Pro-opiomelanocortin gene expression and protein processing in rat mononuclear leukocytes

Abstract

Although production of pro-opiomelanocortin (POMC) mRNA and POMC-derived peptides in extrapituitary tissues, including immune tissues, has been demonstrated, questions remain concerning the nature of POMC transcripts and peptide products. With regard to POMC gene expression in lymphocytes, the expression of full-length mRNA and POMC has been questioned. In the present report, we have tested for the existence of these molecules. Western blot analysis with an antibody against POMC-derived adrenocorticotropic hormone (ACTH) specifically identified identical immunoreactive (ir) species in both rat anterior pituitary (AP) and splenocyte cell extracts. The relative molecular weights were those expected for nonglycosylated ACTH, as well as its biosynthetic intermediate, and POMC. Mitogen stimulation of splenic mononuclear cells (MNC) enhanced the levels of these three molecular species. Primer extension analysis identified a band which migrated with a size equivalent to a full-length POMC transcript (~816 nt) in both mitogen-stimulated MNC and AP mRNA. Macrophages produced POMC protein and mRNA among unstimulated splenocytes, while lymphocytes could be induced to produce POMC mRNA upon stimulation. 5′ RACE-tailed PCR products were cloned and sequenced. A mRNA encoding all three POMC exons was identified in Concanavalin A (ConA)-stimulated MNC and was identical to that from the anterior pituitary. These results unequivocally demonstrate that mononuclear cells produce full-length POMC transcripts. Its regulation in lymphocytes is distinct from that in macrophages which constitutively produce POMC-derived peptides and mRNA. Also, the biosynthetic pathway of ACTH from POMC in splenic MNC stimulated with ConA appears to be identical to that in rat corticotrophs.