Phillip J. Lowry

Biosynthesis of corticotropin-releasing hormone in human T-lymphocytes

Corticotropin-releasing hormone (CRH) is a 41-amino acid neuropeptide which increases the transcription of the proopiomelanocortin (POMC) gene, as well as the biosynthesis and secretion of POMC-derived peptides. Using a specific human CRH radioimmunoassay we have shown that human T-lymphocytes contain immunoreactive CRH. We studied the effects of phytohemagglutinin (PHA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the biosynthesis of CRH in human T-lymphocyte cell cultures. A significant increase in CRH mRNA levels was observed in human lymphocytes after 12 h of PHA/TPA treatment, while the levels decreased after 22 h. These findings could imply an immunomodulatory role for CRH that could be due to autocrine and/or paracrine interactions.

Mitogenic effects and nuclear localisation 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.

Expression and partial purification of human prolactin in escherichia coli

1. Human prolactin has been expressed in Escherichia coli. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a beta-galactosidase prolactin fusion protein when expressed in E. coli. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.

Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: Characterization and purification

1. Human pre-procorticotropin releasing hormone (CRH) was expressed in E. coli strain TG2 as a fusion protein with beta-galactosidase. 2. A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) [ph PPC (10-196)] after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining. The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay. 3. Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea. 4. When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).

Anatomical Localization of Corticotropin‐Releasing Factor and Arginine Vasopressin in the Human Hypothalamus; the Effect of Corticosteroids on their Concentrations in Human and Rat Hypothalami

In this study, we have determined the distribution of corticotropin‐releasing factor and vasopressin in the human hypothalamus, and investigated the effect of glucocorticoid administration on the concentrations of both peptides. Corticotropin‐releasing factor and vasopressin were measured by a two‐site immunoradiometric assay and/or radioimmunoassay. The presence of both peptides was studied in extracts of eleven areas of the human hypothalamus as well as in the pituitary stalk from autopsied patients who had been free of chronic steroid administration (n = 14) or had received Corticosteroids (n = 5). Unlike vasopressin, corticotropin‐releasing factor was detected in all extracts: the highest concentration was found in the pituitary stalk, whilst the lowest detectable amounts occurred in the supraoptic and lateral areas and in the mammillary bodies. This pattern of distribution is similar to that reported for the rat hypothalamus. The excellent correlation (R = 0.994) between corticotropin‐releasing factor data obtained by immunoradiometric assay and by radioimmunoassay renders the presence of a corticotropin‐releasing factor precursor molecule in the extracts highly unlikely. In the human brain extracts, glucocorticoid treatment affected neither the content, nor the distribution of corticotropin‐releasing factor and vasopressin. In the rats, dexamethasone administration produced a 50% decrease in the vasopressin content (P < 0.05) of the basomedial and dorsal parts of the hypothalamus and had no effect on the corticotropin‐releasing factor content of these areas. These results show that the distribution of corticotropin‐releasing factor is similar in both human and rat hypothalami. The rat data suggest that negative feedback effects of glucocorticoids involve changes in hypothalamic vasopressin content.