Vol. 8, 1225-1240, November 1997 Cell Growth & Differentiation 1225 CGP 41251 and Tamoxifen Selectively Inhibit Mitogen- activated Protein Kinase Activation and c-Fos Phosphoprotein Induction by Substance P in Human Astrocytoma Cells1 Weihua Luo, Taraneh R. Sharif, Peter J. Houghton, and Mohammed Sharif Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 Abstract The substance P (SP) receptor (NK-1 subtype) is widely expressed in primary human astrocytomas and glioblastomas and many brain tumor-derived cell lines. SP receptor activation stimulates the mitogen- activated protein (MAP) kinase pathway and the expression of immediate-early genes (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 the role of protein kinase C (PKC) in SP receptor activation of the MAP kinase pathway. SP peptide, epidermal growth factor, and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the tyrosine phosphorylation of the Erki and Erk2 MAP kinases in a concentration-dependent manner in U-373 MG cells. Pretreatment of the cells with PKC inhibitors, CGP 41251 or tamoxifen, inhibited tyrosine phosphorylation of Erki and Erk2 MAP kinases induced by low concentrations of SP or TPA and significantly attenuated phosphorylation at high concentrations of SP or TPA. The inhibitory effect exhibited by tamoxifen on SP-induced MAP kinase activation is similar to that exhibited by the selective PKC inhibitor CGP 41251 , suggesting that the PKC enzyme is the in sftu target for both inhibitors. Furthermore, SP-induced c-Fos phosphoprotein expression is inhibited by CGP 41251 or tamoxifen with similar efficacy. Importantly, neither CGP 41251 nor tamoxifen has any detectable effect on the MAP kinase activation by epidermal growth factor, consistent with the ability of this growth factor to activate the MAP kinase pathway by a PKC-independent mechanism. Received 3/27/97; revised 8/14/97; accepted 9/23/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdi- cate this fact. 1 This work was supported in part by the Cancer Center CORE Grant CA21765 from the NIH, Grants CA71756 and CA23099 from the National Cancer Institute, and funds from the American Lebanese Syrian Associ- ated Charities. 2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: (901) 495-2793; Fax: (901) 521 -1668; E-mail: mohammed.sharif@stjude.org. Prolonged treatment with TPA resulted in down- regulation of PKC and selective inhibition of TPA- and SP-induced Erki and Erk2 tyrosine phosphorylation in U-373 MG cells. Consistent with the in situ results, CGP 41251 and tamoxifen significantly inhibited endogenous PKC enzymatic activity from U-373 MG cells in vitro. In contrast to CGP 41251 and tamoxifen, Go 6976, a highly selective inhibitor for PKCa and PKCI3I isozymes, did not inhibit SP- or TPA-induced tyrosine phosphorylation of Erki and Erk2 MAP kinases; rather, it inhibited a signaling pathway leading to the phosphorylation of cAMP-responsive element binding protein in U-373 MG cells. To investigate whether selective PKC isozyme(s) are involved in the activation of the MAP kinase pathway by SP, we determined the expression of PKC isozymes in U-373 MG cells. We found that U-373 MG cells express nine different PKC isozymes (a, fil, 1311,a, , g, , &, and t) and that stimulation with SP results in significant and selective translocation of PKC#{128} isozyme from cytosolic to membrane fraction. This establishes a correlation between the ability of SP to activate the MAP kinase pathway and its ability to translocate PKC#{128}. In conclusion, the results presented in this study demonstrate that SP receptor activation of PKC, possibly PKCe, leads to the activation of the MAP kinase pathway, and that this pathway can be inhibited by known PKC inhibitors. Introduction The SP3 neuropeptide preferentially binds to and acts through the SP receptor (NK-1 subtype; Refs. 1 and 2). In mammals, SP peptide regulates various important functions such as neurotransmitter release, smooth muscle contrac- tion, and cell growth (1 , 2). Activation of the SP receptor, which belongs to the superfamily of G protein-coupled re- ceptors (3, 4), induces the hydrolysis of phosphoinositides, leading to the mobilization of intracellular Ca2 and activa- tion of PKC (5). PKC is a phospholipid-dependent serine/ threonine kinase family that can be activated by Ca2 , phos- pholipids, and DAG (6-8). The involvement of SP peptide in stimulating mitogenesis in various cell types has been documented by our group 3 The abbreviations used are: SP, substance P; PKC, protein kinase C; DAG, diacylglycerol; MAP, mitogen-activated protein; 5K, substance K; EGF, epidermal growth factor; TPA, i2-O-tetradecanoylphorbol-i3-ace- tate; MBP, myelin basic protein; CaM, calmodulin; CRE, cAMP-respon- sive element; CREB, cAMP-responsive element binding.