Integrin α6A splice variant regulates proliferation and the Wnt/ß-catenin pathway in human colorectal cancer cells.

The integrin α6 subunit pre-messenger RNA undergoes alternative splicing to generate two different splice variants, named α6A and α6B, having distinct cytoplasmic domains. In the human colonic gland, these splice variants display different patterns of expression suggesting specific functions for each variant. We have previously found an up-regulation of the α6ß4 integrin in colon adenocarcinomas as well as an increase in the α6A/α6B ratio, but little is known about the involvement of α6Aß4 versus α6Bß4 in this context. The aim of this study was to elucidate the function of the α6Aß4 integrin in human colorectal cancer (CRC) cells. Expression studies on a panel of primary CRCs confirmed that the up-regulation of the α6 subunit in CRC is a direct consequence of the increase of the α6A variant. To investigate the functional significance of an α6A up-regulation in CRC, we specifically knocked down its expression in well-established CRC cell lines using a small-hairpin RNA approach. Results showed a growth rate reduction in all α6A knockdown CRC cell lines studied. The α6A silencing was also found to be associated with a significant repression of a number of Wnt/ß-catenin pathway end points. Moreover, it was accompanied by a reduction in the capacity of these cells to develop tumours in xenografts. Taken together, these results demonstrate that the α6A variant is a pro-proliferative form of the α6 integrin subunit in CRC cells and appears to mediate its effects through the Wnt/ß-catenin pathway.

Integrin-linked kinase regulates migration and proliferation of human intestinal cells under a fibronectin-dependent mechanism.

Integrin-linked kinase (ILK) plays a role in integrin signaling-mediated extracellular matrix (ECM)-cell interactions and also acts as a scaffold protein in functional focal adhesion points. In the present study, we investigated the expression and roles of ILK in human intestinal epithelial cells (IECs) in vivo and in vitro. Herein, we report that ILK and its scaffold-function interacting partners, PINCH-1, alpha-parvin, and beta-parvin, are expressed according to a decreasing gradient from the bottom of the crypt (proliferative/undifferentiated) compartment to the tip of the villus (non-proliferative/differentiated) compartment, closely following the expression pattern of the ECM/basement membrane component fibronectin. The siRNA knockdown of ILK in human IECs caused a loss of PINCH-1, alpha-parvin, and beta-parvin expression, along with a significant decrease in cell proliferation via a loss of cyclin D1 and an increase in p27 and hypophosphorylated pRb expression levels. ILK knockdown severely affected cell spreading, migration, and restitution abilities, which were shown to be directly related to a decrease in fibronectin deposition. All ILK knockdown-induced defects were rescued with exogenously deposited fibronectin. Altogether, our results indicate that ILK performs crucial roles in the control of human intestinal cell and crypt-villus axis homeostasis-especially with regard to basement membrane fibronectin deposition-as well as cell proliferation, spreading, and migration.

Integrin alpha8beta1 confers anoikis susceptibility to human intestinal epithelial crypt cells.

We previously reported that integrin alpha8beta1 is expressed in human intestinal epithelial crypt cells (HIECs) and represents one of the major RGD-binding integrins expressed by these cells. Moreover, the depletion of alpha8beta1 affects vinculin, but not paxillin, localization at focal adhesion points. In the present study, we show that the integrin alpha8 shRNA-mediated knockdown in HIECs leads to a decrease in anoikis susceptibility under cell suspension culture conditions, marked by a reduction in PARP cleavage and propidium iodide incorporation. Moreover, alpha8beta1-depleted HIECs exhibited an illicitly sustained activation of Fak and PI3-K/Akt-1 under anoikis conditions, rendering them refractory to anoikis. To this effect, colon cancer cells exhibiting resistance to anoikis not only displayed a loss of alpha8beta1 expression, but forced expression of alpha8beta1 in these cells decreased their resistance to anoikis. Consequently, alpha8beta1 is a prerequisite for the proper conduct of anoikis in normal HIECs, whereas its loss contributes to the illicit acquisition of anoikis resistance.

ILK supports RhoA/ROCK-mediated contractility of human intestinal epithelial crypt cells by inducing the fibrillogenesis of endogenous soluble fibronectin during the spreading process.

BACKGROUND: Fibronectin (FN) assembly into an insoluble fibrillar matrix is a crucial step in many cell responses to extracellular matrix (ECM) properties, especially with regards to the integrin-related mechanosensitive signaling pathway. We have previously reported that the silencing of expression of integrin-linked kinase (ILK) in human intestinal epithelial crypt (HIEC) cells causes significant reductions in proliferation and spreading through concomitantly acquired impairment of soluble FN deposition. These defects in ILK-depleted cells are rescued by growth on exogenous FN. In the present study we investigated the contribution of ILK in the fibrillogenesis of FN and its relation to integrin-actin axis signaling and organization. RESULTS: We show that de novo fibrillogenesis of endogenous soluble FN is ILK-dependent. This function seemingly induces the assembly of an ECM that supports increased cytoskeletal tension and the development of a fully spread contractile cell phenotype. We observed that HIEC cell adhesion to exogenous FN or collagen-I (Col-I) is sufficient to restore fibrillogenesis of endogenous FN in ILK-depleted cells. We also found that optimal engagement of the Ras homolog gene family member A (RhoA) GTPase/Rho-associated kinase (ROCK-1, ROCK-2)/myosin light chain (MLC) pathway, actin ventral stress fiber formation, and integrin adhesion complex (IAC) maturation rely primarily upon the cell's capacity to execute FN fibrillogenesis, independent of any significant ILK input. Lastly, we confirm the integrin α5ß1 as the main integrin responsible for FN assembly, although in ILK-depleted cells αV-class integrins expression is needed to allow the rescue of FN fibrillogenesis on exogenous substrate. CONCLUSION: Our study demonstrates that ILK specifically induces the initiation of FN fibrillogenesis during cell spreading, which promotes RhoA/ROCK-dependent cell contractility and maturation of the integrin-actin axis structures. However, the fibrillogenesis process and its downstream effect on RhoA signaling, cell contractility and spreading are ILK-independent in human intestinal epithelial crypt cells.

Differential expression of the integrins alpha6Abeta4 and alpha6Bbeta4 along the crypt-villus axis in the human small intestine.

The integrin alpha6 subunit exists as two different variants, termed alpha6A and alpha6B. These two variants have been shown to harbor potentially distinct biochemical properties but little is known about their cellular function. The aim of this work was to characterize the expression of the integrin alpha6A and B variants in relation to cell proliferation and differentiation in the human small intestinal epithelium. The results showed distinct expression patterns for the two variants along the crypt-villus axis. Indeed, proliferative cells of the crypt were found to predominantly express alpha6A, while differentiated enterocytes and Paneth cells expressed the alpha6B variant. A similar relationship was observed in intestinal cell models by competitive RT-PCR. Further studies in the Caco-2 cell model showed that manipulating the cellular balance of the two alpha6 variants can influence transcriptional activities related to cell proliferation but not differentiation. This suggests that differential expression of the alpha6 subunits is involved in the intestinal epithelial cell renewal process. Further studies will be needed to substantiate this hypothesis.

Integrin alpha6Bbeta4 inhibits colon cancer cell proliferation and c-Myc activity.

BACKGROUND: Integrins are known to be important contributors to cancer progression. We have previously shown that the integrin beta4 subunit is up-regulated in primary colon cancer. Its partner, the integrin alpha6 subunit, exists as two different mRNA splice variants, alpha6A and alpha6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these alpha6 splice variants is still lacking. METHODS: In this work, we first analyzed the expression of integrin alpha6A and alpha6B at the protein and transcript levels in normal human colonic cells as well as colorectal adenocarcinoma cells from both primary tumors and established cell lines. Then, using forced expression experiments, we investigated the effect of alpha6A and alpha6B on the regulation of cell proliferation in a colon cancer cell line. RESULTS: Using variant-specific antibodies, we observed that alpha6A and alpha6B are differentially expressed both within the normal adult colonic epithelium and between normal and diseased colonic tissues. Proliferative cells located in the lower half of the glands were found to predominantly express alpha6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed alpha6B. A relative decrease of alpha6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the alpha6A/alpha6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the alpha6A/alpha6B balance in favor of alpha6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc. CONCLUSION: The findings that the alpha6Bbeta4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its alpha6Abeta4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this alpha6Bbeta4 integrin. Taken together, these findings point out the importance of integrin variant expression in colon cancer cell biology.

MYC Regulates α6 Integrin Subunit Expression and Splicing Under Its Pro-Proliferative ITGA6A Form in Colorectal Cancer Cells.

The α6 integrin subunit (ITGA6) pre-mRNA undergoes alternative splicing to form two splicing variants, named ITGA6A and ITGA6B. In primary human colorectal cancer cells, the levels of both ITGA6 and ß4 integrin subunit (ITGB4) subunits of the α6ß4 integrin are increased. We previously found that the upregulation of ITGA6 is a direct consequence of the increase of the pro-proliferative ITGA6A variant. However, the mechanisms that control ITGA6 expression and splicing into the ITGA6A variant over ITGA6B in colorectal cancer cells remain poorly understood. Here, we show that the promoter activity of the ITGA6 gene is regulated by MYC. Pharmacological inhibition of MYC activity with the MYC inhibitor (MYCi) 10058-F4 or knockdown of MYC expression by short hairpin RNA (shRNA) both lead to a decrease in ITGA6 and ITGA6A levels in colorectal cancer cells, while overexpression of MYC enhances ITGA6 promoter activity. We also found that MYC inhibition decreases the epithelial splicing regulatory protein 2 (ESRP2) splicing factor at both the mRNA and protein levels. Chromatin immunoprecipitation revealed that the proximal promoter sequences of ITGA6 and ESRP2 were occupied by MYC and actively transcribed in colorectal cancer cells. Furthermore, expression studies in primary colorectal tumors and corresponding resection margins confirmed that the up-regulation of the ITGA6A subunit can be correlated with the increase in MYC and ESRP2. Taken together, our results demonstrate that the proto-oncogene MYC can regulate the promoter activation and splicing of the ITGA6 integrin gene through ESRP2 to favor the production of the pro-proliferative ITGA6A variant in colorectal cancer cells.

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy.

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.

Laminin receptor 37/67LR regulates adhesion and proliferation of normal human intestinal epithelial cells.

Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR) is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.

RGD-Dependent Epithelial Cell-Matrix Interactions in the Human Intestinal Crypt.

Interactions between the extracellular matrix (ECM) and integrin receptors trigger structural and functional bonds between the cell microenvironment and the cytoskeleton. Such connections are essential for adhesion structure integrity and are key players in regulating transduction of specific intracellular signals, which in turn regulate the organization of the cell microenvironment and, consequently, cell function. The RGD peptide-dependent integrins represent a key subgroup of ECM receptors involved in the maintenance of epithelial homeostasis. Here we review recent findings on RGD-dependent ECM-integrin interactions and their roles in human intestinal epithelial crypt cells.

Autophagy is active in normal colon mucosa.

Recently, autophagy has been found to be strongly activated in colon cancer cells, but few studies have addressed the normal colon mucosa. The aim of this study was to characterize autophagy in normal human intestinal cells. We used the expression of LC3-II and BECN1 as well as SQSTM1 as markers of autophagy activity. Using the normal human intestinal epithelial crypt (HIEC) cell experimental model, we found that autophagy was much more active in undifferentiated cells than in differentiated cells. In the normal adult colonic mucosa, BECN1 was found in the proliferative epithelial cells of the lower part of the gland while SQSTM1 was predominantly found in the differentiated cells of the upper part of the gland and surface epithelium. Interestingly, the weak punctate pattern of SQSTM1 expression in the lower gland colocalized with BECN1-labeled autophagosomes. The usefulness of SQSTM1 as an active autophagy marker was confirmed in colon cancer specimens at the protein and transcript levels. In conclusion, our results show that autophagy is active in the colonic gland and is associated with the intestinal proliferative/undifferentiated and progenitor cell populations.

Collagen VI is a basement membrane component that regulates epithelial cell-fibronectin interactions.

Collagen VI is a heterotrimer composed of three α chains (α1, α2, α3) widely expressed throughout various interstitial matrices. Collagen VI is also found near the basement membranes of many tissues where it serves as an anchoring meshwork. The aim of this study was to investigate the distribution and role of collagen VI at the epithelial-stromal interface in the intestine. Results showed that collagen VI is a bona fide epithelial basal lamina component and constitutes the major collagen type of epithelial origin in this organ. In vitro, collagen VI co-distributes with fibronectin. Targeted knockdown of collagen VI expression in intestinal epithelial cells was used to investigate its function. Depletion of collagen VI from the matrix led to a significant increase in cell spreading and fibrillar adhesion formation coinciding with an upregulation of fibronectin expression, deposition and organization as well as activation of myosin light chain phosphorylation by the myosin light chain kinase and Rho kinase dependent mechanisms. Plating cells deficient for collagen VI on collagen VI rescued the phenotype. Taken together, these data demonstrate that collagen VI is an important basal lamina component involved in the regulation of epithelial cell behavior most notably as a regulator of epithelial cell-fibronectin interactions.