Leukotriene D₄ activates MAPK through a Ras-independent but PKCϵ-dependent pathway in intestinal epithelial cells

We have recently shown that leukotriene D₄ (LTD₄)increases cell survival in intestinal epithelial cells. Here we report and explore the complementary finding that LTD₄ also enhances proliferation in these cells. This proliferative response was approximately half of that induced by epidermal growth factor (EGF) and its required activation of protein kinase C (PKC), Ras and the mitogen-activated protein kinase (MAPK) Erk-1/2. EGF also activated Erk-1/2 in these cells; however the EGF-receptor inhibitor PD153035 did not affect the LTD₄-induced activation of Erk-1/2. In addition, LTD₄ did not induce phosphorylation of the EGF receptor, nor did pertussis toxin (PTX) block EGF-induced activation of Erk-1/2, thus refuting a possible crosstalk between the receptors. Furthermore, LTD₄-induced, but not EGF-induced,activation of Erk-1/2 was sensitive to PTX, PKC inhibitors and downregulation of PKCϵ. A definite role for PKCϵ in LTD₄-induced stimulation of Erk-1/2 was documented by the inability of LTD₄ to activate Erk-1/2 in cells transfected with either the regulatory domain of PKCϵ (an isoform specific dominant-negative inhibitor) or a kinase-dead PKCϵ. Although Ras and Raf-1 were both transiently activated by LTD₄, only Raf-1 activation was abolished by abrogation of the PKC signal. Furthermore, the LTD₄-induced activation of Erk-1/2 was unaffected by transfection with dominant-negative N17 Ras but blocked by transfection with kinase-dead Raf-1. Consequently, LTD₄ regulates the proliferative response by a distinct Ras-independent, PKCϵ-dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway.