Analysis of proopiomelanocortin (POMC) messenger ribonucleic acid and POMC-derived peptides in human peripheral blood mononuclear cells: no evidence for a lymphocyte-derived POMC system.
A number of recent studies suggest that cells of the immune system, e.g. peripheral blood mononuclear cells (PBMC), can synthesize and process POMC and secrete POMC-derived peptides, such as ACTH and endorphins, upon immune and hormonal challenges. From this, it has been proposed that POMC-derived peptides originating from lymphoid cells can function as hormones, for instance in a lymphoid-adrenal axis. In view of the important physiological implications of this proposal, the present study was designed to investigate the expression of the POMC gene in human PBMC and the production by these cells of alpha-, beta-, and gamma-endorphins (alpha E, beta E, and gamma E) peptides that are established end products of the posttranslational processing of POMC. PBMC of individual donors were used uncultured (fresh cells) or cultured for 24 and 48 h in the presence and absence of Concanavalin-A (Con-A), bacterial lipopolysaccharide, phytohemagglutinin, or CRH, and vasopressin, conditions that reportedly stimulate POMC activity in those cells, to investigate the presence of POMC transcripts by analysis of total RNA with Northern blotting and the reverse transcriptase polymerase chain reaction (RT-PCR). Large scale preparations containing over 10(9) cells (fresh, cultured with and without Con-A) originating from several donors were examined for the presence of POMC transcripts by analysis of poly(A)+ RNA on Northern blots and for the presence of alpha E, beta E, and gamma E by gel filtration over Sephadex G-75 and reverse phase HPLC, followed by assay of the fractions in four endorphin RIA systems with different specificities. On the Northern blots of total RNA, no POMC transcripts were detectable. In poly(A)+ RNA preparations, no full-length POMC mRNA was found, and it was estimated that the concentration of POMC mRNA, if present, was below approximately 0.005 transcript/cell in Con-A-stimulated cells and still lower in unstimulated cells. In accord with literature data, an 800- to 900-nucleotide POMC transcript was detected in cultured PBMC, and the levels of this transcript were stimulated by Con-A. In all samples analyzed with RT-PCR, a transcript spanning most of exons 2 and 3 was detectable only on Southern blots of the RT-PCR product, but not on agarose gels stained with ethidium bromide. Chromatographic analysis of endorphin immunoreactivities in cell extracts revealed no qualitative differences between the immunoreactive profiles of fresh PBMC or PBMC cultured with or without Con-A.(ABSTRACT TRUNCATED AT 400 WORDS)
van Woudenberg
A D
AD
Rudolf Magnus Institute, Department of Medical Pharmacology, University Hospital, Utrecht University, The Netherlands.
Metzelaar
M J
MJ
van der Kleij
A A
AA
de Wied
D
D
Burbach
J P
JP
Wiegant
V M
VM
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002402:Cathepsin D / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D015342:DNA Probes; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D012333:RNA, Messenger / Q000097:blood*; D001615:beta-Endorphin / Q000032:analysis*
D015342:DNA Probes; D010446:Peptide Fragments; D012333:RNA, Messenger; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D002402:Cathepsin D