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Regulator of G-protein signalling expression and function in ovarian cancer cell lines

Cellular & Molecular Biology Letters volume 14, pages 153–174 (2009)Cite this article

Abstract

Regulator of G-protein signalling (RGS)2 proteins critically regulate signalling cascades initiated by G-protein coupled receptors (GPCRs) by accelerating the deactivation of heterotrimeric G-proteins. Lysophosphatidic acid (LPA) is the predominant growth factor that drives the progression of ovarian cancer by activating specific GPCRs and G-proteins expressed in ovarian cancer cells. We have recently reported that RGS proteins endogenously expressed in SKOV-3 ovarian cancer cells dramatically attenuate LPA stimulated cell signalling. The goal of this study was twofold: first, to identify candidate RGS proteins expressed in SKOV-3 cells that may account for the reported negative regulation of G-protein signalling, and second, to determine if these RGS protein transcripts are differentially expressed among commonly utilized ovarian cancer cell lines and non-cancerous ovarian cell lines. Reverse transcriptase-PCR was performed to determine transcript expression of 22 major RGS subtypes in RNA isolated from SKOV-3, OVCAR-3 and Caov-3 ovarian cancer cell lines and non-cancerous immortalized ovarian surface epithelial (IOSE) cells. Fifteen RGS transcripts were detected in SKOV-3 cell lines. To compare the relative expression levels in these cell lines, quantitative real time RT-PCR was performed on select transcripts. RGS19/GAIP was expressed at similar levels in all four cell lines, while RGS2 transcript was detected at levels slightly lower in ovarian cancer cells as compared to IOSE cells. RGS4 and RGS6 transcripts were expressed at dramatically different levels in ovarian cancer cell lines as compared to IOSE cells. RGS4 transcript was detected in IOSE at levels several thousand fold higher than its expression level in ovarian cancer cells lines, while RGS6 transcript was expressed fivefold higher in SKOV-3 cells as compared to IOSE cells, and over a thousand fold higher in OVCAR-3 and Caov-3 cells as compared to IOSE cells. Functional studies of RGS 2, 6, and 19/GAIP were performed by measuring their effects on LPA stimulated production of inositol phosphates. In COS-7 cells expressing individual exogenous LPA receptors, RGS2 and RSG19/GAIP attenuated signalling initiated by LPA1, LPA2, or LPA3, while RGS6 only inhibited signalling initiated by LPA2 receptors. In SKOV-3 ovarian cancer cells, RGS2 but not RGS6 or RGS19/GAIP, inhibited LPA stimulated inositol phosphate production. In contrast, in CAOV-3 cells RGS19/GAIP strongly attenuated LPA signalling. Thus, multiple RGS proteins are expressed at significantly different levels in cells derived from cancerous and normal ovarian cells and at least two candidate RGS transcripts have been identified to account for the reported regulation of LPA signalling pathways in ovarian cancer cells.

Abbreviations

CT :

threshold cycle

ELISA:

enzyme-linked immunosorbent assay

GPCR:

G-protein coupled receptor

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

GROα:

growth-regulated oncogene α

IOSE:

immortalized ovarian surface epithelial

IP:

inositol phosphates

LPA:

lysophosphatidic acid

PLC:

phospholipase C

RGS:

regulator of G-protein signalling

RT-PCR:

reverse transcriptase polymerase chain raction

siRNA:

small interfering RNA

References

  1. Xu, Y., Fang, X.J., Casey, G. and Mills, G.B. Lysophospholipids activate ovarian and breast cancer cells. Biochem. J. 309 (1995) 933–940.

    PubMed  CAS  Google Scholar 

  2. Mills, G.B., May, C., McGill, M., Roifman, C.M. and Mellors, A. A putative new growth factor in ascitic fluid from ovarian cancer patients: identification, characterization, and mechanism of action. Cancer Res. 48 (1988) 1066–1071.

    PubMed  CAS  Google Scholar 

  3. Frankel, A. and Mills, G.B. Peptide and lipid growth factors decrease cis-diamminedichloroplatinum-induced cell death in human ovarian cancer cells. Clin Cancer Res. 2 (1996) 1307–1313.

    PubMed  CAS  Google Scholar 

  4. Sengupta, S., Xiao, Y.J. and Xu, Y. A novel laminin-induced LPA autocrine loop in the migration of ovarian cancer cells. FASEB J. 17 (2003) 1570–1572.

    PubMed  CAS  Google Scholar 

  5. Sengupta, S., Kim, K.S., Berk, M.P., Oates, R., Escobar, P., Belinson, J., Li, W., Lindner, D.J., Williams, B. and Xu, Y. Lysophosphatidic acid downregulates tissue inhibitor of metalloproteinases, which are negatively involved in lysophosphatidic acid-induced cell invasion. Oncogene 26 (2007) 2894–2901.

    Article  PubMed  CAS  Google Scholar 

  6. Fang, X., Gaudette, D., Furui, T., Mao, M., Estrella, V., Eder, A., Pustilnik, T., Sasagawa, T., Lapushin, R., Yu, S., Jaffe, R.B., Wiener, J.R., Erickson, J.R. and Mills, G.B. Lysophospholipid growth factors in the initiation, progression, metastases, and management of ovarian cancer. Ann. N. Y. Acad. Sci. 905 (2000) 188–208.

    PubMed  CAS  Google Scholar 

  7. Anliker, B. and Chun, J. Lysophospholipid G protein-coupled receptors. J. Biol. Chem. 279 (2004) 20555–20558.

    Article  PubMed  CAS  Google Scholar 

  8. Noguchi, K., Ishii, S. and Shimizu, T. Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family. J. Biol. Chem. 278 (2003) 25600–25606.

    Article  PubMed  CAS  Google Scholar 

  9. Lee, C.W., Rivera, R., Gardell, S., Dubin, A.E. and Chun, J. GPR92 as a new G12/13- and Gq-coupled lysophosphatidic acid receptor that increases cAMP, LPA5. J. Biol. Chem. 281 (2006) 23589–23597.

    Article  PubMed  CAS  Google Scholar 

  10. Oldham, W.M. and H, E.H. Structural basis of function in heterotrimeric G proteins. Q. Rev. Biophys. (2006) 1–50.

  11. Heximer, S.P., Knutsen, R.H., Sun, X., Kaltenbronn, K.M., Rhee, M.H., Peng, N., Oliveira-dos-Santos, A., Penninger, J.M., Muslin, A.J., Steinberg, T.H., Wyss, J.M., Mecham, R.P. and Blumer, K.J. Hypertension and prolonged vasoconstrictor signalling in RGS2-deficient mice. J. Clin. Invest. 111 (2003) 445–452.

    PubMed  CAS  Google Scholar 

  12. Chen, C.K., Burns, M.E., He, W., Wensel, T.G., Baylor, D.A. and Simon, M.I. Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1. Nature 403 (2000) 557–560.

    Article  PubMed  CAS  Google Scholar 

  13. Fu, Y., Huang, X., Piao, L., Lopatin, A.N. and Neubig, R.R. Endogenous RGS proteins modulate SA and AV nodal functions in isolated heart: implications for sick sinus syndrome and AV block. Am. J. Physiol. Heart Circ. Physiol. 292 (2007) H2532–2539.

    Article  PubMed  CAS  Google Scholar 

  14. Hurst, J.H., Henkel, P.A., Brown, A.L. and Hooks, S.B. Endogenous RGS proteins attenuate Galpha(i)-mediated lysophosphatidic acid signalling pathways in ovarian cancer cells. Cell. Signal. 20 (2008) 381–389.

    Article  PubMed  CAS  Google Scholar 

  15. Livak, K.J. and Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif. 25 (2001) 402–408.

    CAS  Google Scholar 

  16. Hepler, J.R., Nakahata, N., Lovenberg, T.W., DiGuiseppi, J., Herman, B., Earp, H.S. and Harden, T.K. Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells. J. Biol. Chem. 262 (1987) 2951–2956.

    PubMed  CAS  Google Scholar 

  17. Hollinger, S. and Hepler, J.R. Cellular regulation of RGS proteins: modulators and integrators of G protein signalling. Pharmacol. Rev. 54 (2002) 527–559.

    Article  PubMed  CAS  Google Scholar 

  18. Chatterjee, T.K., Liu, Z. and Fisher, R.A. Human RGS6 gene structure, complex alternative splicing, and role of N terminus and G protein gamma-subunit-like (GGL) domain in subcellular localization of RGS6 splice variants. J. Biol. Chem. 278 (2003) 30261–30271.

    Article  PubMed  CAS  Google Scholar 

  19. Fukushima, N., Kimura, Y. and Chun, J. A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid. Proc. Natl. Acad. Sci. USA 95 (1998) 6151–6156.

    Article  PubMed  CAS  Google Scholar 

  20. Ishii, I., Contos, J.J., Fukushima, N. and Chun, J. Functional comparisons of the lysophosphatidic acid receptors, LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7 in neuronal cell lines using a retrovirus expression system. Mol. Pharmacol. 58 (2000) 895–902.

    PubMed  CAS  Google Scholar 

  21. Contos, J.J., Ishii, I. and Chun, J. Lysophosphatidic acid receptors. Mol. Pharmacol. 58 (2000) 1188–1196.

    PubMed  CAS  Google Scholar 

  22. Lee, C.W., Rivera, R., Dubin, A.E. and Chun, J. LPA(4)/GPR23 is a lysophosphatidic acid (LPA) receptor utilizing G(s)-, G(q)/G(i)-mediated calcium signalling and G(12/13)-mediated Rho activation. J. Biol. Chem. 282 (2007) 4310–4317.

    Article  PubMed  CAS  Google Scholar 

  23. Waldo, G.L., Boyer, J.L., Morris, A.J. and Harden, T.K. Purification of an AlF4- and G-protein beta gamma-subunit-regulated phospholipase C-activating protein. J. Biol. Chem. 266 (1991) 14217–14225.

    PubMed  CAS  Google Scholar 

  24. Boyer, J.L., Waldo, G.L. and Harden, T.K. Beta gamma-subunit activation of G-protein-regulated phospholipase C. J. Biol. Chem. 267 (1992) 25451–25456.

    PubMed  CAS  Google Scholar 

  25. Hains, M.D., Wing, M.R., Maddileti, S., Siderovski, D.P. and Harden, T.K. Galpha12/13- and rho-dependent activation of phospholipase C-epsilon by lysophosphatidic acid and thrombin receptors. Mol. Pharmacol. 69 (2006) 2068–2075.

    Article  PubMed  CAS  Google Scholar 

  26. Chen, C.K., Eversole-Cire, P., Zhang, H., Mancino, V., Chen, Y.J., He, W., Wensel, T.G. and Simon, M.I. Instability of GGL domain-containing RGS proteins in mice lacking the G protein beta-subunit Gbeta5. Proc. Natl. Acad. Sci. USA 100 (2003) 6604–6609.

    Article  PubMed  CAS  Google Scholar 

  27. Ujioka, T., Russell, D.L., Okamura, H., Richards, J.S. and Espey, L.L. Expression of regulator of G-protein signalling protein-2 gene in the rat ovary at the time of ovulation. Biol. Reprod. 63 (2000) 1513–1517.

    Article  PubMed  CAS  Google Scholar 

  28. Heximer, S.P., Watson, N., Linder, M.E., Blumer, K.J. and Hepler, J.R. RGS2/G0S8 is a selective inhibitor of Gqalpha function. Proc. Natl. Acad. Sci. USA 94 (1997) 14389–14393.

    Article  PubMed  CAS  Google Scholar 

  29. Heximer, S.P., Srinivasa, S.P., Bernstein, L.S., Bernard, J.L., Linder, M.E., Hepler, J.R. and Blumer, K.J. G protein selectivity is a determinant of RGS2 function. J. Biol. Chem. 274 (1999) 34253–34259.

    Article  PubMed  CAS  Google Scholar 

  30. Ingi, T., Krumins, A.M., Chidiac, P., Brothers, G.M., Chung, S., Snow, B.E., Barnes, C.A., Lanahan, A.A., Siderovski, D.P., Ross, E.M., Gilman, A.G. and Worley, P.F. Dynamic regulation of RGS2 suggests a novel mechanism in G-protein signalling and neuronal plasticity. J. Neurosci. 18 (1998) 7178–7188.

    PubMed  CAS  Google Scholar 

  31. Tosetti, P., Parente, V., Taglietti, V., Dunlap, K. and Toselli, M. Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels. J. Physiol. 549 (2003) 157–169.

    Article  PubMed  CAS  Google Scholar 

  32. De Vries, L., Mousli, M., Wurmser, A. and Farquhar, M.G. GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain. Proc. Natl. Acad. Sci. USA 92 (1995) 11916–11920.

    Article  PubMed  Google Scholar 

  33. Hepler, J.R., Berman, D.M., Gilman, A.G. and Kozasa, T. RGS4 and GAIP are GTPase-activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio-GTP-Gq alpha. Proc. Natl. Acad. Sci. USA 94 (1997) 428–432.

    Article  PubMed  CAS  Google Scholar 

  34. Berman, D.M., Wilkie, T.M. and Gilman, A.G. GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell 86 (1996) 445–452.

    Article  PubMed  CAS  Google Scholar 

  35. Huang, C., Hepler, J.R., Gilman, A.G. and Mumby, S.M. Attenuation of Gi-and Gq-mediated signalling by expression of RGS4 or GAIP in mammalian cells. Proc. Natl. Acad. Sci. USA 94 (1997) 6159–6163.

    Article  PubMed  CAS  Google Scholar 

  36. Hooks, S.B., Waldo, G.L., Corbitt, J., Bodor, E.T., Krumins, A.M. and Harden, T.K. RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity. J. Biol. Chem. 278 (2003) 10087–10093.

    Article  PubMed  CAS  Google Scholar 

  37. Seki, N., Hattori, A., Hayashi, A., Kozuma, S., Hori, T. and Saito, T. The human regulator of G-protein signalling protein 6 gene (RGS6) maps between markers WI-5202 and D14S277 on chromosome 14q24.3. J. Hum. Genet. 44 (1999) 138–140.

    Article  PubMed  CAS  Google Scholar 

  38. Gunthert, A.R., Grundker, C., Bottcher, B. and Emons, G. Luteinizing hormone-releasing hormone (LHRH) inhibits apoptosis induced by cytotoxic agent and UV-light but not apoptosis mediated through CD95 in human ovarian and endometrial cancer cells. Anticancer Res. 24 (2004) 1727–1732.

    PubMed  Google Scholar 

  39. Lee, Z., Swaby, R.F., Liang, Y., Yu, S., Liu, S., Lu, K.H., Bast, R.C.Jr., Mills, G.B. and Fang, X. Lysophosphatidic acid is a major regulator of growth-regulated oncogene alpha in ovarian cancer. Cancer Res. 66 (2006) 2740–2748.

    Article  PubMed  CAS  Google Scholar 

  40. Choi, J.H., Choi, K.C., Auersperg, N. and Leung, P.C. Gonadotropins activate proteolysis and increase invasion through protein kinase A and phosphatidylinositol 3-kinase pathways in human epithelial ovarian cancer cells. Cancer Res. 66 (2006) 3912–3920.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA, 30602, USA

    Jillian H. Hurst, Nisha Mendpara & Shelley B. Hooks

  2. 377 Wilson Pharmacy Building, 250 West Green Street, Athens, GA, 30602-2352, USA

    Shelley B. Hooks

Authors
  1. Jillian H. Hurst
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  2. Nisha Mendpara
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  3. Shelley B. Hooks
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Correspondence to Shelley B. Hooks.

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Hurst, J.H., Mendpara, N. & Hooks, S.B. Regulator of G-protein signalling expression and function in ovarian cancer cell lines. Cell Mol Biol Lett 14, 153–174 (2009). https://doi.org/10.2478/s11658-008-0040-7

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Cellular & Molecular Biology Letters

ISSN: 1689-1392

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