P2Y nucleotide receptor signaling through MAPK/ERK is regulated by extracellular matrix: involvement of beta3 integrins.

Extracellular matrix influences cell behavior through receptors such as integrins and through transmission of mechanical forces. Nucleotides are released in response to mechanical stimuli and bind to P2 nucleotide receptors. As chondrocytes are subjected to frequent mechanical stimulation within a rich extracellular matrix, they are an excellent model for studying integration of signals induced by matrix and nucleotides. We investigated signaling of G protein-coupled P2Y receptors to MAPK/ERK and how this is influenced by matrix. Rat articular chondrocytes expressed transcripts for P2Y1, P2Y2, P2Y4, and P2Y6 receptors and responded to extracellular nucleotides by transient elevation of cytosolic calcium and MAPK/ERK phosphorylation. ERK1/2 activation was suppressed by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and rottlerin, and by the phospholipase D inhibitor 1-butanol. Thus, nucleotides stimulate P2Y receptors to activate ERK1/2 through a mechanism dependent on PKC and phospholipase D. We next examined the involvement of integrins. Both an RGD-containing pentapeptide and a beta3 integrin blocking antibody, but not a beta1 integrin blocking antibody, abolished nucleotide-induced ERK1/2 phosphorylation. Moreover, chondrocytes adhering to fibronectin (which binds to beta1 and beta3 containing integrins in an RGD-dependent manner) displayed prolonged ERK1/2 signaling compared to cells grown on type I or II collagen (which bind to beta1-containing integrins in an RGD-independent manner). In conclusion, P2Y receptor signaling through ERK1/2 is gated selectively by matrix proteins. Thus, nucleotides released in response to mechanical stimulation will have differing effects on cell function due to changes in the composition of the extracellular matrix during development and disease.