Molecular mediators of alphavbeta3-induced endothelial cell survival.

The alphavbeta3 integrin interaction with the extracellular matrix (ECM) plays an essential role in inhibiting apoptosis in endothelial cells. We have recently shown that alphavbeta3 ligation on rat aortic endothelial cells (RAECs) specifically activates the transcription factor nuclear factor kappaB (NF-kappaB) and promotes cell survival. Inhibiting NF-kappaB nuclear translocation abolished the protective effect of alphavbeta3 ligands. Here, we report that ligation of alphavbeta3 by its ligand, osteopontin (OPN), induces the phosphorylation and activation of inhibitory kappa B kinase beta IKKbeta and promotes the specific degradation of inhibitory kappa Balpha (IkappaBalpha) in RAECs. Overexpression of a dominant negative (DN) IKKbeta protein prevents IkappaBalpha phosphorylation, NF-kappaB activation, and inhibits the protective effects of OPN. The NF-kappaB-inducing kinase (NIK) has been shown to be one of the upstream kinases involved in IKK activation. OPN-mediated NF-kappaB activity is increased upon NIK wild-type (WT) overexpression and blocked following NIK DN overexpression. In addition, NIK-/-mouse embryonic fibroblasts (MEFs) plated on OPN display reduced NF-kappaB activity and decreased IkappaBalpha phosphorylation compared to NIK+/+MEFs. Finally, functional inhibition of integrin beta3-dependent NF-kappaB signaling decreases OPN-induced IkappaBalpha, IKKbeta and NIK phosphorylation. These studies for the first time show that the alphavbeta3-NF-kappaB-dependent endothelial survival pathway is dependent on IkappaBalpha, IKKbeta, and NIK.

Src kinase activity is required for integrin alphaVbeta3-mediated activation of nuclear factor-kappaB.

Integrin adhesion to extracellular matrix proteins protects adhesion-dependent cells from suspension-induced apoptosis. Previous studies indicate that activation of the transcription factor nuclear factor-kappaB was necessary for the integrin alpha(v)beta3 ligand osteopontin to protect endothelial cells from apoptosis caused by serum withdrawal. In this study, beta3 integrins were overexpressed in smooth muscle cells. When plated on osteopontin, cells overexpressing wild-type beta3 had enhanced cell adhesion, cell spreading, and nuclear factor-kappaB activation compared with vector control. Removal of four amino acids (759X) from the C terminus of beta3 eliminated the ability of the integrin to promote these processes. Single amino acid substitutions indicated that phosphorylation at tyrosine 759 was not required for activation of the transcription factor, however this residue appeared to play a structural role, because mutation to alanine significantly inhibited nuclear factor-kappaB activation. The Src family of tyrosine kinases represents important transducers during integrin signaling, and the C terminus of beta3 has been implicated as the binding site for Src. Immunoprecipitations demonstrated that Src associated with wild-type beta3 integrins, but Src and integrins lacking the C terminus (759X) did not form a complex. Pharmacological inhibition with the Src inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) or overexpression of kinase-dead c-Src blocked nuclear factor-kappaB activation. Mouse embryonic fibroblasts deficient for Src failed to activate nuclear factor-kappaB when plated on osteopontin, in contrast to control fibroblasts. Together, these experiments indicate that the C terminus of beta3 and Src activity are required for integrin alpha(v)beta3-mediated nuclear factor-kappaB activation.

Differential effects of growth factors on tissue-engineered cartilage.

The effects of four regulatory factors on tissue-engineered cartilage were examined with specific focus on the ability to increase construct growth rate and concentrations of glycosaminoglycans (GAG) and collagen, the major extracellular matrix (ECM) components. Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in medium with or without supplemental insulin-like growth factor (IGF-I), interleukin-4 (IL-4), transforming growth factor-beta1 (TGF-beta1) or platelet-derived growth factor (PDGF). IGF-I, IL-4, and TGF-beta1 increased construct wet weights by 1.5-2.9-fold over 4 weeks of culture and increased amounts of cartilaginous ECM components. IGF-I (10-300 ng/mL) maintained wet weight fractions of GAG in constructs seeded at high cell density and increased by up to fivefold GAG fractions in constructs seeded at lower cell density. TGF-beta1 (30 ng/mL) increased wet weight fractions of total collagen by up to 1.4-fold while maintaining a high fraction of type II collagen (79 plus minus 11% of the total collagen). IL-4 (1-100 ng/mL) minimized the thickness of the GAG-depleted region at the construct surfaces. PDGF (1-100 ng/mL) decreased construct growth rate and ECM fractions. Different regulatory factors thus elicit significantly different chondrogenic responses and can be used to selectively control the growth rate and improve the composition of engineered cartilage.