MAB2253Z Sigma-AldrichAnti-Integrin β1 Antibody, clone 6S6, Azide Free

We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.

Wnt5a is a representative ligand that activates the ?-catenin-independent pathway of Wnt signaling in mammals. This pathway might be related to planar cell polarity signaling in Drosophila. Because reliable biochemical assays to measure Wnt5a pathway activity have not yet been established, we examined whether Wnt5a signaling stimulates focal adhesion turnover in migrating cells using live immunofluorescence imaging and immunocytochemical analysis. These assays demonstrated that the Wnt5a pathway cooperates with integrin signaling to regulate cell migration and adhesion through focal adhesion dynamics.

Rab GTPases play an essential role in vesicular transport by coordinating the movement of various types of cargo from one cellular compartment to another. Individual Rab GTPases are distributed to specific organelles and thus serve as markers for discrete types of endocytic vesicles. Mammalian reovirus binds to cell surface glycans and junctional adhesion molecule-A (JAM-A) and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within organelles of the endocytic compartment, reovirus undergoes stepwise disassembly catalyzed by cathepsin proteases, which allows the disassembly intermediate to penetrate endosomal membranes and release the transcriptionally active viral core into the cytoplasm. The pathway used by reovirus to traverse the endocytic compartment is largely unknown. In this study, we found that reovirus particles traffic through early, late, and recycling endosomes during cell entry. After attachment to the cell surface, reovirus particles and JAM-A codistribute into each of these compartments. Transfection of cells with constitutively active and dominant-negative Rab GTPases that affect early and late endosome biogenesis and maturation influenced reovirus infectivity. In contrast, reovirus infectivity was not altered in cells expressing mutant Rab GTPases that affect recycling endosomes. Thus, reovirus virions localize to early, late, and recycling endosomes during entry into host cells, but only those that traverse early and late endosomes yield a productive infection.

Reovirus cell entry is initiated by viral attachment to cell surface glycans and junctional adhesion molecule A. Following receptor engagement, reovirus is internalized into cells by receptor-mediated endocytosis using a process dependent on β1 integrin. Endocytosed virions undergo stepwise disassembly catalyzed by cathepsin proteases, followed by endosomal membrane penetration and delivery of transcriptionally active core particles into the cytoplasm. Cellular factors that mediate reovirus endocytosis are poorly defined. We found that both genistein, a broad-spectrum tyrosine kinase inhibitor, and PP2, a specific Src-family kinase inhibitor, diminish reovirus infectivity by blocking a cell entry step. Although neither inhibitor impedes internalization of reovirus virions, both inhibitors target virions to lysosomes. Reovirus colocalizes with Src during cell entry, and reovirus infection induces phosphorylation of Src at the activation residue, tyrosine 416. Diminished Src expression by RNA interference reduces reovirus infectivity, suggesting that Src is required for efficient reovirus entry. Collectively, these data provide evidence that Src kinase is an important mediator of signaling events that regulate the appropriate sorting of reovirus particles in the endocytic pathway for disassembly and cell entry.

Ameloblastin, the most abundant nonamelogenin enamel matrix protein, plays a role in ameloblast differentiation. Here, we found that ameloblastin was expressed in osteosarcoma cells; to explore the potential functions of ameloblastin in osteoblasts, we investigated whether this protein is involved in osteogenic differentiation and bone formation on the premise that CD63, a member of the transmembrane-4 glycoprotein superfamily, interacts with integrins in the presence of ameloblastin. Ameloblastin bound to CD63 and promoted CD63 binding to integrin β1. The interaction between CD63 and integrin β1 induced Src kinase inactivation via the binding of CD63 to Src. The reduction of Src activity and osteogenic differentiation mediated by ameloblastin were abrogated by treatment with anti-CD63 antibody and overexpression of constitutively active Src, respectively. Therefore, our results suggest that ameloblastin is expressed in osteoblasts and functions as a promoting factor for osteogenic differentiation via a novel pathway through the interaction between CD63 and integrin β1.

Cell migration through connective tissue, or cell invasion, is a fundamental biomechanical process during metastasis formation. Cell invasion usually requires cell adhesion to the extracellular matrix through integrins. In some tumors, increased integrin expression is associated with increased malignancy and metastasis formation. Here, we have studied the invasion of cancer cells with different α5β1 integrin expression levels into loose and dense 3D collagen fiber matrices. Using a cell sorter, we isolated from parental MDA-MB-231 breast cancer cells two subcell lines expressing either high or low amounts of α5β1 integrins (α5β1(high) or α5β1(low) cells, respectively). α5β1(high) cells showed threefold increased cell invasiveness compared to α5β1(low) cells. Similar results were obtained for 786-O kidney and T24 bladder carcinoma cells, and cells in which the α5 integrin subunit was knocked down using specific siRNA. Knockdown of the collagen receptor integrin subunit α2 also reduced invasiveness, but to a lesser degree than knockdown of integrin subunit α5. Fourier transform traction microscopy revealed that the α5β1(high) cells generated sevenfold greater contractile forces than α5β1(low) cells. Cell invasiveness was reduced after addition of the myosin light chain kinase inhibitor ML-7 in α5β1(high) cells, but not in α5β1(low) cells, suggesting that α5β1 integrins enhance cell invasion through enhanced transmission and generation of contractile forces.

Calreticulin (CRT) is a multi-functional Ca(2+) -binding molecular chaperone in the endoplasmic reticulum. We previously reported that kidney epithelial cell-derived Madin-Darby Canine Kidney cells were transformed into mesenchymal-like cells by gene transfection of CRT. In this study, we investigated the altered characteristics of cell adhesion in these epithelial-mesenchymal transition (EMT)-like cells. Several extracellular matrix substrata were tested, and cell adhesion to fibronectin was found to be specifically increased in the CRT-overexpressing cells compared to controls. The expression of integrins was significantly up-regulated in subunits ?5 and ?V, resulting in an increase in the formation of complexes such as ?5?1 and ?V?3. These integrins also contributed to the enhanced binding of fibronectin. In the CRT-overexpressing cells, the phosphorylation of Akt, a downstream target of integrin-linked kinase (ILK), was up-regulated on attachment to fibronectin or collagen IV. Integrin-associated signaling through ILK was also promoted on attachment to fibronectin, suggesting some of the correlation between ILK and Akt in the CRT-overexpressing cells. Furthermore, on treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester, a membrane-permeable Ca(2+) chelator, the enhanced Akt signaling was suppressed with a concomitant decrease in the formation of complexes between integrins and ILK in the CRT-overexpressing cells. In conclusion, these findings demonstrate that CRT regulates cell-substratum adhesion by modulating integrin-associated signaling through altered Ca(2+) homeostasis in the CRT-overexpressing EMT-like cells, suggesting a novel regulatory role for CRT in EMT.

By interacting with components of the human host, including extracellular matrix (ECM) proteins, Streptococcus pneumoniae has evolved various strategies for colonization. Here, we characterized the interaction of pneumococci with the adhesive glycoprotein vitronectin and the contribution of this protein to pneumococcal uptake by host cells in an integrin-dependent manner. Specific interaction of S. pneumoniae with the heparin-binding sites of purified multimeric vitronectin was demonstrated by flow cytometry analysis. Host-cell-bound vitronectin promoted pneumococcal adherence to and invasion into human epithelial and endothelial cells. Pneumococci were trapped by microspike-like structures, which were induced upon contact of pneumococci with host-cell-bound vitronectin. Alphavbeta3 integrin was identified as the major cellular receptor for vitronectin-mediated adherence and uptake of pneumococci. Ingestion of pneumococci by host cells via vitronectin required a dynamic actin cytoskeleton and was dependent on integrin-linked kinase (ILK), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt), as demonstrated by gene silencing or in inhibition experiments. In conclusion, pneumococci exploit the vitronectin-alphavbeta3-integrin complex as a cellular receptor for invasion and this integrin-mediated internalization requires the cooperation between the host signalling molecules ILK, PI3K and Akt.

We have designed multifunctional peptide fibrils using bioactive laminin-derived peptides and evaluated their potential as a biomedical material for tissue engineering. The Leu-Arg-Gly-Asp-Asn (LRGDN) peptide derived from laminin-111, which contains an RGD sequence bound to integrin alphavbeta3, was added to the N-terminus of the four amyloidogenic cell-adhesive laminin-derived peptides (A119: LSNIDYILIKAS, AG97: SAKVDAIGLEIV, B133: DISTKYFQMSLE, and B160: VILQQSAADIAR). The RGD-conjugated peptides were stained with Congo red and exhibited amyloid-like fibril formation in the electron microscopic. The RGD-conjugated peptides promoted human dermal fibroblasts spreading with well-organized actin stress fibers and focal contacts. Human dermal fibroblast attachment to the RGD-conjugated peptides was inhibited by anti-alphav integrin antibody. Further, cell attachment to B133 was inhibited by anti-alpha2 and anti-beta1 integrin antibodies, whereas attachment to RGD-B133 was inhibited by anti-alphav and anti-beta1 integrin antibodies. These results suggest that the RGD-conjugated peptides interact with integrin alphavbeta3 and that RGD-B133 interacts with both integrin alphavbeta3 and integrin beta1. The RGD-conjugated peptide fibrils promoted neurite outgrowth in a peptide-dependent manner. These results support that biologically active sequence-conjugated peptide fibrils interact in a receptor-specific manner with cells and promote multifunctional activities. These fibrils may have use as biological supports for cell-specific tissue engineering.

Host interactions with tumor cells contribute to tumor progression by several means. This study was done to determine whether mammary epithelium could interact with breast carcinoma by producing substances capable of inducing motility in the cancer cells. Conditioned medium of immortalized 184A1 mammary epithelium collected in serum-free conditions induced dose-dependent motility in the MCF-7 breast carcinoma cell line by both a semiquantitative scattering assay and a Boyden chamber assay. Purification of the motility factor revealed that it was laminin 332 (formerly laminin 5) by mass spectroscopy. A Western blot of the 184A1 conditioned medium using a polyclonal antibody confirmed the presence of laminin 332 in the conditioned medium. Blockage of the motility with antibodies to the laminin 332 and its receptor components, alpha(3) and beta(1) integrins, provided further evidence that tumor cell motility was caused by the laminin 332 in the conditioned medium. Invasion of MCF-7, BT-20, and MDA-MB-435 S was induced by purified laminin 332 and 184A1 conditioned medium and blocked by an anti-alpha(3) integrin antibody. Staining of carcinoma in situ from breast cancer specimens revealed that laminin 332 in the myoepithelium adjacent to the preinvasive cells provided a source of laminin 332 that could potentially encourage the earliest steps of stromal invasion. In metaplastic breast carcinomas, the presence of laminin 332-producing cells coexpressing alpha(3) integrin and the greater metastatic potential of tumors with higher laminin 332 levels suggest that laminin 332 expression is associated with aggressive features in these human breast cancers.