Biomedical subjects

S Dedhar

Publications and source records attributed to S Dedhar.

At least 19 recordsLinked to original sources

PubMed2006

Modulation of Wnt3a-mediated nuclear beta-catenin accumulation and activation by integrin-linked kinase in mammalian cells.

The Wnt gene family encodes secreted signaling molecules that play important roles in tumorgenesis and embryogenesis. The canonical Wnt signaling pathway regulates target gene expression via the stabilization and nuclear translocation of the cytoplasmic pool of beta-catenin. The activation of integrin-linked kinase (ILK) is also known to regulate the stabilization and subsequent nuclear translocation of beta-catenin in several epithelial cell models. We now report that molecular and pharmacological inhibition of ILK activity in mammalian cells directly modulates Wnt signaling by suppressing the stabilization and nuclear translocation of beta-catenin, as well as beta-catenin/Lef-mediated transcription. Inhibition of ILK activity, but not phosphatidylinositol-3 kinase (PI3K) or MEK activities suppresses nuclear beta-catenin stabilization in cells stably expressing Wnt3a as well as in cells exposed to either Wnt3a conditioned media or purified Wnt3a. Furthermore, ILK inhibition reverses the Wnt3a-induced suppression of beta-catenin phosphorylation that accompanies beta-catenin stabilization. In addition, we show that ILK can be identified in a complex with Wnt pathway components such as adenomatous polyposis coli and GSK-3. Upon treatment of L cells with Wnt3a-CM, glycogen synthase kinase-3 (GSK-3beta) becomes highly phosphorylated on Ser 9, which is completely abolished upon inhibition of ILK activity. However, acute exposure of L cells to purified Wnt3a does not result in the stimulation of GSK-3beta Ser 9 phosphorylation, despite beta-catenin stabilization. Together our data demonstrate that ILK activity can modulate acute Wnt3a mediated beta-catenin phosphorylation, stabilization and nuclear activation in a PI3K-independent manner, as well as the more prolonged PI3K-dependent secondary effects of Wnt signaling on GSK-3 phosphorylation. Finally, we suggest that a novel small molecule inhibitor of ILK, QLT-0267, may be a useful tool in the regulation of pathological Wnt signaling.

Animals↗

PubMed2006

PTEN and GSK3beta: key regulators of progression to androgen-independent prostate cancer.

Prostate cancer (PrCa) is characterized by progression from an androgen-dependent phenotype to one that is inevitably androgen independent (AI) and lethal. Recent evidence strongly suggests that the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) and androgen receptor (AR) signalling pathways provide prostatic epithelium with the necessary signalling events to escape the apoptotic response associated with androgen withdrawal therapy. Silencing of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and glycogen synthase kinase beta (GSK3beta) are frequently associated with advanced PrCa systems and likely serve critical roles in promoting AR and PI3K/Akt gain-of-function. That PTEN negatively regulates AR and is sufficient to promote metastatic PrCa in murine models strongly implies its role as a gatekeeper of progressive PrCa. In human PrCa, PTEN loss is correlated with substantial increases in Akt(Ser473) and integrin-linked kinase expression, both of which promote Ser(9) phospho-inhibition of GSK3beta and inactivation of apoptotic factors. Sufficient evidence also suggests that GSK3beta is not only a critical regulator of proproliferative signalling but also a promiscuous one as PI3K/Akt pools of GSK3beta are, at least in part, functionally interchangeable with those of the Wnt/beta-catenin pathway. Thus, GSK3beta may serve not only as a mediator of PI3K/Akt activation but may also regulate the potent transactivation and proproliferative effects that Wnt3a and beta-catenin confer upon AR. These data suggest that prostate-specific activation of GSK3beta may serve as a viable pharmacological option. Thus, in this review, we emphasize that temporal changes in GSK3beta and PTEN expression during progression to AI PrCa are important factors when considering the potential for therapies targeting the oncogenic contributions of PI3K/Akt and AR signalling pathways.

Androgens↗

PubMed2006

Development and assessment of conventional and targeted drug combinations for use in the treatment of aggressive breast cancers.

Combination chemotherapy has been at the forefront of cancer treatment for over 40 years. However, the rationale for selecting drug combinations and the process used to demonstrate clinical effectiveness has primarily followed trial and error methodology. Typically, the selection and assessment of combined drug therapies has been based on the effectiveness of each agent as monotherapy in treating the neoplasm and avoiding overlapping toxicities, followed by clinical trials to establish dose scheduling, toxicity, and efficacy. Unfortunately, this scheme is inefficient in terms of the time required to complete and revise these clinical trials based on the outcome to optimize the drug combination. A more rational approach for the development of combination oncology products should consider (i) in vitro assays for assessing therapeutic effects of drug combinations (antagonistic, additive or synergistic interactions) when added simultaneously; (ii) methods for measuring these interactions in vivo; (iii) the importance of understanding pharmacokinetic and biodistribution parameters when using drug combinations; (iv) the need to assess pathways known to contribute to cancer cell survival as well as metastasis; and (iv) the need to assess the fate of different cell populations (cancer and stroma) contributing to the development of cancer. Therefore, the goal of this article is to provide a road map for the preclinical development of drug combination products that will have improved therapeutic activity and a high likelihood of providing beneficial therapeutic outcomes in patients with aggressive cancers with a specific focus on patients with breast cancer.

Animals↗

PubMed2003

Characterisation of integrin-linked kinase signalling in sporadic human colon cancer.

The putative oncogene, integrin-linked kinase (ILK) is a protein serine/threonine kinase that has been reported to regulate a number of biological properties including anchorage-independent cell cycle progression, tumour cell invasion and apoptosis. Overexpression of ILK has been documented in a wide variety of human malignancies including Ewing's sarcoma (ES), primitive neural ectodermal tumours (PNETs) and prostate tumours (PT). We recently reported that ILK signalling was also dysregulated in patients with the genetic condition familial adenomatous polyposis (FAP), a precursor to colon cancer. In this study, we extended our previous work by investigating the ILK-signalling pathway in sporadic human colon cancer and representative lymph node metastases. The data indicate that the ILK protein is significantly hyperexpressed in malignant acini in relation to normal crypts. Moreover, overexpression of ILK not only coincided with increased MBP phosphotransferase activity but as well with effects on downstream targets like GSK3beta. Based upon the presented data, we propose that ILK signalling is dysregulated early during the development of human colon cancer, and that selective inhibition of this molecule alone or in combination with the standard therapeutic modality might be a more effective means of treating colon cancer.

Colon↗

PubMed2001

Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes.

How intracellular cytoskeletal and signaling proteins connect and communicate with the extracellular matrix (ECM) is a fundamental question in cell biology. Recent biochemical, cell biological, and genetic studies have revealed important roles of cytoplasmic integrin-linked kinase (ILK) and its interactive proteins in these processes. Cell adhesion to ECM is an important process that controls cell shape change, migration, proliferation, survival, and differentiation. Upon adhesion to ECM, integrins and a selective group of cytoskeletal and signaling proteins are recruited to cell matrix contact sites where they link the actin cytoskeleton to the ECM and mediate signal transduction between the intracellular and extracellular compartments. In this review, we discuss the molecular activities and cellular functions of ILK, a protein that is emerging as a key component of the cell-ECM adhesion structures.

Actin Cytoskeleton↗

PubMed2001

Mammary epithelial-specific expression of the integrin-linked kinase (ILK) results in the induction of mammary gland hyperplasias and tumors in transgenic mice.

The integrin linked kinase (ILK) is a cytoplasmic effector of integrin receptors, involved in the regulation of integrin binding properties as well as the activation of cell survival and proliferative pathways, including those involving MAP kinase, PKB/Akt and GSK-3beta. Overexpression of ILK in cultured intestinal and mammary epithelial cells has been previously shown to induce changes characteristic of oncogenic transformation, including anchorage-independent growth, invasiveness, suppression of anoikis and tumorigenicity in nude mice. In order to determine if ILK overexpression can result in the formation of mammary tumors in vivo, we generated transgenic mice expressing ILK in the mammary epithelium, under the transcriptional control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). By the age of 6 months, female MMTV/ILK mice developed a hyperplastic mammary phenotype, which was accompanied by the constitutive phosphorylation of PKB/Akt, GSK-3beta and MAP kinase. Focal mammary tumors subsequently appeared in 34% of the animals at an average age of 18 months. Given the focal nature and long latency of the tumors, however, additional genetic events are likely required for tumor induction in the MMTV/ILK mice. These results provide the first direct demonstration of a potential oncogenic role for ILK, which is upregulated in human tumors and tumor cell lines.

Adenocarcinoma, Papillary↗

PubMed2001

Dysregulation of integrin-linked kinase (ILK) signaling in colonic polyposis.

Mutation of the adenomatous polyposis coli (APC) gene and the subsequent dysregulation of beta-catenin are well-documented abnormalities in familial adenomatous polyposis (FAP), as well as sporadic polyposis. Intriguingly, overexpression of the integrin-linked kinase (ILK) has been shown to modulate beta-catenin subcellular localization and function. However, the significance of this finding for human carcinogenesis remains unclear. Here, we report the increased biochemical activity and expression of ILK protein in polyps from FAP patients. Furthermore, dramatic increases in ILK immunoreactivity were observed in all abnormal crypts from sporadic polyps, when compared with the normal appearing crypts within the same resected specimens. As sulindac and aspirin are the two most important therapeutic/chemopreventative agents demonstrated in colorectal carcinogenesis, in both humans and animals, further investigation revealed that these non-steroidal anti-inflammatory drugs (NSAIDs) target ILK and ILK-mediated events in vivo. These include inhibition of, both the biochemical activation of ILK, inhibition of serine 9 GSK3beta phosphorylation and the enhancement of TCF-4 transcriptional activity. In conclusion, ILK protein hyperexpression appears to be an early event in colonic polyposis. Additionally, ILK signaling is shown to undergo modulation by sulindac (and aspirin) for the first time, indicating that it is likely to be one of the targets affected by these agents in vivo.

Adenomatous Polyposis Coli↗

PubMed2001

Inhibition of tumor cell invasion and angiogenesis by motuporamines.

Tissue invasion is an important determinant of angiogenesis and metastasis and constitutes an attractive target for cancer therapy. We have developed an assay to identify agents that inhibit invasion by mechanisms other than inhibition of cell attachment or cytotoxicity. A screen of marine sponge extracts identified motuporamines as micromolar inhibitors of invasion of basement membrane gels by MDA-231 breast carcinoma, PC-3 prostate carcinoma, and U-87 and U-251 glioma cells. Motuporamine C inhibits cell migration in monolayer cultures and impairs actin-mediated membrane ruffling at the leading edge of lamellae. Motuporamine C also reduces beta1-integrin activation, raising the possibility that it interferes with "inside-out" signaling to integrins. In addition, motuporamine C inhibits angiogenesis in an in vitro sprouting assay with human endothelial cells and an in vivo chick chorioallantoic membrane assay. The motuporamines show little or no toxicity or inhibition of cell proliferation, and they are structurally simple and easy to synthesize, making them attractive drug candidates.

Alkaloids↗

PubMed2001

Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

Cadherins↗

PubMed2001

Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343.

Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB- serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.

Amino Acid Sequence↗

PubMed2001

Inhibition of integrin linked kinase (ILK) suppresses beta-catenin-Lef/Tcf-dependent transcription and expression of the E-cadherin repressor, snail, in APC-/- human colon carcinoma cells.

Loss of functional adenomatous polyposis coli (APC) protein results in the stabilization of cytosolic beta-catenin and activation of genes that are responsive to Lef/Tcf family transcription factors. We have recently shown that an independent cell adhesion and integrin linked kinase (ILK)-dependent pathway can also activate beta-catenin/LEF mediated gene transcription and downregulate E-cadherin expression. In addition, ILK activity and expression are elevated in adenomatous polyposis and colon carcinomas. To examine the role of this pathway in the background of APC mutations we inhibited ILK activity in APC-/- human colon carcinoma cell lines. In all cases, inhibition of ILK resulted in substantial inhibition of TCF mediated gene transcription and inhibition of transcription and expression of the TCF regulated gene, cyclin D1. Inhibition of ILK resulted in decreased nuclear beta-catenin expression, and in the inhibition of phosphorylation of GSK-3 and stimulation of its activity, leading to accelerated degradation of beta-catenin. In addition, inhibition of ILK suppressed cell growth in culture as well as growth of human colon carcinoma cells in SCID mice. Strikingly, inhibition of ILK also resulted in the transcriptional stimulation of E-cadherin expression and correlated with the inhibition of gene transcription of snail, a repressor of E-cadherin gene expression. Overexpression of ILK caused a stimulation of expression of snail, but snail expression was found not to be regulated by beta-catenin/Tcf. These data demonstrate that ILK can regulate beta-catenin/TCF and snail transcription factors by distinct pathways. We propose that inhibition of ILK may be a useful strategy in the control of progression of colon as well as other carcinomas. Oncogene (2001) 20, 133 - 140.

Adenomatous Polyposis Coli↗

PubMed2001

Rat seminiferous epithelium contains a unique junction (Ectoplasmic specialization) with signaling properties both of cell/cell and cell/matrix junctions.

The seminiferous epithelium contains unique actin related cell-cell junctions, termed ectoplasmic specializations (ESs). Turnover of these junctions is fundamental to sperm release and to movement of spermatocytes from basal to adluminal compartments of the epithelium during spermatogenesis. In this study we report several novel observations related to the spatial and temporal distribution of integrin-related signaling molecules at ESs. We confirm the presence of beta(1)-integrin at these sites and further demonstrate co-localization of integrin linked kinase (ILK). beta(1)-Integrin and ILK were shown by immunoprecipitation to associate in whole cell lysates of seminiferous epithelium. This observation provides the first evidence for a direct beta(1)-integrin/ILK interaction in noncultured epithelium. Pan-cadherin and beta-catenin antibodies did not react at ESs. Rather, antibodies reacted with desmosome-like junctions that are present both at basal junctional complexes between Sertoli cells and at sites of attachment to spermatogenic cells. Focal adhesion kinase (FAK), a known integrin-associated molecule, did not codistribute with beta(1)-integrins and did not associate with these adhesion molecules in immunoprecipitation studies. Although FAK was expressed in the epithelium, it appeared to be limited to the cytoplasm of early spermatogenic cells. Significantly, polyclonal antibodies against phosphotyrosine-containing residues reacted strongly at ESs, with highest levels detected during sperm release and turnover of basal junction complexes. Our observations indicate that ESs share cell signaling features both of cell-cell junctions and of cell-extracellular matrix junctions.

Actins↗

PubMed2001

Overexpression of the integrin-linked kinase mesenchymally transforms mammary epithelial cells.

Signals generated by the interaction of beta1 integrins with laminin in the basement membrane contribute to mammary epithelial cell morphogenesis and differentiation. The integrin-linked kinase (ILK) is one of the signaling moieties that associates with the cytoplasmic domain of beta1 integrin subunits with some specificity. Forced expression of a dominant negative, kinase-dead form of ILK subtly altered mouse mammary epithelial cell morphogenesis but it did not prevent differentiative milk protein expression. In contrast, forced overexpression of wild-type ILK strongly inhibited both morphogenesis and differentiation. Overexpression of wild-type ILK also caused the cells to lose the cell-cell adhesion molecule E-cadherin, become invasive, reorganize cortical actin into cytoplasmic stress fibers, and switch from an epithelial cytokeratin to a mesenchymal vimentin intermediate filament phenotype. Forced expression of E-cadherin in the latter mesenchymal cells rescued epithelial cytokeratin expression and it partially restored the ability of the cells to differentiate and undergo morphogenesis. These data demonstrate that ILK, which responds to interactions between cells and the extracellular matrix, induces a mesenchymal transformation in mammary epithelial cells, at least in part, by disrupting cell-cell junctions.

Adherens Junctions↗

PubMed2000

The integrin linked kinase (ILK) induces an invasive phenotype via AP-1 transcription factor-dependent upregulation of matrix metalloproteinase 9 (MMP-9).

Overexpression of Integrin Linked Kinase (ILK) in intestinal and mammary epithelial cells results in a highly invasive phenotype, associated with increased levels of expression of the matrix metalloproteinase MMP-9. This increase was at the transcriptional level as determined by MMP-9 promoter-CAT reporter assays. Mutations in the two AP-1 binding sites within the MMP-9 promoter completely inhibited the reporter activity. We have previously shown that ILK inhibits glycogen synthase kinase-3 (GSK-3) activity. Transient transfection of wild-type GSK-3beta in ILK-overexpressing cells decreased MMP-9 promoter activity and AP-1 activity, indicating that ILK can stimulate MMP-9 expression via GSK-3beta and AP-1 transcription factor. A small molecule inhibitor of the ILK kinase reduced the in vitro invasiveness of ILK-overexpressing cells as well as the invasiveness of several human brain tumor cell lines. Furthermore, both MMP-9 promoter and AP-1 activities were inhibited by the ILK inhibitor. Invasiveness of ILK-overexpressing cells was also reduced by inhibition of MMP-9. These data demonstrate that ILK can induce an invasive phenotype via AP-1-dependent upregulation of MMP-9.

Animals↗

PubMed2000

The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20-30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the beta-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of beta(1) and beta(3) integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide -54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3beta (GSK-3beta) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.

Activating Transcription Factor 2↗

PubMed2000

Integrin-linked kinase (ILK): a "hot" therapeutic target.

Integrin-mediated cell adhesion is known to regulate gene expression through the activation of transcription factors. We have recently revealed that these activations are mediated through integrin-linked kinase (ILK). ILK is an ankyrin repeat-containing serine-threonine protein kinase that can interact directly with the cytoplasmic domain of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. We have shown that ILK overexpression results in the translocation of beta-catenin to the nucleus, which then forms a complex formation with the lymphoid enhancer binding factor 1 (LEF-1) transcription factor, subsequently activating the transcriptional activity of promoters containing LEF-1 response elements. ILK phosphorylates the glycogen synthase kinase-3 (GSK-3), which inhibits GSK-3 activity. We have demonstrated that ILK stimulates activator protein-1 transcriptional activity through GSK-3 and the subsequent regulation of the c-Jun-DNA interaction. ILK also phosphorylates protein kinase B (PKB/Akt) and stimulates its activity. We have shown that ILK is an upstream effector of the phosphatidylinositol 3-kinase-dependent regulation of PKB/Akt. ILK has been shown to phosphorylate PKB/Akt on Ser-473 in vitro and in vivo. Our results clearly indicate that ILK is a key element in the regulation of integrin signaling as well as growth factor and Wnt signaling pathways. PTEN (phosphatase and tensin homolog detected on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. It is now estimated that inactivation mutants of PTEN exist in 60% of all forms of solid tumors. Loss of expression or mutational inactivation of PTEN leads to the constitutive activation of PKB/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We have demonstrated that the activity of ILK is constitutively elevated in PTEN mutant cells. A small molecule ILK inhibitor suppresses the phosphorylation of PKB at the Ser-473 but not the Thr-308 site in the PTEN mutant cells. These results indicate that inhibition of ILK may be of significant value in solid tumor therapy.

Animals↗

PubMed2000

The integrin-linked kinase (ILK) suppresses anoikis.

Disruption of integrin-extracellular matrix interactions in normal epithelial cells induces apoptosis, a process termed anoikis. Reduced sensitivity to anoikis appears to be an important hallmark of oncogenic transformation, particularly in the process of metastasis. Several pathways have been implicated in the suppression of anoikis, however, the events which take place proximal to the integrin receptors remain unclear. Integrin-linked kinase (ILK) is an integrin-interacting protein kinase which has been identified as a potential PDK-2, as it is capable of phosphorylating PKB/Akt on Ser-473, and stimulating its activity. Here, we show that ILK activity is stimulated upon adhesion of SCP2 mouse mammary epithelial cells to fibronectin, and inhibited in suspended cells. Overexpression of ILK in the anoikis-sensitive SCP2 cells results in a profound inhibition of anoikis, as determined by annexin V binding and activation of caspases 8 and 3. This effect is reversible by the transfection and expression of a dominant-negative, kinase deficient ILK (ILK KD), as well as by a dominant negative PKB/Akt (PKB AAA). On the other hand, transfection of a dominant negative form of FAK (FRNK) failed to reverse the suppression of anoikis by ILK. Furthermore, inhibition of ILK activity induced anoikis in two anoikis-resistant human breast cancer cell lines. These findings suggest that ILK plays a major role in the suppression of anoikis.

Animals↗

PubMed2000

Heart, brain, and body wall defects in mice lacking calreticulin.

Calreticulin is a ubiquitously expressed protein, which has been implicated in a large number of cellular functions, including calcium storage and signaling, protein folding, and cell attachment. To examine the role of calreticulin during in vivo development, mice deficient in calreticulin were generated by targeted inactivation of the calreticulin gene. Calreticulin-deficient mutants die in utero, mostly in late gestation. Half of these embryos had decreased cardiac cell mass, associated with increased apoptosis of cardiac myocytes. In vitro differentiation cultures of calreticulin-deficient embryonic stem cells resulted in fewer embryoid bodies with contractile activity than cultures derived from calreticulin +/- stem cells (P < 0.001). Sixteen percent of the mutants exhibited exencephaly secondary to a defect in neural tube closure. Embryos surviving until Embryonic Day 16.5 had omphalocele. Lack of calreticulin did not influence survival of embryonic fibroblasts under various endoplasmic reticulum stress conditions. However, calreticulin did influence cell migration in a calcium- and substrate-dependent manner. We conclude that calreticulin is not essential during the early stages of embryonic development, but is important for the development of heart and brain and for ventral body wall closure. The observed abnormalities are compatible with a role of calreticulin in the modulation of cellular calcium signaling.

Abnormalities, Multiple↗