Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq-coupled receptors

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Abstract

GPR54 is a receptor for peptides derived from the metastasis suppressor gene KiSS-1. To investigate the intracellular mechanisms involved in the reduction of the metastatic potential of MDA-MB-435S cells expressing GPR54, a time course stimulation by kisspeptin-10 over a period of 25 h was performed using cDNA microarrays. Comparison with the bradykinin B2 receptor revealed a distinct pattern of gene regulation despite a common coupling to the Gq/11 class of G-proteins. Inhibitors of PLC and PK-C abolished the transcriptional regulation of all tested genes, while an inhibitor of p42/44 affected a subset of genes controlled both by GPR54 and B2. Among the genes specifically up-regulated by GPR54, we found several proapoptotic genes. Stimulation of GPR54 promoted apoptosis while no significant change was observed after B2 receptor activation. Our results suggest that the metastasis suppressor properties of GPR54 are mediated in part by cell cycle arrest and induction of apoptosis in malignant cells.

Section snippets

Materials and methods

Cell lines. The human breast cancer cell line MDA-MB-435S was obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Kisspeptin-10 and bradykinin were purchased from Calbiochem and Neosystem, respectively. The cDNA coding regions of human GPR54 and bradykinin B2 receptors [3], [6] cloned in the pEFIN3 vector, as well as the empty vector pEFIN3, were transfected into MDA-MB-435S cells using Fugene 6 as

Expression of recombinant GPR54 in a human breast carcinoma cell line

We established a recombinant expression system in the human MDA-MB-435S cell line, originating from a metastatic ductal breast carcinoma, allowing to compare in a rigorous way cells expressing or not GPR54, as well as other receptors reported to be coupled to the same intracellular pathways. This cell line was tested negative for the presence of GPR54 transcripts, and it did not respond functionally to kisspeptin-10. The full-length GPR54 coding sequence was inserted into the bicistronic

Discussion

The involvement of GPR54 and its ligands in the control of the metastatic potential of tumoral cells has been described previously [4], [17], [18]. However, the mechanisms by which KiSS-1 expression or GPR54 activation restrains the metastatic properties of cells are largely unknown. In order to investigate this process, we have analyzed the transcriptional program regulated by GPR54, in a human breast carcinoma cell line. In agreement with previous reports [2], [3], [4], stimulation of human

Acknowledgments

This work was supported by a grant from the Actions de Recherche Concertées of the Communauté Française de Belgique, the Centre de Recherche Interuniversitaire en Vaccinologie, the Belgian program on Interuniversity Poles of attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming, the Cell Factory (Grant QLK3-CT2000-00237) and LifeSciHealth (Grant LSHB-CT-2003-503337) programs of the European Community, the Fonds de la Recherche Scientifique Médicale of

References (28)

  • N. de Roux et al.

    Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54

    Proc. Natl. Acad. Sci. USA

    (2003)
  • J.F. Mirjolet et al.

    G (1)/S but not G (0)/G (1) cell fraction is related to 5-fluorouracil cytotoxicity

    Cytometry

    (2002)
  • C. Workman et al.

    A new non-linear normalization method for reducing variability in DNA microarray experiments

    Genome Biol.

    (2002)
  • M.D. Ringel et al.

    Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells

    J. Clin. Endocrinol. Metab

    (2002)
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