Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by beta1 integrins.

The anti-angiogenic effect of thrombospondin-1 has been shown to be mediated through binding of the type-1 repeat (TSR) domain to the CD36 transmembrane receptor. We now report that the TSR domain can inhibit VEGF-induced migration in human umbilical vein endothelial cells (HUVEC), cells that lack CD36. Moreover, we identified beta1 integrins as a critical receptor in TSR-mediated inhibition of migration in HUVEC. Using pharmacological inhibitors of downstream VEGF receptor effectors, we found that phosphoinositide 3-kinase (PI3k) was essential for TSR-mediated inhibition of HUVEC migration, but that neither PLCgamma nor Akt was necessary for this response. Furthermore, beta1 integrins were critical for TSR-mediated inhibition of microvascular endothelial cells, cells that express CD36. Together, our results indicate that beta1 integrins mediate the anti-migratory effects of TSR through a PI3k-dependent mechanism.

Mechanical control of cAMP signaling through integrins is mediated by the heterotrimeric Galphas protein.

Mechanical stresses that are preferentially transmitted across the cell surface via transmembrane integrin receptors activate gene transcription by triggering production of intracellular chemical second messengers, such as cAMP. Here we show that the sensitivity of the cAMP signaling pathway to mechanical stresses transferred across beta1 integrins is mediated by force-dependent activation of the heterotrimeric G protein subunit Galphas within focal adhesions at the site of stress application. Galphas is recruited to focal adhesions that form within minutes following clustering of beta1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and beta1 integrin and Galphas co-precipitate when analyzed biochemically. Stress application to activated beta1 integrins using magnetic twisting cytometry increases Galphas recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azido-anilido-GTP, whereas application of similar stresses to inactivated integrins or control histocompatibility antigens has little effect. This response is relevant physiologically as application of mechanical strain to cells bound to flexible extracellular matrix-coated substrates induce translocation of phospho-CREB to the nucleus, which can be attenuated by inhibiting Galphas activity, either using the inhibitor melittin or suppressing its expression using siRNA. Although integrins are not typical G protein-coupled receptors, these results show that integrins focus mechanical stresses locally on heterotrimeric G proteins within focal adhesions at the site of force application, and transduce mechanical stimuli into an intracellular cAMP signaling response by activating Galphas at these membrane signaling sites.

B2A peptide induces chondrogenic differentiation in vitro and enhances cartilage repair in rats.

This study investigated whether the synthetic peptide B2A (B2A2-K-NS) could induce in vitro chondrogenic differentiation and enhance the in vivo repair of damaged cartilage in an osteoarthritis model. In vitro, micromass cultures of murine and human stem cells with and without B2A were used as models of chondrogenic differentiation. Micromasses were evaluated for gene expression using microarray analysis and quantitative PCR; and for extracellular matrix production by Alcian blue staining for sulfated glycosaminoglycan and immunochemical detection of collagen type II. In vivo, osteoarthritis was chemically induced in knees of adult rats by an injection of mono-iodoacetate (MIA) into the synovial space. Treatment was administered at 7- and 14 days after the MIA by injection into the synovial space of B2A or saline and terminated at 21 days, after which knee cartilage damage was determined and scored by histological analysis. In murine C3H10T1/2 micromass culture, B2A induced the expression of more than 11 genes associated with growth factors/receptors, transcription, and the extracellular matrix, including PDGF-AA. B2A also significantly increased the sulfated glycosaminoglycan and collagen of murine- and human micromass cultures. In the knee osteoarthritis model, B2A treatment enhanced cartilage repair compared to untreated knees as determined histologically by a decrease in damage indicators. These findings suggest that B2A induces stem cells chondrogenic differentiation in vitro and enhances cartilage repair in vivo. The results suggest that B2A might be useful to promote cartilage repair.

A synthetic, bioactive PDGF mimetic with binding to both alpha-PDGF and beta-PDGF receptors.

A multi-domain peptide, PAB2-1c, was designed and synthesized as a bioactive mimic of PDGF. PBA2-1c bound to both alpha- and beta-PDGF receptors as determined by surface plasmon resonance (SPR). The equilibrium dissociation constant (Kd) of binding to alpha-PDGF receptors by PAB2-1c (1.7 x 10(-8) M) compared favorably rhPDGF-AA (1.34 x 10(-8) M). Binding to -PDGF receptor by PAB2-1c (2.2 x 10(-8) M) was less favorable than, that of recombinant human PDGFBB (1.59 x 10(-9) M). Interestingly, PBA2-1c bound to these two receptors with similar affinity suggesting that, PBA2-1c was not PDGF receptor selective. In a murine myoblast cell line C2C12, PBA2-1c increased the tyrosine phosphorylation on PDGF receptors and the phosphorylation of AKT and ERK1/2 in a concentration-related manner. PBA2-1c also stimulated an increase in cell proliferation, cell migration, and collagen gel contraction. In these cell-based assays, PAB2-1c was effective at 1 microg/ml or lesser. The results support the hypothesis that PBA2-1c is a mimetic of PDGF, although it has a more promiscuous receptor interaction.

Src-mediated RGS16 tyrosine phosphorylation promotes RGS16 stability.

The amplitude of signaling evoked by stimulation of G protein-coupled receptors may be controlled in part by the GTPase accelerating activity of the regulator of G protein signaling (RGS) proteins. In turn, subcellular targeting, protein-protein interactions, or post-translational modifications such as phosphorylation may shape RGS activity and specificity. We found previously that RGS16 undergoes tyrosine phosphorylation on conserved tyrosine residues in the RGS box. Phosphorylation on Tyr(168) was mediated by the epidermal growth factor receptor (EGFR). We show here that endogenous RGS16 is phosphorylated after epidermal growth factor stimulation of MCF-7 cells. In addition, p60-Src or Lyn kinase phosphorylated recombinant RGS16 in vitro, and RGS16 underwent phosphorylation in the presence of constitutively active Src (Y529F) in EGFR(-) CHO-K1 cells. Blockade of endogenous Src activity by selective inhibitors attenuated RGS16 phosphorylation induced by pervanadate or receptor stimulation. Furthermore, the rate of RGS16 degradation was reduced in cells expressing active Src or treated with pervanadate or a G protein-coupled receptor ligand (CXCL12). Induction of RGS16 tyrosine phosphorylation was associated with increased RGS16 protein levels and enhanced GAP activity in cell membranes. These results suggest that Src mediates RGS16 tyrosine phosphorylation, which may promote RGS16 stability.