Abstract
Mouse bone marrow derived mast cells (BMMCs) migrate toward histamine through H4 receptor (H4R), which involves pertussis toxin-sensitive G protein and phospholipase C. We have previously shown that Rho family small G proteins Rac1 and Rac2 physically and functionally associate with H4R and that histamine-induced chemotaxis of BMMCs requires an activation of these small G proteins.
Transglutaminase is an enzyme that catalyzes crosslinks between proteins or between a protein and a small molecule containing primary amino group such as histamine. A previous report showed that the activation of transglutaminase in stimulated mast cells increased the incorporation of histamine into proteins, or histaminylation. Another report showed that cdc42, a Rho family small G protein that is highly homologous to Rac1/2, became constitutively active upon in vitro histaminylation by transglutaminase. Although these findings imply possible roles of histaminlylation in mast cell signaling, there is no direct evidence showing significance of histaminylation in cellular function. In this study possible roles of transglutaminase in H4R signaling were investigated in histamine-induced chemotaxis of BMMCs.
We first examined whether histamine-induced Rac1/2 activation required calcium because transglutaminase is a calcium-dependent enzyme. A phospholipase C inhibitor U73122 significantly suppressed the histamine-induced Rac1/2 activation. Furthermore intracellular calcium chelator BAPTA abolished histamine-induced Rac1/2 activation. These results suggest that Rac1/2 activation induced by histamine requires calcium mobilization. Next the effect of cystamine, a transglutaminase inhibitor, on histamine-induced chemotaxis of BMMCs was investigated. Cystamine attenuated the migration of BMMCs toward histamine in concentration-dependent manner. Interestingly, cystamine had no effect on migration toward other attractants such as stem cell factor or adenosine. In addition, Rac1/2 activation induced by histamine but not by stem cell factor or adenosine was inhibited by cystamine, suggesting that histamine-induced Rac1/2 activation specifically requires transglutaminase activation. Finally the effect of specific knockdown of transglutaminase 2 (TG2) by shRNA was examined on histamine-induced chemotaxis. BMMCs with TG2 knockdown resulted in decreased migration toward histamine, confirming the role of transglutaminase in histamine-induced chemotaxis.
Our findings suggest that transglutaminase is involved in histamine-induced chemotaxis of mouse BMMC by regulating H4R-mediated Rac1/2 activation.
