CGP-41251 AND TAMOXIFEN SELECTIVELY INHIBIT MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION AND C-FOS PHOSPHOPROTEIN INDUCTION BY SUBSTANCE-P IN HUMAN ASTROCYTOMA-CELLS

The substance P (SP) receptor (NK-1 subtype) is widely expressed in pr imary human astrocytomas and glioblastomas and many brain tumor-derive d cell lines. SP receptor activation stimulates the mitogen-activated protein (MAP) kinase pathway and the expression of immediate-early gen es (e.g., c-Fos and c-Myc), resulting in an increase in DNA synthesis in human astrocytoma U-373 MG cells. In this study, we investigated th e role of protein kinase C (PKC) in SP receptor activation of the MAP kinase pathway. SP peptide, epidermal growth factor, and the PKC activ ator 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the tyrosine p hosphorylation of the Erk1 and Erk2 MAP kinases in a concentration-dep endent manner in U-373 MG cells. Pretreatment of the cells with PKC in hibitors, CGP 41251 or tamoxifen, inhibited tyrosine phosphorylation o f Erk1 and Erk2 MAP kinases induced by low concentrations of SP or TPA and significantly attenuated phosphorylation at high concentrations o f SP or TPA. The inhibitory effect exhibited by tamoxifen on SP-induce d MAP kinase activation is similar to that exhibited by the selective PKC inhibitor CGP 41251, suggesting that the PKC enzyme is the in situ target for both inhibitors. Furthermore, SP-induced c-Fos phosphoprot ein expression is inhibited by CGP 41251 or tamoxifen with similar eff icacy. Importantly, neither CGP 41251 nor tamoxifen has any detectable effect on the MAP kinase activation by epidermal growth factor, consi stent with the ability of this growth factor to activate the MAP kinas e pathway by a PKC-independent mechanism. Prolonged treatment with TPA resulted in down-regulation of PKC and selective inhibition of TPA- a nd SP-induced Erk1 and Erk2 tyrosine phosphorylation in U-373 MG cells . Consistent with the in site results, CGP 41251 and tamoxifen signifi cantly inhibited endogenous PKC enzymatic activity from U-373 MG cells in vitro. In contrast to CGP 41251 and tamoxifen, G(o) double over do t 6976, a highly selective inhibitor for PKC alpha and PKC beta I isoz ymes, did not inhibit SP- or TPA-induced tyrosine phosphorylation of E rk1 and Erk2 MAP kinases; rather, it inhibited a signaling pathway lea ding to the phosphorylation of cAMP-responsive element binding protein in U-373 MG cells. To investigate whether selective PKC isozyme(s) ar e involved in the activation of the MAP kinase pathway by SP, we deter mined the expression of PKC isozymes in U-373 MG cells. We found that U-373 MG cells express nine different PKC isozymes (alpha, beta I, bet a II, epsilon, delta, eta, zeta, iota, and mu)) and that stimulation w ith SP results in significant and selective translocation of PKC epsil on isozyme from cytosolic to membrane fraction. This establishes a cor relation between the ability of SP to activate the MAP kinase pathway and its ability to translocate PKC epsilon. In conclusion, the results presented in this study demonstrate that SP receptor activation of PK C, possibly PKC epsilon, leads to the activation of the MAP kinase pat hway, and that this pathway can be inhibited by known PKC inhibitors.