Characterization of the Promoter-Directing Expression of Growth Hormone in a Monocyte Cell Line

Abstract

Previous work from our laboratory has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that produced by the pituitary. In the present study, using Southern analysis of RT-PCR products and sequencing of cloned cDNA molecules, we demonstrate that lymphoid cell lines utilize the same promoter and first exon as the pituitary somatotrope. To identify the cis-elements involved in transcriptional regulation of immune cell-derived GH, we have coupled rat GH promoter fragments to a luciferase reporter gene and transfected a monocyte cell line (P-388) by electroporation. The results suggest the presence of both positive (–299/–193 bp) and negative (–193/–107 bp) regulatory elements. The same constructs transfected in the pituitary cell line, GH3, in contrast to the monocyte cell line, showed a gradual decrease in luciferase expression. The overexpression of GHF-1 or GHF-2 resulted in a modest but significant reduction in rat GH promoter activity in the P-388 cell line. Taken together, the data suggest that immune cells utilize the same first exon and promoter sequence for the expression of monocyte GH as that reported for the expression of pituitary GH. Further, it appears that sequences between –299 and –107 bp are important in the regulation of the promoter where different transcription factors may be recruited to promote GH expression in a monocyte cell line.

Hypoxia and cytoplasmic alkalinization upregulate growth hormone expression in lymphocytes

“…Rat GH promoter luciferase plasmid DNA (pGL2-B luciferase vector, Promega) containing a fragment (−417 to +13 bp) of the GH promoter [29] was cotransfected by electroporation with the pCR3.1 vector (Invitrogen) containing G418 resistance at a ratio of 10:1. Cells were resuspended at 30 × 10 6 cells/ml in RPMI 1640 (no serum) + 10 mM dextrose, 0.1 mM dithiothreitol (DTT) containing plasmid DNA.…”