MAB1959 Sigma-AldrichAnti-Integrin β1 Antibody, clone P5D2

Studies of cellular mechanotransduction have converged upon the idea that cells sense extracellular matrix (ECM) elasticity by gauging resistance to the traction forces they exert on the ECM. However, these studies typically utilize purely elastic materials as substrates, whereas physiological ECMs are viscoelastic, and exhibit stress relaxation, so that cellular traction forces exerted by cells remodel the ECM. Here we investigate the influence of ECM stress relaxation on cell behaviour through computational modelling and cellular experiments. Surprisingly, both our computational model and experiments find that spreading for cells cultured on soft substrates that exhibit stress relaxation is greater than cells spreading on elastic substrates of the same modulus, but similar to that of cells spreading on stiffer elastic substrates. These findings challenge the current view of how cells sense and respond to the ECM.

Insulin-like growth factor binding protein-2 (IGFBP-2) is significantly increased in the serum of patients with malignant gliomas. High plasma IGFBP-2 levels are correlated with poor prognosis in glioma patients. However, the exact role of exogenous IGFBP-2 in gliomas is unclear.Using the MTT cell viability assay, cell cycle analysis, and the transwell migration assay, it was demonstrated that IGFBP-2 treatment stimulated proliferation and invasion in U87 and U251 cell lines and primary SU3 glioma cells. Western blot analysis and immunofluorescence staining revealed that IGFBP-2 promoted ERK phosphorylation and nuclear translocation. Moreover, blocking ERK activation using the inhibitor PD98059 markedly reduced the effects of IGFBP-2 in glioma cells. As IGFBP-2 has an integrin-binding domain, the contribution of integrin β1 to these IGFBP-2-mediated processes was examined. Neutralisation or knockdown of the expression of integrin β1 inhibited IGFBP-2-induced ERK activation, cell proliferation, and cell invasion. Significantly, IGFBP-2 induced temozolomide resistance in glioma cells in an integrin β1/ERK-dependent manner.Exogenous IGFBP-2 induces proliferation, invasion, and chemoresistance in glioma cells via integrin β1/ERK signaling, suggesting that targeting this pathway could represent a potential therapeutic strategy for the treatment of gliomas. The identification of this pathway in glioma progression provides insight into the mechanism by which serum IGFBP-2 levels can predict the prognosis of glioma patients.

Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif ((329)RGD(331)). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5β1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the (329)RGD(331) motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5β1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines.Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral fusion (F) protein mediates both attachment and fusion activity. Recently, it was suggested that hMPV F protein utilizes integrins as receptors for entry via a poorly understood mechanism. Here, we show that α5β1 and αv integrins are essential for hMPV infectivity and F protein-mediated cell-cell fusion and that the integrin-binding motif in the F protein plays a crucial role in these functions. Our results also identify the integrin-binding motif to be a new, attenuating target for the development of a live vaccine for hMPV. These findings not only will facilitate the development of antiviral drugs targeting viral entry steps but also will lead to the development new live attenuated vaccine candidates for hMPV.

Rab25 is a tumor suppressor for colon cancer in humans and mice. To identify elements of intestinal polarity regulated by Rab25, we developed Caco2-BBE cell lines stably expressing short hairpin RNA for Rab25 and lines rescuing Rab25 knockdown with reexpression of rabbit Rab25. Rab25 knockdown decreased α2-, α5-, and β1-integrin expression. We observed colocalization and direct association of Rab25 with α5β1-integrins. Rab25 knockdown also up-regulated claudin-1 expression, increased transepithelial resistance, and increased invasive behavior. Rab25-knockdown cells showed disorganized brush border microvilli with decreases in villin expression. All of these changes were reversed by reintroduction of rabbit Rab25. Rab25 knockdown altered the expression of 29 gene transcripts, including the loss of α5-integrin transcripts. Rab25 loss decreased expression of one transcription factor, ETV4, and overexpression of ETV4 in Rab25-knockdown cells reversed losses of α5β1-integrin. The results suggest that Rab25 controls intestinal cell polarity through the regulation of gene expression.

Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.

Background: Pancreatic cancer shows a highly aggressive and infiltrative growth pattern and is characterized by an abundant tumor stroma known to interact with the cancer cells, and to influence tumor growth and drug resistance. Cancer cells actively take part in the production of extracellular matrix proteins, which then become deposited into the tumor stroma. Type IV collagen, an important component of the basement membrane, is highly expressed by pancreatic cancer cells both in vivo and in vitro. In this study, the cellular effects of type IV collagen produced by the cancer cells were characterized. <br />Methods: The expression of type IV collagen and its integrin receptors were examined in vivo in human pancreatic cancer tissue. The cellular effects of type IV collagen were studied in pancreatic cancer cell lines by reducing type IV collagen expression through RNA interference and by functional receptor blocking of integrins and their binding-sites on the type IV collagen molecule. <br />Results: We show that type IV collagen is expressed close to the cancer cells in vivo, forming basement membrane like structures on the cancer cell surface that colocalize with the integrin receptors. Furthermore, the interaction between type IV collagen produced by the cancer cell, and integrins on the surface of the cancer cells, are important for continuous cancer cell growth, maintenance of a migratory phenotype, and for avoiding apoptosis. <br />Conclusion: We show that type IV collagen provides essential cell survival signals to the pancreatic cancer cells through an autocrine loop.

Syntenin is a scaffolding-PDZ domain-containing protein. Although it is reported that syntenin is associated with melanoma growth and metastasis, the possible role of syntenin in breast cancer has not been well elucidated. The present study investigated the expression and function of syntenin in breast cancer.Real-time polymerase chain reaction (PCR) and Western blots were used to determine the mRNA and protein expression of syntenin. With a combination of overexpression and RNA interference, the effect of syntenin on migration, invasion, and ERK1/2 activation was examined in breast cancer cell lines. The effect of syntenin in vivo was assessed with an orthotropic xenograft tumor model in BALB/c nu/nu mice. In addition, the expression level of syntenin in clinical breast cancer tissues was evaluated with immunohistochemistry. The Kaplan-Meier survival curve was used to evaluate patient survival, and the Cox proportional hazards model was used for multivariate analysis.Our study showed that syntenin expression was upregulated in high-metastasis breast cancer cell lines and breast cancer tissues. Overexpression of syntenin in breast cancer cells promoted cell migration and invasion in vitro. Moreover, overexpression of syntenin promoted breast tumor growth and lung metastasis in vivo. We further showed that activation of integrin β1 and ERK1/2 was required for syntenin-mediated migration and invasion of breast cancer cells. The correlation between syntenin expression and tumor size (P = 0.011), lymph node status (P = 0.001), and recurrence (P = 0.002) was statistically significant. More important, syntenin expression in primary tumors was significantly related to patients' overall survival (OS; P = 0.023) and disease-free survival (DFS; P = 0.001). Its status was an independent prognostic factor of OS (P = 0.049) and DFS (P = 0.002) in our cohort of patients.These results suggest that syntenin plays a significant role in breast cancer progression, and it warrants further investigation as a candidate molecular marker of breast cancer metastasis and a potential therapeutic target.

The syndecans are a family of heparan-sulfate-decorated cell surface proteoglycans, matrix receptors with roles in cell adhesion and growth factor signaling. Their heparan sulfate chains recognize "heparin-binding" motifs ubiquitously present in the extracellular matrix (ECM), providing the means for syndecans to constitutively bind and cluster to sites of cell-matrix adhesion. Emerging evidence suggests that specialized docking sites in the syndecan extracellular domains may serve to localize other receptors to these sites as well, including integrins and growth factor receptor tyrosine kinases. A prototypic example of this mechanism is the capture of the αvβ3 integrin and insulin-like growth factor-1 receptor (IGF1R) by syndecan-1 (Sdc1) - forming a ternary receptor complex in which signaling downstream of IGF1R activates the integrin. This Sdc1-coupled ternary receptor complex is especially prevalent on tumor cells and activated endothelial cells undergoing angiogenesis, reflecting the upregulated expression of αvβ3 integrin in such cells. As such, much effort has focused on developing therapeutics that target this integrin in various cancers. Along these lines, the site in the Sdc1 ectodomain responsible for capture and activation of the αvβ3 or αvβ5 integrins by IGF1R can be mimicked by a short peptide called "synstatin" (SSTN), which competitively displaces the integrin and IGF1R kinase from the syndecan and inactivates the complex. This review summarizes our current knowledge of the Sdc1-coupled ternary receptor complex and the efficacy of SSTN as an emerging therapeutic to target this signaling mechanism. © 2013 The Authors Journal compilation © 2013 FEBS.

Cancer cell invasion is a key element in metastasis that requires integrins for adhesion/de-adhesion, as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Herein we show that MMP-2 is up-regulated in resected colorectal tumors and degrades β1 integrins with the release of fragments containing the β1 I-domain. The β1 cleavage pattern is similar to that produced by digestion of α5β1 and α2β1 with MMP-2. Two such fragments, at 25 and 75 kDa, were identified after immunoprecipitation, with monoclonal antibody BD610468 reacting with the NH(2)-terminal I-like ectodomain followed by SDS-PAGE and microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometry. Cleavage of the β1 integrin can be abolished by inhibition of MMP-2 activity; it can be induced by up-regulation of MMP-2 expression, as exemplified by HT29 colon cancer cells transfected with pCMV6-XL5-MMP-2. Co-immunoprecipitation studies of colon cancer cells showed that the β1 integrin subunit is associated with MMP-2. The MMP-2-mediated shedding of the I-like domain from β1 integrins resulted in decreased adhesion of colon cancer cells to collagen and fibronectin, thus abolishing their receptivity. Furthermore, such cells showed enhanced motility as evaluated by a "wound healing-like" assay and time-lapse microscopy, indicating their increased invasiveness. Altogether, our data demonstrate that MMP-2 amplifies the motility of colon cancer cells, not only by digesting the extracellular matrix components in the vicinity of cancer cells but also by inactivating their major β1 integrin receptors.

In mouse and human skin, HIF-1α is constitutively expressed in the epidermis, mainly in the basal layer. HIF-1α has been shown to have crucial systemic functions: regulation of kidney erythropoietin production in mice with constitutive HIF-1α epidermal deletion, and hypervascularity following epidermal HIF-1α overexpression. However, its local role in keratinocyte physiology has not been clearly defined. To address the function of HIF-1α in the epidermis, we used the mouse model of HIF-1α knockout targeted to keratinocytes (K14-Cre/Hif1a(flox/flox)). These mice had a delayed skin phenotype characterized by skin atrophy and pruritic inflammation, partly mediated by basement membrane disturbances involving laminin-332 (Ln-332) and integrins. We also investigated the relevance of results of studies in mice to human skin using reconstructed epidermis and showed that HIF-1α knockdown in human keratinocytes impairs the formation of a viable reconstructed epidermis. A diminution of keratinocyte growth potential, following HIF-1α silencing, was associated with a decreased expression of Ln-322 and α6 integrin and β1 integrin. Overall, these results indicate a role of HIF-1α in skin homeostasis especially during epidermal aging.