Anti-Integrin beta 1 Antibody [MEM-101A] (A85541)

An essential phase of skeletal myogenesis is the fusion of mononucleated myoblasts to form multinucleated myotubes. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, but the mechanisms by which these proteins regulate cell fusion remain mostly unknown. Here, we focused on the role of focal adhesion kinase (FAK), an important nonreceptor protein tyrosine kinase involved in integrin signaling, as a potential mediator by which integrins may regulate myoblast fusion. To test this hypothesis in vivo, we generated mice in which the Fak gene was disrupted specifically in muscle stem cells ("satellite cells") and we found that this resulted in impaired myotube formation during muscle regeneration after injury. To examine the role of FAK in the fusion of myogenic cells, we examined the expression of FAK and the effects of FAK deletion on the differentiation of myoblasts in vitro. Differentiation of mouse primary myoblasts was accompanied by a rapid and transient increase of phosphorylated FAK. To investigate the requirement of FAK in myoblast fusion, we used two loss-of-function approaches (a dominant-negative inhibitor of FAK and FAK small interfering RNA [siRNA]). Inhibition of FAK resulted in markedly impaired fusion but did not inhibit other biochemical measures of myogenic differentiation, suggesting a specific role of FAK in the morphological changes of cell fusion as part of the differentiation program. To examine the mechanisms by which FAK may be regulating fusion, we used microarray analysis to identify the genes that failed to be normally regulated in cells that were fusion defective due to FAK inhibition. Several genes that have been implicated in myoblast fusion were aberrantly regulated during differentiation when FAK was inhibited. Intriguingly, the normal increases in the transcript of caveolin 3 as well as an integrin subunit, the beta1D isoform, were suppressed by FAK inhibition. We confirmed this also at the protein level and show that direct inhibition of beta1D subunit expression by siRNA inhibited myotube formation with a prominent effect on secondary fusion. These data suggest that FAK regulation of profusion genes, including caveolin 3 and the beta1D integrin subunit, is essential for morphological muscle differentiation.

This study appears from an experiment previously carried out in New Zealand white rabbits. Allogenic mesenchymal stem cells (MSCs) were transplanted into an iatrogenically-created defect in the lateral section of the distal physis of the left femur in 10 miniature pigs. The right femur with the same defect served as a control. To transfer MSCs, a freshly prepared porous scaffold was used, based on collagen and chitosan, constituting a compact tube into which MSCs were implanted. The pigs were euthanized four months after the transplantation. On average, the left femur with transplanted MSCs grew more in length (0.56+/-0.14 cm) compared with right femurs with physeal defect without transplanted MSCs (0.14+/-0.3 cm). The average angular (valgus) deformity of the left femur had an angle point of 0.78 degrees , following measurement and X-ray examination, whereas in the right femur without transplantation it was 3.7 degrees. The initial results indicate that preventive transplantation of MSCs into a physeal defect may prevent valgus deformity formation and probably also reduce disorders of the longitudinal bone growth. This part of our experiment is significant in the effort to advance MSCs application in human medicine by using pig as a model, which is the next step after experimenting on rabbits.

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.