Transcriptional regulation of the leptin gene promoter in rat GH3 pituitary and C6 glioma cells.
Leptin was originally believed to be an exclusively adipocyte-derived hormone regulating appetite and energy balance. It has recently become apparent that leptin is actively expressed in a number of other tissues including the CNS and pituitary, as well as brain- and pituitary-derived cell lines. However, the factors controlling leptin expression in cells of neuroectodermal origin are unknown. The mouse leptin gene 5'-flanking DNA contains multiple AP-1 and SRF-1 binding sites as well as a consensus CRE site at -491 to -482 bp. In addition, a number of potential PIT1 and Oct-1 binding sites may contribute to leptin gene transcription in pituitary and brain. We have used leptin promoter-luciferase reporter constructs to examine the regulation of the leptin promoter in 3T3-L1 preadipocytes, C6 glioma cells, and GH3 pituitary cells in response to serum and hormonal stimuli. Cells were transiently transfected with reporter constructs containing either the proximal 500 bp of the leptin promoter (-500-luc) or 6000 bp of the leptin gene 5' flanking region (-6000-luc). Functional analysis indicates that the leptin promoter is constitutively active in all 3 cell lines. Transcriptional activity was significantly higher with a -500 to +9 promoter than with a construct containing -6000 to +9 bp of 5' flanking DNA, indicating the presence of repressor elements which may contribute to the tissue-specific regulation of leptin expression. However, qualitatively similar results were observed with both constructs in response to serum and hormonal manipulation. Leptin promoter activity was significantly stimulated by serum in all cell lines, although to varying extents. In contrast, the response of the leptin promoter to insulin, IGF-1 and dibutyryl cAMP was cell-type specific and dependent on the presence or absence of FBS in the culture medium. Insulin, IGF-1 and dibutyryl cAMP each caused an approximately two-fold stimulation of leptin promoter activity in 3T3-L1 cells under serum-free conditions, but had no significant effect in the presence of 10% FBS. In contrast, dibutyryl cAMP markedly stimulated leptin promoter activity (5-8-fold) in C6 or GH3 cells in the presence or absence of FBS, whereas insulin or IGF-1 had minimal effects. These findings support our previous studies on the regulation of leptin steady state mRNA levels in C6 cells and demonstrate tissue-specific differences in the regulation of leptin gene transcription in adipose vs. neuroectodermal tissues.
Li
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AW
Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
Morash
B
B
Hollenberg
A N
AN
Ur
E
E
Wilkinson
M
M
Murphy
P R
PR
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D005910:Glioma / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D020738:Leptin / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D007328:Insulin; D020738:Leptin; D007334:Insulin-Like Growth Factor I; D000242:Cyclic AMP