RasGRP4 regulates the expression of prostaglandin D2 in human and rat mast cell lines

Abstract

Mast cells (MCs) are a major source of prostaglandin (PG) D2 in connective tissues, and the expression of this eicosanoid has been linked to asthma and other inflammatory disorders. While it is known that the surface receptor c-kit controls PGD2 expression in MCs by regulating the levels of a synthase that converts PGH2 to PGD2, the intracellular signaling proteins that act downstream of c-kit in this cyclooxygenase pathway have not been identified. We recently cloned a new cation-dependent, guanine nucleotide exchange factor/phorbol ester receptor (designated RasGRP4) that is required for the efficient expression of granule proteases in the human MC line HMC-1. GeneChip analysis of ∼12,600 transcripts in RasGRP4- and RasGRP4+ HMC-1 cells revealed a >100-fold difference in the levels of hematopoietic PGD2 synthase mRNA. No other transcript in the eicosanoid pathway was influenced by RasGRP4 in a comparable manner. As assessed by SDS-PAGE immunoblot analysis, RasGRP4+ HMC-1 cells contained substantial amounts of PGD2 synthase protein. RasGRP4+ MCs also produced ∼15-fold more PGD2 than did RasGRP4- MCs when both cell populations were activated by calcium ionophore. The induced transcript is therefore translated, and substantial amounts of functional PGD2 synthase accumulate in RasGRP4+ MCs. In support of the conclusion that RasGRP4 controls PGD2 expression in MCs, inhibition of RasGRP4 expression in the rat MC line RBL-2H3 using a siRNA approach resulted in low levels of PGD2 synthase protein.