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 hormo ne regulating appetite and energy balance. It has recently become apparent that leptin is actively expressed in a number of other tissues including th e CNS and pituitary, as well as brain- and pituitary-derived cell lines. Ho wever, the factors controlling leptin expression in cells of neuroectoderma l origin are unknown. The mouse leptin gene 5'-flanking DNA contains multip le AP-I 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 ma y contribute to leptin gene transcription in pituitary and brain. We have u sed leptin promoter-luciferase reporter constructs to examine the regulatio n of the leptin promoter in 3T3-L1 preadipocytes, C6 glioma cells, and GH3 pituitary cells in response to serum and hormonal stimuli. Cells were trans iently 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 (- 6000-1uc). Functional analysis indicates that the leptin promoter is constitutively active in all 3 cell lines. Transcriptional act ivity was significantly higher with a - 500 to + 9 promoter than with a con struct containing - 6000 to + 9 bp of 5' flanking DNA, indicating the prese nce of repressor elements which may contribute to the tissue-specific regul ation of leptin expression. However, qualitatively similar results were obs erved 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. Ill contrast, the response of the lepti n promoter to insulin, ICF-I 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, b ut had no significant effect in the presence of 10% FBS. In contrast, dibut yryl 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 m inimal effects. These findings support our previous studies on the regulati on of leptin steady state mRNA levels in C6 cells and demonstrate tissue-sp ecific differences in the regulation of leptin gene transcription in adipos e vs. neuroectodermal tissues. (C) 2001 Elsevier Science Ireland Ltd. All r ights reserved.