not demonstrate any ILK staining. PanIN grades 1 (n = 16), 2 (n = 11), and 3 (n = 19) lesions demonstrated only focal positivity (<10%) for ILK. E-cadherin showed a reciprocal staining pattern to ILK in 21 of 25 cases, with only focal expression of the marker in pancreatic adenocarcinoma. Interestingly, 15 of 19 PanIN-3 lesions expressed extensive E-cadherin staining. N-cadherin, however, was moderately expressed in the majority of cases (n = 18). Snail expression (n = 22) correlated with ILK expression in ductal pancreatic adenocarcinoma (rho = 0.8168, p = 0.02), but only minimal Snail staining activity was detected in PanIN lesions. The increase in expression of the E-cadherin repressor Snail, as well as the related increase in the ILK expression, may point towards an ILK-mediated induction, opening possible avenues for targeted drug therapy.

Investigation of interleukin 1beta-mediated regulation of NF-kappaB activation in colonic cells reveals divergence between PKB and PDK-transduced events.

Recent work has highlighted a role for PDK1 in adaptive immunity, however its contribution to innate immunity has not been addressed. We have investigated the role of PKB and PDK1 in IL-1beta-induced NF-kappaB activation. Over-expression of either in HCT 116 and HEK 293T cells, effected a reproducible NF-kappaB activation. This was validated in a one-hybrid assay utilizing Gal4-RelA and Gal4-luciferase assay. N-tosyl phenylalanyl chloromethyl ketone (TPCK), wortmannin and Ly294002 inhibited IL-1beta-induced NF-kappaB activation in both systems indicating involvement of the PI3K axis in this response. p65 (Rel A) Ser536 phosphorylation was not affected by the PI3K inhibitors but was dose-dependently attenuated by TPCK. Evaluation of IKK-associated activity using GST-p65 substrate phosphorylation in immune complex assays, revealed that whilst TPCK attenuated this, neither of the PI3K inhibitors had any effect. Furthermore whilst TPCK inhibited IL-1beta-induced p65 DNA binding, this was not apparent with either of wortmannin or Ly294002. Similarly, over-expression of PDK1 but not PKB resulted in promotion of p65 DNA binding. Using a p65-S536A reporter construct, we found inhibition of only PDK1 over-expression-induced, but not PKB over-expression-induced NF-kappaB activation. This was supported using biochemical analysis in which immunoprecipitated IKKgamma from IL-1beta-activated cells was unable to phosphorylate a p65-S536A substrate, confirming this as the dominant IKK-dependent site. In further support of a dissociated response, we observed an attenuation of the Ser177/181 IKK phosphorylation by TPCK but not in response to PI3K inhibition. Our data reveals for the first time that PDK1 and PKB may differentially activate NF-kappaB, and that TPCK may subserve a useful anti-inflammatory function by inhibiting IKKbeta.

Expression of integrin-linked kinase is not a useful prognostic marker in resected hepatocellular cancer.

BACKGROUND: Hepatocellular cancer (HCC) is one of the most common malignancies worldwide, and is known to be associated with a poor prognosis. Unfortunately there are no available reliable markers of prognosis. The aim of this study was to determine whether integrin-linked kinase (ILK) expression correlates with post-resection survival from HCC. PATIENTS AND METHODS: A tissue microarray was constructed using HCC samples, and immunohistochemical analysis for ILK was then carried out and scored by three independent observers. Clinical chart review was performed to determine survival parameters. RESULTS: Of the 52 cases of HCC, 22 cases were associated with hepatitis B (HBV), 18 with hepatitis C (HCV), 2 with HBV and HCV co-infection; 81% of all patients were male and 19% female. Western immunoblotting showed a highly significant correlation between levels of expression of ILK and ser473-PKBphosphorylation, both in control and tumor sections (Spearman rank correlation, r=0.8155, p=0.0004), however, there was no direct correlation between the levels of expression of ILK with patient survival (log-rank test, p= 0.864). CONCLUSION: ILK expression does not appear to have a role in predicting outcome in patients with resected HCC.

The specific JNK inhibitor SP600125 targets tumour necrosis factor-alpha production and epithelial cell apoptosis in acute murine colitis.

Stress-activated protein kinases (SAPKs) are activated in human inflammatory bowel disease (IBD). Recently it has been demonstrated that p38MAPK (mitogen-activated protein kinase) inhibition using SB203580 is effective in reducing disease in both dextran sulphate sodium (DSS)-induced and 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced murine colitides, underscoring the importance of this pathway in gastrointestinal inflammation. However, the contribution of c-Jun N-terminal kinase (JNK) in intestinal inflammation is unknown. Based on the known involvement of JNK in tumour necrosis factor-alpha (TNF-alpha) expression and in mediating the effects of oxidant stress, we hypothesized that JNK inhibition would also affect colitis. Our studies in mice with DSS-induced colitis treated with the JNK inhibitor SP600125, indicate that there is a significant reduction in wasting as well as a significant reduction in histological damage scores. Both total colonic and mesenteric lymphocyte CD3/CD28-stimulated TNF-alpha levels were dramatically reduced under the same circumstances. This was associated with a reduction in JNK protein expression and activity, as well as a reduction in AP-1 DNA binding with SP600125. Interestingly, there were no apparent changes in either p38MAPK or p42/44ERKs. Immunofluorescence of the colon for the active form of JNK revealed a prominent signal arising from the infiltrating inflammatory cells. SP600125 reduced this as well as, specifically, macrophage infiltration. Strikingly, we also demonstrate reduced epithelial cell apoptosis in response to treatment with SP600125. We conclude that specific inhibition of JNK is beneficial in the DSS model of colitis, and may be of value in human IBD.

Curcumin mediates ceramide generation via the de novo pathway in colon cancer cells.

A wealth of evidence supports the notion that curcumin, a phytochemical present in turmeric, is a potent chemopreventive agent for colon cancer. Its mechanism of action remains incompletely understood. Here we report that curcumin's apoptosis-inducing effects in colon cancer cell lines are accompanied by robust ceramide generation. This occurs through de novo synthesis as the increase in ceramide could be attenuated by pre-incubation of the cells with myriocin, and no changes were observed in sphingomyelin levels, or in either acidic or neutral sphingomyelinase activities. Furthermore, cell death could in part be reversed by myriocin, indicating, for the first time, that endogenous ceramide generation by this agent contributes towards its biological activity. We then investigated the role of reactive oxygen species (ROS) in this phenomenon and demonstrated that curcumin induced robust oxidant generation in the cell lines tested, and its reversal by N-acetylcysteine, completely attenuated apoptosis. We next confirmed that curcumin could activate c-jun N-terminal kinase (JNK) and that its modulation could reverse cell death; however, this intervention could not block ceramide generation, or ROS production. Conversely, however, the inhibition of ROS using N-acetylcysteine led to an inhibition of JNK activation. Hence, we conclude that curcumin induces apoptosis via a ROS-associated mechanism that converges on JNK activation, and to a lesser extent via a parallel ceramide-associated pathway.