Abstract
Insulin has been shown to inhibit rat growth hormone (GH) gene transcription. The effects of insulin were therefore tested on the expression of a transfected human GH gene. A 2.6-kilobase EcoRI fragment of the human GH gene was propagated in pUC18 and transfected by calcium-phosphate shock into HeLa and GC cells, respectively. Transfected cells grown in serum-free medium for 72 h expressed human GH measured by specific radioimmunoassay, incorporation of [35S] methionine into newly synthesized GH, and the presence of the appropriately sized protected transcripts seen after RNase protection assay. Immunoprecipitation analysis showed that insulin (0.7-7 nM) suppressed both the basal as well as the hydrocortisone (100 nM)-stimulated expression of newly synthesized 22-kDa GH in a dose-dependent fashion. Insulin (7 nM) also suppressed the basal and hydrocortisone-stimulated GH mRNA transcripts in these cells. Control nontransfected cells did not express human GH. Cells transfected with the truncated pOGH gene and pTKGH gene failed to respond to insulin treatment, whereas the human GH promoter was able to confer insulin responsiveness to the chloramphenicol acetyltransferase reporter gene. cis-Acting regulatory sequences residing on the 497-base pair 5'-flanking region of the human GH gene therefore appear to be a requirement for human GH gene response to the insulin signal.
Highlights
Insulin has been shown to inhibit rat growth horinsulin (1.7 MM)suppressed dexamethasone-induced GH gene mone (GH) gene transcription
Insulin (7 nM) did not significantly alter thealready low basal hGH secretion by untreated transfected cells, but did markedly suppress the hGH expression induced by treatment with hydrocortisone during 72 h incubation
Immunoprecipitation and Gel Analysis-HeLa and GC cells were treated for 72 h and labeled with [35S]methionine for a further 4 h
Summary
Immunoprecipita- To further delineate the transcriptional regulation of GH tion analysis showedthatinsulin (0.7-7 nM) sup- by insulin, a human GH (EcoRI fragment) recombinant in pressed both the basal as well as the hydrocortisone pUC18 plasmid [20] was transfected into human HeLa and (100 nM)-stimulated expression of newly synthesized rat GC cellscontaining abundantinsulin receptors. Insulin has been shown human growth hormone gene construct, was confirmed by apto regulate the mRNA levels and transcriptional activity for propriate restriction endonuclease digestion, according to the pubseveral proteins [6,7,8,9,10,11]. In rat pituitary the hGH gene intothe HindIIIsite of pSVOCAT [33] utilizing cell cultures, insulin (10 nM) maximally inhibited [3H]leucine HindIII lin‘kers.
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