MITOGENIC EFFECTS AND NUCLEAR-LOCALIZATION OF PROCORTICOTROPHIN-RELEASING HORMONE EXPRESSED WITHIN STABLY TRANSFECTED FIBROBLAST CELLS (CHO-K1)

To investigate the intracellular localisation and biological activity of procorticotrophin-releasing hormone (proCRH), we have established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA. Using immunoblot analysis of cell lysates of transfected CHO-K1 cells, we detected a major CRH immunoreactive band with an apparent molecular weight of approximately 19 kDa. This 19 kDa band could account for full length proCRH molecule which has not undergone post-translational modifications. Metabolic labelling followed by immunoprecipitation, SDS-PAGE and autoradiography indicated that no endoproteolytic processing of proCRH takes place within the transfected CHO-K1 cells. Immunofluorescence staining localises the CRH precursor to both the cytoplasm and to the nucleus in transfected CHO-K1 cells. This result was confirmed using subcellular fractionation techniques on radiolabelled CHO-K1 cells expressing immunoreactive CRH. A major CRH-immunoreactive band of 19 kDa was detected both in the microsomal and secreted fractions, indicating the presence of proCRH within the secretory pathway of these cells. This was also evident in the nuclear fraction, therefore confirming the nuclear localisation of proCRH. Analysis of DNA concentration, cell number and DNA synthesis showed that stably transfected CHO-K1 cells expressing proCRH have a higher proliferation and DNA synthesis rate than wildtype CHO-K1 cells or CHO-K1 cells transfected with pEE14 alone. Our results therefore suggest a mitogenic role for the intact proCRH molecule within CHO-K1 cells. Furthermore, treatment of mouse corticotrophic tumour cells (AtT20/D16-16) with conditioned medium from transfected CHO-K1 cells expressing proCRH, stimulated both DNA synthesis and cell proliferation above basal levels. Our results constitute the first reported direct evidence of a mitogenic role for proCRH acting on a corticotrophic cell population.