A peptide of the α3(IV) chain of type IV collagen modulates stimulated neutrophil function via activation of cAMP-dependent protein kinase and Ser/Thr protein phosphatase

Abstract

Previous reports from our laboratories showed that type IV collagen from anterior lens capsule (ALC) inhibited stimulated neutrophil function. This property was shown to reside in the region comprising residues 185–203 of the non-collagenous domain (NC1) of the α3(IV) chain. We also reported that ALC-type IV collagen or the synthetic α3(IV) 185–203 peptide, induced a rise in intracellular cAMP which persisted for up to 60 minutes. In the present work we extend our previous studies on signal transduction by α3(IV) 185–203 and we provide new data showing the involvement of cAMP-dependent PKA and protein phosphatases. The data also show that the α3(IV) peptide triggered a rise in intracellular calcium that was dependent on phospholipase C activation. Inhibitors of the Ca 2+/calmodulin system suppressed both the α3(IV) 185–203 peptide-induced cAMP increase and the inhibitory activity of the peptide on f-Met-Leu-Phe triggered O 2 − generation. When α3(IV) 185–203 peptide-induced calcium mobilization was blocked by U-73122, an inhibitor of phospholipase C activation, or by BAPTA/AM, a chelator of intracellular calcium, the inhibitory effect of the peptide on PMA-triggered O 2 − production was also abolished. These findings provide evidence that signal transduction by the α3(IV) peptide occurs via pathways which involve calcium. Indeed, the cAMP increase was shown to be mediated by adenosine and adenosine A2 receptors and required calcium elevation, since adenosine deaminase, theophilline, dimethylpropargylxanthine, trifluoperazine or autocamtide-2 related inhibitory peptide, suppressed the activity of the α3(IV) peptide. The inhibitory effect of the peptide on f-Met-Leu-Phe-induced O 2 − generation was slightly affected by 1 μM KT5720 or H89, two inhibitors of cAMP-dependent PKA, but was completely suppressed by 10 nM calyculin A or 10 μM okadaic acid, two inhibitors of ser/thr phosphatases. These results suggest that Ser/Thr protein phosphatases and/or cAMP-dependent PKA are involved in signal transduction by the α3(IV) 185–203 peptide and is consistent with the concept that adenosine receptor occupancy modulates neutrophil function.