Expression of porcine pro-opiomelanocortin cDNA in an established fibroblastic cell line: constitutive secretion of the precursor without proteolytic processing.
Pro-opiomelanocortin (POMC) is the common precursor of several pituitary hormones including alpha-melanotropic hormone, adrenocorticotropic hormone, beta-lipotropin and beta-endorphin. The porcine POMC cDNA was inserted downstream from the late promoter of an SV40-derived expression vector and co-transfected in NIH 3T3 cells with a marker plasmid carrying the neomycin resistance gene. Colonies resistant to the neomycin analog G418 were selected and analyzed for the production of POMC-related peptides by radioimmunoassay. Three clones were found to produce from 350 to 1750 pg of POMC-related peptides per 10(6) cells in 16 h and selected for further analysis. The number of POMC cDNA copies integrated in the host cell genome was determined and the levels of transcription were compared. POMC-related material released in the culture medium by the best producing clone (NJP 4-4) was further analyzed by gel filtration and reversed-phase high-pressure liquid chromatography combined with radioimmunoassays. POMC was found to be synthesized and secreted without further processing or degradation. Negligible amounts of POMC-immunoreactive species were found in cellular extracts indicating that the prohormone is secreted from the NIH 3T3 cells without storage, presumably through a constitutive pathway. Our results suggest that NIH 3T3 fibroblasts do not contain the enzymatic machinery to process complex precursors such as POMC.
Zollinger
L
L
Département de Biochimie, Faculté de Médecine, Université de Montréal, Qc, Canada.
Noël
G
G
Des Parois
L
L
Sales
V
V
Crine
P
P
Boileau
G
G
D000818:Animals; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D051379:Mice; D009355:Neomycin / Q000235:genetics; D010957:Plasmids; D011333:Pro-Opiomelanocortin / Q000276:immunology / Q000502:physiology*; D012313:RNA / Q000032:analysis; D013539:Simian virus 40 / Q000502:physiology; D013552:Swine
D012313:RNA; D011333:Pro-Opiomelanocortin; D004247:DNA; D009355:Neomycin