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Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6β1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis., (© 2013 Published by Elsevier Inc.)

The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics., (Copyright © 2013 Orthopaedic Research Society.)

Background: The cellular and molecular mechanisms that mediate interactions between tumour cells and the surrounding bone stroma are to date largely undetermined in prostate cancer (PCa) progression. The purpose of this study was to evaluate the role of alpha 6 and beta 1 integrin subunits in mediating tumour-stromal interactions., Methods: Utilising 3D in vitro assays we evaluated and compared 1. Monocultures of prostate metastatic PC3, bone stromal derived HS5 and prostate epithelial RWPE-1 cells and 2. Tumour-stromal co-cultures (PC3 + HS5) to ascertain changes in cellular phenotype, function and expression of metastatic markers., Results: In comparison to 3D monocultures of PC3 or HS5 cells, when cultured together, these cells displayed up-regulated invasive and proliferative qualities, along with altered expression of epithelial-to-mesenchymal and chemokine protein constituents implicated in metastatic dissemination. When co-cultured, HS5 cells were found to re-express N-Cadherin and chemokine receptor CXCR7. Alterations in N-Cadherin expression were found to be mediated by soluble factors secreted by PC3 tumour cells, while chemokine receptor re-expression was dependent on direct cell-cell interactions. We have also shown that integrins beta 1 and alpha 6 play an integral role in maintaining cell homeostasis and mediating expression of E-Cadherin, N-Cadherin and vimentin, in addition to chemokine receptor CXCR7., Conclusions: Collectively our results suggest that both PC3 and HS5 cells provide a "protective" and reciprocal milieu that promotes tumour growth. As such 3D co-cultures may serve as a more complex and valid biological model in the drug discovery pipeline.

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and β1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and β1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and β1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6β1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.

Background: Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (α1β1γ1) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α2, α4, or α5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown., Methods and Results: Using an in vitro flow assay, we show that laminin-411 (α4β1γ1), laminin-511 (α5β1γ1), and laminin-521 (α5β2γ1), but not laminin-211 (α2β1γ1), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6β1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α5-containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α6. Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6β1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis., Conclusions: This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin α6β1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Liver fibrosis occurs as a wound-healing response to chronic hepatic injuries irrespective of the underlying etiology and may progress to life-threatening cirrhosis. Here we show that CCN1, a matricellular protein of the CCN (CYR61/CTGF/NOV) family, is accumulated in hepatocytes of human cirrhotic livers. CCN1 is not required for liver development or regeneration, since these processes are normal in mice with hepatocyte-specific Ccn1 deletion. However, Ccn1 expression is upregulated upon liver injuries and functions to inhibit liver fibrogenesis induced by either carbon tetrachloride intoxication or bile duct ligation and promote fibrosis regression. CCN1 acts by triggering cellular senescence in activated hepatic stellate cells and portal fibroblasts by engaging integrin α6β1 to induce reactive oxygen species accumulation through the RAC1-NADPH oxidase 1 enzyme complex, whereupon the senescent cells express an antifibrosis genetic program. Mice with hepatocyte-specific Ccn1 deletion suffer exacerbated fibrosis with a concomitant deficit in cellular senescence, whereas overexpression of hepatic Ccn1 reduces liver fibrosis with enhanced senescence. Furthermore, tail vein delivery of purified CCN1 protein accelerates fibrosis regression in mice with established fibrosis. These findings reveal a novel integrin-dependent mechanism of fibrosis resolution in chronic liver injury and identify the CCN1 signaling pathway as a potential target for therapeutic intervention.

The characterization of cells with tumour initiating potential is significant for advancing our understanding of cancer and improving therapy. Aggressive, triple-negative breast cancers (TNBCs) are enriched for tumour-initiating cells (TICs). We investigated that hypothesis that VEGF receptors expressed on TNBC cells mediate autocrine signalling that contributes to tumour initiation. We discovered the VEGF receptor neuropilin-2 (NRP2) is expressed preferentially on TICs, involved in the genesis of TNBCs and necessary for tumour initiation. The mechanism by which NRP2 signalling promotes tumour initiation involves stimulation of the α6β1 integrin, focal adhesion kinase-mediated activation of Ras/MEK signalling and consequent expression of the Hedgehog effector GLI1. GLI1 also induces BMI-1, a key stem cell factor, and it enhances NRP2 expression and the function of α6β1, establishing an autocrine loop. NRP2 can be targeted in vivo to retard tumour initiation. These findings reveal a novel autocrine pathway involving VEGF/NRP2, α6β1 and GLI1 that contributes to the initiation of TNBC. They also support the feasibility of NRP2-based therapy for the treatment of TNBC that targets and impedes the function of TICs., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells., (Copyright © 2012 AlphaMed Press.)

Laminins are major constituents of basement membranes. At least 16 isoforms have now been described, each with distinct spatio-temporal expression patterns and functions. The laminin-511 heterotrimer (α5β1γ1) is one of the more recent isoforms to be identified and a potent adhesive and pro-migratory substrate for a variety of normal and tumor cell lines in vitro. As our understanding of its precise function in normal tissues and in pathologies is rapidly unraveling, current evidence suggests an important regulatory role in cancer. This review describes published data on laminin-511 expression in several malignancies and experimental evidence from both in vitro and in vivo studies supporting its functional role during tumor progression. A particular emphasis is put on more recent studies from our laboratory and that of others indicating that laminin-511 contributes to tumor dissemination and metastasis in advanced breast carcinomas and other tumor types. Collectively, the experimental evidence suggests that high expression of laminin-511 has prognostic significance and that targeting tumor-laminin-511 interactions may have therapeutic potential in advanced cancer patients.

CD151 is a member of the tetraspanin family that is implicated as a promoter of the tumor metastasis of malignant cells. Tetraspanins form membrane complexes with integrins. In the present study, we constructed a CD151-AAA mutant to assess the roles of Rac, cdc42 and phospho-Rac/cdc42 (P-Rac/cdc42) and the effects of CD151‑integrin complexes on the proliferation, migration and invasion of HepG2 cells. The pAAV-CD151 and pAAV‑CD151‑AAA mutant plasmids were constructed and used to transiently transfect HepG2 cells using the Qiagen Attractene transfection reagent. Following transfection, the expression of CD151 was determined by western blotting. A cell proliferation assay was performed using the cell counting kit-8 (CCK-8) method, cell migration was assessed by a cell wound-healing assay and cell invasion was evaluated in microchemotaxis chambers using FBS as the chemotactic stimulus. The potential involvement of various signaling pathways was explored using relevant antibodies. The association between CD151 and integrins was evaluated by immunoblotting analysis. We found that CD151 promoted cell proliferation, migration and chemotaxis and increased P-Rac/cdc42 activity. The CD151-AAA mutant had reduced cellular proliferation, migration and invasion compared with the CD151 mutant. Moreover, the CD151-AAA mutant abrogated the association between CD151 and integrins. These data suggest that CD151 forms complexes by interacting with integrins, particularly α3β1 and α6β1, and thereby affects the functioning of the HepG2 cells. The mechanism is possibly related to the Rac, cdc42 and P-Rac/cdc42 signaling pathways.

This review advances the hypothesis that the ability of integrins to engage their extracellular matrix ligands and signal can be regulated in tumor cells by vascular endothelial growth factor (VEGF), a major angiogenic factor that also has direct effects on the function of tumor cells. More specifically, we will discuss how neuropilins (NRPs), a distinct class of VEGF receptors, enable the function of specific integrins that contribute to tumor initiation and progression.

Chronic cerebral hypoxia leads to a strong vascular remodeling response, though little is known about which part of the vascular tree is modified, or whether this response includes formation of new arterial vessels. In this study, we examined this process in detail, analyzing how hypoxia (8% O(2) for 14 days) alters the size distribution of vessels, number of arteries/arterioles, and expression pattern of endoglin (CD105), a marker of angiogenic endothelial cells in tumors. We found that cerebral hypoxia promoted the biggest increase in the number of medium to large size vessels, and this correlated with increased numbers of alpha smooth muscle actin (α-SMA)-positive arterial vessels. Surprisingly, hypoxia induced a marked reduction in CD105 expression on brain endothelial cells (BECs) within remodeling arterial vessels, and these BECs also displayed an angiogenic switch in β1 integrins (from α6 to α5), previously described for developmental angiogenesis. In vitro, transforming growth factor (TGF)-β1 also promoted this switch of BEC β1 integrins. Together, these results show that cerebral hypoxia promotes arteriogenesis, and identify reduced CD105 expression as a novel marker of arteriogenesis. Furthermore, our data suggest a mechanistic model whereby BECs in remodeling arterial vessels downregulate CD105 expression, which alters TGF-β1 signaling, to promote a switch in β1 integrins and arteriogenic remodeling.

Cell-cell interactions are critical to understanding functional tissues. A number of stem cell populations have been shown to receive key regulatory information from endothelial cells (ECs); however, the role of ECs in the retinal stem and progenitor cell (RSPC) niche has been largely unexplored. To gain greater insight into the role of ECs on RSPC fate, a three-dimensional (3D) co-culture model, incorporating cell-cell interactions, was designed by covalently-modifying agarose hydrogels with growth factors and cell-adhesive peptides in defined volumes. Therein ECs adopted tubular-like morphologies similar to those observed in vivo, but not observed in two-dimensional (2D) cultures. Unexpectedly, ECs inhibited proliferation and differentiation of RSPCs, revealing, for the first time, the possible role of ECs on RSPC fate. This 3D hydrogel scaffold provides a simple, reproducible and versatile method with which to answer biological questions related to the cellular microenvironment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvβ5 or α6β1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvβ3 or α6β1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway., (Copyright © 2012 Wiley Periodicals, Inc.)

Activation of TNF receptor 1 (TNF-R1) can generate signals that promote either apoptosis or survival. In this study, we show that these signals can be determined by the character of the extracellular matrix in the tumor microenvironment. Specifically, through studies of glioblastoma, we showed that TNFα stimulation induced apoptosis of primary brain endothelial cells (EC) attached to collagen or fibronectin (which engage integrins α2β1/α3β1 and α5β1, respectively), but did not induce apoptosis of ECs attached to laminin (which engages integrins α6β1 and α3β1). TNF-R1 expression was significantly higher in ECs in glioblastoma (GBM) tumors compared with ECs in normal brain specimens. TNFα was also expressed in GBM tumor-associated ECs, which was associated with longer patient survival. ECs plated on anti-integrin α2 or α3 antibody were susceptible to TNFα-induced apoptosis, whereas those plated on anti-integrin α6 antibody were not. Moreover, the ECs plated on laminin, but not collagen, expressed cellular FLICE inhibitory protein (cFLIP) and TNFα stimulation of laminin-attached cells in which cFLIP had been downregulated resulted in the induction of apoptosis. In contrast, attachment to laminin did not induce cFLIP expression in GBM tumor stem cells. Together, our findings indicate that the laminin receptor integrin α6β1 promotes the survival of brain ECs by inhibiting prodeath signaling by TNF-R1, in part by inducing cFLIP expression.

The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2(high) and NRP2(low) populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2(high) population adhered more avidly to laminin and expressed high levels of the α6β1 integrin than the NRP2(low) population. The NRP2(high) population formed numerous focal adhesions on laminin that were not seen in the NRP2(low) population. These results were substantiated using breast carcinoma cell lines that express NRP2 and α6β1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and α6β1 integrin was detected by co-immunoprecipitation. NRP2 is necessary for focal adhesion formation on laminin and for the association of α6β1 integrin with the cytoskeleton. NRP2 also facilitates α6β1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin. The mechanism by which NRP2 regulates the interaction of α6β1 integrin with laminin to form focal adhesions involves PKC activation. Together, our data reveal a new VEGF-NRP2 signaling pathway that activates the α6β1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer.

The blood-testis barrier (BTB), similar to other blood-tissue barriers, such as the blood-brain barrier and the blood-retinal barrier, is used to protect the corresponding organ from harmful substances (e.g., xenobiotics) including drugs and foreign compounds. More importantly, the BTB allows postmeiotic spermatid development to take place in an immune privileged site at the adluminal (or apical) compartment to avoid the production of antibodies against spermatid-specific antigens, many of which express transiently during spermiogenesis and spermiation. The BTB, however, also poses an obstacle in developing nonhormonal-based male contraceptives by sequestering drugs (e.g., adjudin) that exert their effects on germ cells in the adluminal compartment. The effects of these drugs include disruption of germ cell cycle progression and development, apoptosis, cell adhesion, metabolism and others. Recent studies have demonstrated that there is a functional axis that operates locally in the seminiferous epithelium to co-ordinate different cellular events across the Sertoli cell epithelium, such as spermiation and BTB restructuring during the seminiferous epithelial cycle of spermatogenesis. Components of this functional axis, such as the apical ectoplasmic specialization (apical ES, a testis-specific atypical anchoring junction type) and the BTB, in particular their constituent protein complexes, such as alpha6beta1-integrin and occludin at the apical ES and the BTB, respectively, can be the target of male contraception. In this chapter, we highlight recent advances regarding the likely mechanism of action of adjudin in this functional axis with emphasis on the use of molecular modeling technique to facilitate the design of better compounds in male contraceptive development.

The laminin-binding integrin α6β1 plays a major role in determining the aggressive phenotype of tumor cells during metastasis. Our previous work has shown that cleavage of the α6β1 integrin to produce the structural variant α6pβ1 on tumor cell surfaces is mediated by the serine protease urokinase plasminogen activator (uPA). Cleavage of α6β1 increases tumor cell motility, invasion, and prostate cancer metastasis, and blockage of uPA inhibits α6pβ1 production. In human tumors, uPA and uPAR are expressed in tumor cells and tumor-associated macrophages (TAM). TAMs localize to solid tumors and contribute to increased tumor growth and the metastatic phenotype. In this study, we utilized a coculture system of PC-3 prostate tumor cells and macrophages [12-O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human leukemia HL-60 cells] to investigate the hypothesis that macrophages stimulate the production of the prometastatic variant α6pβ1 on human prostate cancer cells via the uPA/uPAR axis. Our results indicate that adherent macrophages cocultured with PC-3 cells increased PC-3 uPAR mRNA, uPAR cell surface protein expression and α6 integrin cleavage. The stimulation does not require macrophage/tumor cell contact because macrophage conditioned medium is sufficient for increased uPAR transcription and α6 cleavage-dependent PC-3 cell invasion. The increased cleavage was dependent on uPAR because production was blocked by silencing RNA-targeting uPAR. These results indicate that macrophages can stimulate uPA/uPAR production in tumor cells which results in α6 integrin cleavage. These data suggest that TAMs promote prometastatic integrin-dependent pericellular proteolysis.

Rationale: Netrin-4 regulates vascular development. Identity of netrin-4 endothelial receptor and its subsequent cell functions is controversial. We previously demonstrated that the inhibition of netrin-1 canonical receptors, Unc5B and neogenin, expressed by lymphatic endothelial cells, do not suppress netrin-4-induced cell signaling and functions. Netrin family members were shown to signal through a range of receptors, including integrins (such as α3β1, α6β1, and α6β4) in nonendothelial cells., Objective: We tested whether integrins are netrin-4 receptors in the endothelium., Methods and Results: The α6β1 integrin is expressed by endothelial cells, and binds netrin-4 in a dose-dependent manner. Inhibition of α6 or β1 integrin subunits suppresses netrin-4-induced endothelial cell migration, adhesion, and focal adhesion contact. Netrin-4-stimulated phosphorylation of Src kinase family, effectors of endothelial cell migration, is also abolished by α6 or β1 inhibition. Finally, netrin-4 and α6β1 integrin expression colocalize in mouse embryonic, intestine, and tumor vasculature., Conclusions: The α6β1 integrin is a netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit netrin-4-induced metastatic dissemination.

Rationale: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through "lateral inhibition" in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected., Objective: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells., Methods and Results: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of α2β1 and α6β1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by α2β1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin γ1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins α2 or α6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel., Conclusions: We show that, in endothelial cells, ligation of α2β1 and α6β1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.

In this study, we applied specific blocking antibodies for integrin α6 or β1 subunit, and evaluated the in vitro effects of integrins α6β1 on the adhesion, chemotaxis and migration of hepatocellular carcinoma (HCC) cell line SMMC-7721 to type IV collagen. The adhesion force and cell migration, as measured by a micropipette aspiration system and Boyden chamber assay respectively, was dramatically reduced when either integrin subunits was blocked. The chemotaxis, as determined using a dual-micropipette system, was only affected by the antibody against β1 subunit. This study suggests that integrin α6β1 is an important cell surface receptor that mediates the adhesion of SMMC-7721 to type IV collagen. But the α6 subunit has minimal effect on pseudopod formation in response to type IV collagen. Therefore, the integrin α6β1-mediated cell migration is, at least in part, through the regulation on the cell adhesion step.

Melanoma cells express and interact with laminins (LMs) and other basement membrane components during invasion and metastasis. In the present study we have investigated the production and migration-promoting activity of laminin isoforms in melanoma. Immunohistochemistry of melanoma specimens and immunoprecipitation/western blotting of melanoma cell lines indicated expression of laminin-111/121, laminin-211, laminin-411/421, and laminin-511/521. Laminin-332 was not detected. In functional assays, laminin-111, laminin-332, and laminin-511, but not laminin-211 and laminin-411, strongly promoted haptotactic cell migration either constitutively or following stimulation with insulin-like growth factors. Both placenta and recombinant laminin-511 preparations were highly active, and the isolated recombinant IVa domain of LMα5 also promoted cell migration. Function-blocking antibodies in cell migration assays revealed α6β1 integrin as the major receptor for laminin-111, and both α3β1 and α6β1 integrins for laminin-332 and laminin-511. In contrast, isolated LMα5 IVa domain-promoted melanoma cell migration was largely mediated via αVβ3 integrin and inhibited by RGD peptides. Given the ubiquitous expression of α5 laminins in melanoma cells and in melanoma-target tissues/anatomical structures, as well as the strong migration-promoting activity of these laminin isoforms, the α5 laminins emerge as putative primary extracellular matrix mediators of melanoma invasion and metastasis via α3β1 and other integrin receptors., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin αβ1, α(v)β or α(6)β receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Myelination in the central nervous system provides a unique example of how cells establish asymmetry. The myelinating cell, the oligodendrocyte, extends processes to and wraps multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. Local regulation of protein synthesis represents one mechanism used to control the different requirements for myelin sheath at each axo-glia interaction. Prior work has established that β1-integrins are involved in the axoglial interactions that initiate myelination. Here, we show that integrin activation regulates translation of a key sheath protein, myelin basic protein (MBP), by reversing the inhibitory effect of the mRNA 3'UTR. During oligodendrocyte differentiation and myelination α6β1-integrin interacts with hnRNP-K, an mRNA-binding protein, which binds to MBP mRNA and translocates from the nucleus to the myelin sheath. Furthermore, knockdown of hnRNP-K inhibits MBP protein synthesis during myelination. Together, these results identify a novel pathway by which axoglial adhesion molecules coordinate MBP synthesis with myelin sheath formation.

Adult salivary gland stem cells are promising candidates for cell therapy and tissue regeneration in cases of irreversible damage to salivary glands in head and neck cancer patients undergoing irradiation therapy. At present, the major restriction in handling such cells is their relatively limited life span during in vitro cultivation, resulting in an inadequate experimental platform to explore the salivary gland-originated stem cells as candidates for future clinical application in therapy. We established a spontaneous immortal integrin α6β1-expressing cell line of adult salivary progenitor cells from rats (rat salivary clone [RSC]) and investigated their ability to sustain cellular properties. This line was able to propagate for more than 400 doublings without loss of differentiation potential. RSC could differentiate in vitro to both acinar- and ductal-like structures and could be further manipulated upon culturing on a 3D scaffolds with different media supplements. Moreover, RSC expressed salivary-specific mRNAs and proteins as well as epithelial stem cell markers, and upon differentiation process their expression was changed. These results suggest RSC as a good model for further studies exploring cellular senescence, differentiation, and in vitro tissue engineering features as a crucial step toward reengineering irradiation-impaired salivary glands.

Metastasizing prostate tumor cells invade along nerves innervating the encapsulated human prostate gland in a process known as perineural invasion. The extracellular matrix laminin class of proteins line the neural route and tumor cells escaping from the gland express the laminin binding integrin alpha6beta1 as a prominent cell surface receptor. Integrin alpha6beta1 promotes aggressive disease and supports prostate tumor cell metastasis to bone. Laminins and their integrin receptors are necessary for the development and maintenance of the peripheral nervous system, indicating the potential role for integrin receptors in directing prostate tumor cell invasion on nerves during perineural invasion.

Background: Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential., Methodology/principal Findings: MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of alphaVbeta3, alpha6beta1 and alpha9beta1 integrins in xenograft cells. Treatment with bicistronic constructs reduced alphaVbeta3, alpha6beta1 and alpha9beta1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments., Conclusions/significance: Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma.

Background: Connective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells. An HC11 mammary epithelial cell line expressing CTGF/CCN2 was constructed to dissect the cellular responses to CTGF/CCN2 that contribute to this differentiation program., Results: Tetracycline-regulated expression of CTGF/CCN2 in HC11 cells enhanced multiple markers of lactogenic differentiation including beta-casein transcription and mammosphere formation. In a separate measure of mammary differentiation the addition of CTGF/CCN2 to cultures of MCF10A cells increased the development of acini in vitro. In HC11 cells the elevated levels of CTGF/CCN2 diminished the requirement for extracellular matrix proteins in the activation of beta-casein transcription, indicating that CTGF/CCN2 contributed to lactogenic differentiation through the regulation of matrix dependent cell adhesion. CTGF/CCN2 expression in HC11 cells increased expression of extracellular matrix proteins and integrins, enhanced the formation of focal adhesion complexes, and increased survival signaling. In addition, HC11 cells adhered to immobilized CTGF/CCN2 and this was inhibited by function-blocking antibodies to the integrins alpha6 and beta1, and to a lesser degree by antibody to beta3 integrin., Conclusions: CTGF/CCN2 expression in HC11 cells led to an increase in multiple markers of lactogenic differentiation. The mechanisms by which CTGF/CCN2 contributed to lactogenic differentiation include direct binding of CTGF/CCN2 to integrin complexes and CTGF/CCN2-induced matrix protein expression resulting in elevated integrin functionality.

Laminin is the first extracellular matrix (ECM) component to be expressed in the developing mammalian embryo. However, the roles of laminin or the related signal pathways are not well known in mouse embryonic stem cells (mESCs). Presently, we examined the effect of laminin on mESC migration. Laminin (10 microg/ml) decreased cell aggregation, whereas migration was increased. Laminin bound alpha6beta1 integrin and laminin receptor 1 (LR1), decreasing their mRNA levels. Laminin increased focal adhesion kinase (FAK) and paxillin phosphorylation, cAMP intracellular concentration, and the protein levels of exchange factor directly activated by cAMP (Epac1) and Rap1. These increases were completely blocked by alpha6beta1 integrin and LR1 neutralizing antibody, indicating that laminin-bound LR1 assists laminin-induced alpha6beta1 integrin activity and initiates signal. As a downstream signal molecule, laminin activated small G protein such as Rac1/cdc42 and its effector protein p21-activated kinase (PAK). Subsequently, laminin stimulated E-cadherin complex disruption. Inhibition of each pathway such as those for alpha6beta1 integrin and LR1, FAK, Rap1, and PAK1 blocked laminin-induced migration. We conclude that laminin binds both alpha6beta1 integrin and LR1 and induces signaling FAK/paxillin and cAMP/Epac1/Rap1. These signaling merge at Rac1/cdc42 subsequently activate PAK1. Activated PAK1 enhances E-cadherin complex disruption and finally increases mESCs migration.

Selective targeting of constructs to pathological cells by conjugating one or more ligands for an overexpressed receptor has been proposed to enhance the delivery of therapeutics to and imaging of specific cells of interest. Previous work in our lab has demonstrated the efficacy of targeting glioblastoma cells with a multivalent, biomacromolecular construct targeted to the alpha(6)beta(1)-integrin. However, solid-phase synthesis of this construct was inefficient in terms of cost and number of steps. Here we show proof-of-concept of a two-step synthesis that can be used to create similar constructs targeted to glioblastoma cells. Specifically, a well-defined aldehyde side chain polymer was synthesized and oxime chemistry was employed to conjugate ligands specific for the alpha(6)beta(1)-integrin. These constructs were then tested in competitive binding, fluorescence binding, and toxicity assays, through which we demonstrate that constructs are multivalent, preferentially target glioblastoma cells, and are nontoxic. Rapid, potentially low-cost synthesis of targeting constructs will enable their use in the clinic and for personalized medicine.

The initiator of coagulation, full-length tissue factor (flTF), in complex with factor VIIa, influences angiogenesis through PAR-2. Recently, an alternatively spliced variant of TF (asTF) was discovered, in which part of the TF extracellular domain, the transmembrane, and cytoplasmic domains are replaced by a unique C terminus. Subcutaneous tumors produced by asTF-secreting cells revealed increased angiogenesis, but it remained unclear if and how angiogenesis is regulated by asTF. Here, we show that asTF enhances angiogenesis in matrigel plugs in mice, whereas a soluble form of flTF only modestly enhances angiogenesis. asTF dose-dependently upregulates angiogenesis ex vivo independent of either PAR-2 or VIIa. Rather, asTF was found to ligate integrins, resulting in downstream signaling. asTF-alphaVbeta3 integrin interaction induces endothelial cell migration, whereas asTF-dependent formation of capillaries in vitro is dependent on alpha6beta1 integrin. Finally, asTF-dependent aortic sprouting is sensitive to beta1 and beta3 integrin blockade and a TF-antibody that disrupts asTF-integrin interaction. We conclude that asTF, unlike flTF, does not affect angiogenesis via PAR-dependent pathways but relies on integrin ligation. These findings indicate that asTF may serve as a target to prevent pathological angiogenesis.

Basement membranes have essential structural and signalling roles in tissue morphogenesis during embryonic development, but the mechanisms that control their formation are still poorly understood. Laminins are key components of basement membranes and are thought to be essential for initiation of basement membrane assembly. Here, we report that muscle progenitor cells populating the myotome migrate aberrantly in the ventral somite in the absence of sonic hedgehog (Shh) signalling, and we show that this defect is due to the failure to form a myotomal basement membrane. We reveal that expression of Lama1, which encodes laminin alpha1, a subunit of laminin-111, is not activated in Shh(-/-) embryos. Recovery of Lama1 expression or addition of exogenous laminin-111 to Shh(-/-);Gli3(-/-) embryos restores the myotomal basement membrane, demonstrating that laminin-111 is necessary and sufficient to initiate assembly of the myotomal basement membrane. This study uncovers an essential role for Shh signalling in the control of laminin-111 synthesis and in the initiation of basement membrane assembly in the myotome. Furthermore, our data indicate that laminin-111 function cannot be compensated by laminin-511.

Background: HAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin alpha3beta1. However, it has never been investigated whether alpha3beta1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells., Methods: Human SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin alpha6beta1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin alpha6beta1. Invasion potential was evaluated with an invasion assay and gelatin zymography., Results: We found that integrin alpha6beta1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs) were partially blocked by integrin alpha6beta1 antibodies (P < 0.01). Wortmannin, a specific phosphatidylinositol kinase (PI3K) inhibitor that reverses the effect of HAb18G/CD147 on the regulation of intracellular Ca2+ mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (P < 0.05). Importantly, no additive effect between Wortmannin and alpha6beta1 antibodies was observed, indicating that alpha6beta1 and PI3K transmit the signal in an upstream-downstream relationship., Conclusion: These results suggest that alpha6beta1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.

The understanding of how adhesion molecules mediate the axon-glial interactions in the CNS that ensure target-dependent survival of oligodendrocytes and initiate myelination remains incomplete. Here, we investigate how signals from adhesion molecules can be integrated to regulate these initial steps of myelination. We first demonstrate that the Ig superfamily molecule contactin is associated in oligodendrocytes with integrins, extracellular matrix receptors that regulate target-dependent survival by amplification of growth factor signaling. This amplification is inhibited by small interfering RNA-mediated knockdown of contactin in oligodendrocytes. In contrast, the presence of L1-Fc, the extracellular portion of a contactin ligand expressed on axons, enhanced survival and additionally promoted myelination in cocultures of neurons and oligodendrocytes. We further demonstrate that the signals from contactin and integrin are integrated by differential phosphorylation of the Src family kinase Fyn. Integrin induced dephosphorylation of the inhibitory Tyr-531, whereas contactin increased phosphorylation of both Tyr-531 and the activating Tyr-420. The combined effect is an enhanced activity of Fyn and also a dynamic regulation of the phosphorylation/dephosphorylation balance of Fyn, as required for normal cell adhesion and spreading. We conclude, therefore, that a novel integrin/contactin complex coordinates signals from extracellular matrix and the axonal surface to regulate both oligodendrocyte survival and myelination by controlling Fyn activity.

Human embryonic stem cells (hESC) exist as large colonies containing tightly adherent, undifferentiated cells. Disaggregation of hESC as single cells significantly affects their survival and differentiation, suggesting that adhesion mechanisms are critical for the assembly and maintenance of hESC colonies. The goal of these studies was to determine the key extracellular matrix (ECM) components that regulate assembly and growth of hESC. Our studies demonstrate that undifferentiated hESC express a specific subtype of laminin (laminin-511) and nidogen-1. The addition of a purified protein complex comprised of human laminin-511 and nidogen-1 to single-cell suspensions of hESC is sufficient to restore hESC assembly in the absence of murine embryonic fibroblasts or exogenous chemicals. The mechanism of hESC aggregation is through binding of the alpha6beta1 integrin receptor highly expressed in the membranes of undifferentiated hESC; aggregation can be inhibited by an antibody against alpha6 and almost completely blocked by an antibody against the beta1 subunit. Reassembly of defined numbers of purified hESC with the laminin-nidogen complex allows consistent production of uniform embryoid bodies (EBs) ("LN-EBs") that differentiate into endodermal, ectodermal, and mesodermal derivatives, and are highly efficient in generating hematoendothelial progenitors. These data reveal for the first time the crucial role of the ECM proteins laminin-511 and nidogen-1 in hESC assembly, and provide a novel practical tool to investigate hESC differentiation in a xenogen-free microenvironment.

Although Fas ligand (FasL) is primarily expressed by lymphoid cells, its receptor Fas (CD95/Apo-1) is broadly expressed in numerous nonlymphoid tissues and can mediate apoptosis of parenchymal cells upon injury and infiltration of inflammatory cells. Here we show that CCN1 (CYR61) and CCN2 (CTGF), matricellular proteins upregulated at sites of inflammation and wound repair, synergize with FasL to induce apoptosis by elevating cellular levels of reactive oxygen species (ROS). CCN1 acts through engagement of integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, leading to ROS-dependent hyperactivation of p38 mitogen-activated protein kinase in the presence of FasL to enhance mitochondrial cytochrome c release. We show that CCN1 activates neutral sphingomyelinase, which functions as a key source of CCN1-induced ROS critical for synergism with FasL. Furthermore, Fas-dependent hepatic apoptosis induced by an agonistic monoclonal anti-Fas antibody or intragastric administration of alcohol is severely blunted in knock-in mice expressing an apoptosis-defective Ccn1 allele. These results demonstrate that CCN1 is a physiologic regulator of Fas-mediated apoptosis and that the extracellular matrix microenvironment can modulate Fas-dependent apoptosis through CCN1 expression.

Peritoneal dissemination of ovarian carcinoma is mediated by epithelial-mesenchymal interconversions leading to the disruption of cell-cell contact and modulation of cell-extracellular matrix (ECM) interactions. The present study was designed to evaluate the effects of epidermal growth factor (EGF) as a modulator of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signalling and changes in integrin expression during the process similar to EMT. A fibroblastic morphology with reduced intercellular cell contacts and increased cell motility was observed in ovarian cancer cell lines in response to EGF and was concomitant with the up regulation of EMT-associated N-cadherin and vimentin expression. These changes were accompanied by an increase in alpha2, alpha6 and beta1 integrin subunits and activation of JAK2 and STAT3 signalling which was suppressed by a specific JAK2 inhibitor. Consistent with the suppression of STAT3 activity, N-cadherin and vimentin expression were abrogated and was coherent with the loss of cell motility and the expression of alpha6 and beta1 integrin subunits. Neutralizing antibodies against alpha6 and beta1 subunits inhibited cancer cell migration. A strong correlation between the expression of N-cadherin, vimentin and JAK2/STAT3 levels were detected in high-grade ovarian tumors and was consistent with the previously reported enhanced expression of alpha6 integrin subunit in advanced tumors [Ahmed N, Riley C, Oliva K, Rice G, Quinn M. Ascites induces modulation of alpha6beta1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. British Journal of Cancer 2005;92:1475-85]. Our data incorporating the clinical samples and the cancer cell lines is the first to demonstrate that JAK2/STAT3 pathway may be one of the downstream events in EMT-like process and alpha6beta1 integrin-mediated signalling in ovarian carcinomas.

Interaction of cancer cells with stroma cells facilitates tumor progression by rebuilding the existing extracellular matrix (ECM) microenvironment. In the tumor, upregulation of Tenascin-C (Tn-C) expression potentially can alter tumor behavior. However, the molecular mechanisms by which tumor-stroma interactions affect the tumor microenvironment have not been well characterized. In this study, we analyzed the expression of fibrillar Tn-C (fTn-C) in human metastatic pancreatic cancers. After co-culturing two pancreatic cancer cell lines, highly metastatic BxPc3 cells and non-metastatic PaCa2 cells, with stromal fibroblasts (SF), we evaluated the roles of matrix metalloproteinase 2 (MMP-2) activation and SF in promoting Tn-C organization. Next, we evaluated whether fibrillar Tn-C promotes pancreatic cancer cell movement using cell adhesion and migration assays. Finally, we observed the relationship between MMP-2 activation and fTn-C formation in vivo by injecting the BxPc3 and PaCa2 cells into nude mice. We found that fTn-C was increased in metastatic pancreatic cancer. The fTn-C expression correlated with MMP-2 activity. In the in vitro co-culture, fTn-C organization was found only in BxPc3/SF co-cultures, and required the participation of active MMP-2. The fTn-C reduced cell adhesion and promote pancreatic cancer cell migration by decreasing the adhesive interactions between integrin alpha6beta1 and the ECM. The in vivo tumorigenesis analysis showed that the fTn-C formation and active MMP-2 were significantly increased in the BxPc3 tumors, compared to the PaCa2 tumors. These results demonstrate that Tn-C deposition into the ECM requires participation of active MMP-2 and SF. The deposited Tn-C could promote pancreatic cancer progression.

Spermiation is the final phase of spermatogenesis leading to release of mature spermatids into the lumen of the seminiferous tubules. Morphologically, it involves a series of events, namely removal of excess spermatid cytoplasm, removal of ectoplasmic specialization, formation of tubulobulbar complex, and final disengagement of the spermatid from the Sertoli cell. Previous studies in our laboratory have shown that administration of 17beta-estradiol at a dose of 100 microg/kg body weight for 10 d resulted in failure of spermiation. This was accompanied by a suppression of FSH and intratesticular testosterone with a concomitant rise in intratesticular 17beta-estradiol. The present study was undertaken to determine the cause of failure and subsequently the molecular events in spermiation. Electron microscopic and confocal studies revealed an absence of tubulobulbar complex in step 19 spermatids after estradiol treatment, highlighting the significance of these structures in spermiation. It was further observed that treatment affected the Sertoli cell cytoskeleton and Arp2/3 complex that is critical for de novo polymerization of actin during tubulobulbar complex formation. In conclusion, the present study reports the role of 17beta-estradiol in inhibiting the formation of tubulobulbar complex, which could be one of the mechanism by which environmental estrogens influence male fertility.

Molecular and cellular interactions coordinating the origin and fate of neural stem cells (NSCs) in the adult brain are far from being understood. We present a protein complex that controls proliferation and migration of adult NSCs destined for the mouse olfactory bulb (OB). Combinatorial selection based on phage display technology revealed a previously unrecognized complex between the soluble protein netrin-4 and laminin gamma1 subunit that in turn activates an alpha6beta1 integrin-mediated signaling pathway in NSCs. Differentiation of NSCs is accompanied by a decrease in netrin-4 receptors, indicating that netrin-4 participates in the continual propagation of this stem cell population. Notably, the stem cells themselves do not synthesize netrin-4. Further, we show that netrin-4 is produced by selected GFAP-positive astrocytes positioned close to newborn neurons migrating in the anterior part of the rostral migratory stream (RMS) and within the OB. Our findings present a unique molecular mechanism mediating astrocytic/neuronal crosstalk that regulates ongoing neurogenesis in the adult olfactory system.

In some cases of infertility, implantation failure is due to a lack of expression of specific critical participating proteins such as cell adhesion molecules. The expression of beta 1 (beta1) integrin molecules within endometrial tissue has been proposed as a marker of uterine receptivity during the implantation window. Present study was conducted to assess uterine receptivity in women with unexplained infertility using beta1 integrin molecules within endometrial tissue in comparison with fertile women. This retrospective study was performed using a semiquantitative analysis on the immunohistochemical staining of beta1 integrins (VLA-4, VLA-5, VLA-6) in the mid-secretory phase of endometrium. Specimens were obtained from 30 fertile women and 28 infertile patients with a history of unexplained infertility. Chi-Square test was used to compare the expression and defect of beta1 integrin molecules between two groups. The results showed beta1 integrin molecules were present in fertile and infertile endometrial uterine tissues with different reactivity in different compartments. VLA-5 and VLA-6 expression on endometrial compartments showed an unrelated pattern of staining in either fertile or infertile women. The majority of glandular epithelial cells and stromal cells expressed VLA-4 integrin molecules in fertile endometrium. However, the reactivity with VLA-4 reduced significantly in both glandular epithelial cells and stromal cells in infertile women (p = 0.001). In conclusion differences may explain causes of unexplained infertility and suggests that VLA-4 integrin molecule may contribute in uterine endometrial receptivity at the time of the implantation window which requires more investigations in benign gynecologic diseases.

Re-epithelialization after skin wounding requires both migration and hyperproliferation of keratinocytes. Laminin-332 is deposited during migration over the provisional matrix. To investigate the function of the laminin-332 binding integrin alpha3beta1 in wound re-epithelialization, we generated Itga3flox/flox; K14-Cre mice lacking the alpha3 subunit specifically in the basal layer of the epidermis. These mice are viable but display several skin defects, including local inflammation, hair loss, basement membrane duplication and microblistering at the dermal-epidermal junction, whereas hemidesmosome assembly and keratinocyte differentiation are not impaired. Wound healing is slightly faster in the absence of integrin alpha3beta1, whereas proliferation, the distribution of other integrins and the deposition of basement membrane proteins in the wound bed are unaltered. In vitro, cell spreading is rescued by increased surface expression of alpha6beta1 integrin in the absence of integrin alpha3. The alpha3-deficient keratinocytes migrate with an increased velocity and persistence, whereas proliferation, growth factor signaling, hemidesmosome assembly, and laminin-332 deposition appeared to be normal. We suggest that integrin alpha3beta1 delays keratinocyte migration during wound re-epithelialization, by binding to the laminin-332 that is newly deposited on the wound bed.

The heparin-binding growth factor, MK, promoting tumorigenesis and survival was found to associate with alpha6beta1 integrins. We showed for the first time that MK interacted with TSPAN1 and facilitated the association between TSPAN1 and integrin alpha6beta1 proteins in head and neck squamous cell carcinoma (HNSCC) cells. We found that MK mediated an integrin-dependent tyrosine phosphorylation of FAK and activation of paxillin and Stat1alpha pathways. As result, downstream target genes implicated in cell migration and invasiveness (e.g. MMP-2 and MMP-26) were overexpressed. We observed that RNAi silencing of the critical signaling intermediates led to decrease of MK-induced migration/invasiveness of HNSCC cells. The major finding of this study is a novel MK-triggered signaling mechanism implicated in migration and invasiveness of HNSCC cells.

Integrins govern cellular adhesion and transmit signals leading to activation of intracellular signaling pathways aimed to prevent apoptosis. Herein we report that attachment of oligodendrocytes (OLs) to fibronectin via alpha(v)beta(3) integrin receptors rendered the cells more resistant to apoptosis than the cells attached to laminin via alpha(6)beta(1) integrins. Investigation of molecular mechanisms involved in alpha(v)beta(3) integrin-mediated cell survival revealed that ligation of the integrin with fibronectin results in higher expression of activated Lyn kinase. Both in OLs and in the mouse brain, Lyn selectively associates with alpha(v)beta(3) integrin, not with alpha(v)beta(5) integrin, leading to suppression of acid sphingomyelinase activity and preventing ceramide-mediated apoptosis. In OLs, knockdown of Lyn with small interfering RNA resulted in OL apoptosis with concomitant accumulation of C(16)-ceramide due to activation of acid sphingomyelinase (ASMase) and sphingomyelin hydrolysis. Knocking down ASMase partially protected OLs from apoptosis. In the brain, ischemia/reperfusion (IR) triggered rearrangements in the alpha(v)beta(3) integrin-Lyn kinase complex leading to disruption of Lyn kinase-mediated suppression of ASMase activity. Thus, co-immunoprecipitation studies revealed an increased association of alpha(v)beta(3) integrin-Lyn kinase complex with ionotropic glutamate receptor subunits, GluR2 and GluR4, after cerebral IR. Sphingolipid analysis of the brain demonstrated significant accumulation of ceramide and sphingomyelin hydrolysis. The data suggest a novel mechanism for regulation of ASMase activity during cell adhesion in which Lyn acts as a key upstream kinase that may play a critical role in cerebral IR injury.

There is an emerging understanding of the importance of the vascular system within stem cell niches. Here, we examine whether neural stem cells (NSCs) in the adult subventricular zone (SVZ) lie close to blood vessels, using three-dimensional whole mounts, confocal microscopy, and automated computer-based image quantification. We found that the SVZ contains a rich plexus of blood vessels that snake along and within neuroblast chains. Cells expressing stem cell markers, including GFAP, and proliferation markers are closely apposed to the laminin-containing extracellular matrix (ECM) surrounding vascular endothelial cells. Apical GFAP+ cells are admixed within the ependymal layer and some span between the ventricle and blood vessels, occupying a specialized microenvironment. Adult SVZ progenitor cells express the laminin receptor alpha6beta1 integrin, and blocking this inhibits their adhesion to endothelial cells, altering their position and proliferation in vivo, indicating that it plays a functional role in binding SVZ stem cells within the vascular niche.

During extravasation and within lymph nodes (LNs), blood lymphocytes interact with laminins (Lms), major components of vascular basement membranes (BMs) and of reticular fibers (RFs), a fibrillar extracellular matrix. However, the identity and role of these laminin isoform(s) are poorly known. By using confocal microscopy examination of human LNs, we show that BMs of high endothelial venules (HEVs) express laminin alpha3, alpha4, alpha5, beta1, beta2, and gamma1 chains and that the same chains, in addition to alpha2, are found in RFs. In functional studies with laminin isoforms covering all Lm alpha chains, alpha5-laminin (Lm-511) was the most adhesion- and migration-promoting isoform for human blood lymphocytes, followed by alpha3- (Lm-332) and alpha4- (Lm-411) laminins, and the lymphocytes used the alpha6beta1 integrin as the primary receptor for the alpha5-laminin. Moreover, Lm-511 strongly co-stimulated T cell proliferation, and blood lymphocytes were able to secrete alpha4- and alpha5-laminins following stimulation. The LN cell number in laminin alpha4-deficient mice compared with wild-type did not differ significantly. This study demonstrates a predominant role for alpha5-laminin(s) in blood lymphocyte biology and identifies LN laminins and their integrin receptors in blood lymphocytes.

We previously showed that a local immune response largely composed of type 1 T cells correlated with a favorable outcome of the peritonitis associated with peritoneal dialysis. To clarify how these subsets are recruited to the peritoneal cavity during inflammation, we measured integrin-mediated interactions between the T cells and human peritoneal mesothelial cells. Direct microscopy showed that lipopolysaccharide or peritoneal dialysis effluent stimulated the adherence of T cells to mesothelial cells, a process mediated by the integrins alpha6beta1 and alpha4beta1. Further, the migration of Th1 cell across human mesothelial cell monolayers grown on transwell surfaces was reduced by anti-alpha6beta1 integrin antibody while that of Th2 cell was inhibited by an anti-alpha4 integrin antibody. Pretreatment with either lipopolysaccharide or rapid response peritoneal dialysis effluent stimulated T cell migration and this was significantly decreased by the alpha6beta1 compared to the alpha4 antibody. These results suggest that integrins may play an important role in mediating selective T cell subset adhesion and migration across human peritoneal mesothelial cell monolayers and differential integrin expression and selective T cell subset recruitment during peritonitis may affect outcome.

A prerequisite for axon regeneration is the interaction between the growth cone and the extracellular matrix (ECM). Laminins are prominent constituents of ECM throughout the body, known to support axon growth in vitro and in vivo. The regenerative capacity of adult neurons is greatly diminished compared to embryonic or early postnatal neurons. Since most lesions in the nervous system occur in the adult, we have examined neurite outgrowth from adult mouse DRG neurons on four laminin isoforms (laminin-1/LM-111, laminin-2/LM-211, laminin-8/LM-411 and laminin-10/LM-511) in vitro. The growth on laminin-1 and -10 was trophic factor-independent and superior to the one on laminin-2 and -8, where growth was very poor in the absence of neurotrophins. Among other ECM proteins, laminins were by far the most active molecules. Using function-blocking antibodies to laminin-binding integrins, we identified non-overlapping functions of integrins alpha3beta1, alpha7beta1 and alpha6beta1 on different laminin isoforms, in that alpha3beta1 and alpha7beta1 integrins appeared to be specific receptors for both laminin-1 and-2, whereas integrin alpha6beta1 was a receptor for laminin-8 and-10. Lastly, by use of immunohistochemistry, expression of subunits of laminin-1, -2, -8 and -10 in sensory organs in the human epidermis could be demonstrated, supporting an important role for these laminins in relation to primary sensory axons.