Abstract
Prostaglandin E(2), which exerts its functions by binding to four G protein-coupled receptors (EP1-4), is implicated in tumorigenesis. Among the four E-prostanoid (EP) receptors, EP3 is unique in that it exists as alternatively spliced variants, characterized by differences in the cytoplasmic C-terminal tail. Although three EP3 variants, alpha, beta, and gamma, have been described in mice, their functional significance in regulating tumorigenesis is unknown. In this study we provide evidence that expressing murine EP3 alpha, beta, and gamma receptor variants in tumor cells reduces to the same degree their tumorigenic potential in vivo. In addition, activation of each of the three mEP3 variants induces enhanced cell-cell contact and reduces cell proliferation in vitro in a Rho-dependent manner. Finally, we demonstrate that EP3-mediated RhoA activation requires the engagement of the heterotrimeric G protein G(12). Thus, our study provides strong evidence that selective activation of each of the three variants of the EP3 receptor suppresses tumor cell function by activating a G(12)-RhoA pathway.
Highlights
Colorectal cancer is the second most frequent cause of cancer-related death in the western world
To evaluate the role of mEP3 receptor variants on tumor cell growth, HCT116 colon cancer cells were stably transfected with mouse EP3 ␣, , and ␥ cDNAs Myc-tagged at the extracellular N terminus
We provide the novel evidence that overexpression of the ␣, , and ␥ mouse EP3 receptor variants in tumor cells reduces their tumorigenic potential in vivo
Summary
Reagents—Prostaglandins were purchased from Cayman Chemicals (Ann Arbor, MI). M&B28767 was a generous gift from Dr M. Flow Cytometry—Empty vector-transfected or mEP3-expressing cells were incubated with anti-Myc antibody (9E10, sc-40; Santa Cruz, CA) followed by phycoerythrin-conjugated anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, PA). Equal volumes of total cell lysates were used to detect total levels of Rho. Western Blot Analysis—To evaluate the effects of mEP3 activation on ERK, p38, and Akt phosphorylation, semi-confluent cells were serum-starved for 24 h and treated with 0.1 M M&B28767 or vehicle for 15 min. To confirm overexpression of constitutively active or dominant negative Rho and/or selective G protein ␣ subunits, 50 g of total cell lysates from untransfected or 48 h transfected cells were analyzed by Western blot analysis using rabbit anti-Rho, anti-G␣i2 (T-19), anti-G␣12 (S-20), or anti-G␣13 (A-20). Statistical Analysis—The Student’s t test was used for comparisons between two groups, and analysis of variance using Sigma-Stat software was used for statistical difference between multiple groups. p Ͻ 0.05 was considered statistically significant
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