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

Mast cells (MCs) are a major source of prostaglandin (PG) D-2 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 PGD(2) expression in MCs by regulating the levels of a synthase that converts PGH(2) to PGD(2), 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 similar to12,600 transcripts in RasGRP4(-) and RasGRP4(+) HMC-1 cells revealed a >100-fold difference in the levels of hematopoietic PGD(2) 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 PGD(2) synthase protein. RasGRP4(+) MCs also produced similar to15-fold more PGD(2) than did RasGRP4(-) MCs when both cell populations were activated by calcium ionophore. The induced transcript is therefore translated, and substantial amounts of functional PGD(2) synthase accumulate in RasGRP4(+) MCs. In support of the conclusion that RasGRP4 controls PGD(2) expression in MCs, inhibition of RasGRP4 expression in the rat MC line RBL-2H3 using a siRNA approach resulted in low levels of PGD(2) synthase protein.