The genomic structure and transcriptional regulation of the rat apelin receptor (APJR) were analysed by rapid amplification of 5′ cDNA ends (5′-RACE), transient expression assays and DNA–protein interaction. Analysis of the 5′-flanking region of a rat genomic clone shows no TATA box, but a putative CAAT box and several putative binding sites for transcription factors are present. Two transcriptional start sites were identified by 5′-RACE, RNase protection and primer extension analyses. Promoter activity was exhibited in the APJR- expressing SH-SY5Y cell line as well as in COS-7 and Chinese hamster ovary (CHO-K1) cells. Consecutive 5′-deletion analysis revealed the highest promoter activity in a region between bp −966 and −165. DNaseI footprint analysis revealed seven protected regions and electrophoretic mobility shift, super-shift and competition assays identified individual DNA–protein complexes capable of binding Sp1, estrogen receptor (ER)α, glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP)γ transcription factors. Site-directed mutagenesis identified an individual Sp1 motif that plays a major role in activation of the APJR promoter and also demonstrated constitutive transcriptional regulation of the promoter by estrogen and glucocorticoid receptors. Promoter regulation by the cAMP-dependent signal cascade was also shown.
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Anne-Marie O’Carroll, Stephen J Lolait, and Gillian M Howell
J. M. Stephens, M. D. Butts, and P. H. Pekala
ABSTRACT
3T3-L1 preadipocytes differentiate into cells having the biochemical properties of adipocytes; tumour necrosis factor-α (TNF) attenuates this process. Inhibition of differentiation by this cytokine, thought to be mediated at the level of transcription, has been investigated by examining the accumulation of mRNA for six transcription factors and three diversely regulated genes during the first 24 h of the differentiation process. Upon induction of differentiation, a rapid and major accumulation of c-fos and jun-B mRNA, which returned to near basal levels within 4–6 h, was observed. In contrast, c-jun mRNA, although rapidly expressed at the induction of differentiation, remained at relatively constant levels throughout the time-course. Exposure of the cells to 5 nm TNF potentiated the accumulation of all three mRNAs but most significantly that of c-jun (12-fold), which remained elevated for at least 24 h after treatment. In control differentiating cells, krox-20 and fos-B were expressed transiently from 30 min to 2 h, while fra-1 mRNA accumulated over an extended period of 1 to 8 h. Again, TNF enhanced the accumulation of these mRNAs. Accumulation of mRNA for C/EBP, a transcription factor proposed to control the expression of genes involved in the terminally differentiated state, was attenuated after exposure of the cells to TNF. Interleukin-6 (IL-6) mRNA was expressed briefly (30 min to 2 h) and again transiently (at 8 h after induction of differentiation). TNF treatment markedly enhanced accumulation of IL-6 message. We propose that an increased cellular content of one or more transcription factors or the suppression of C/EBP may be responsible for the attenuation of differentiation induced by exposure of the cells to TNF.
Ashley A Able, Allison J Richard, and Jacqueline M Stephens
STAT5A (signal transducer and activator of transcription 5A) is a transcription factor that plays a role in adipocyte development and function. In this study, we report DBC1 (deleted in breast cancer 1 – also known as CCAR2) as a novel STAT5A-interacting protein. DBC1 has been primarily studied in tumor cells, but there is evidence that loss of this protein may promote metabolic health in mice. Currently, the functions of DBC1 in mature adipocytes are largely unknown. Using immunoprecipitation and immunoblotting techniques, we confirmed that there is an association between endogenous STAT5A and DBC1 proteins under physiological conditions in the adipocyte nucleus that is not dependent upon STAT5A tyrosine phosphorylation. We used siRNA to knockdown DBC1 in 3T3-L1 adipocytes to determine the impact on STAT5A activity, adipocyte gene expression and TNFα (tumor necrosis factor α)-regulated lipolysis. The loss of DBC1 did not affect the expression of several STAT5A target genes including Socs3, Cish, Bcl6, Socs2 and Igf1. However, we did observe decreased levels of TNFα-induced glycerol and free fatty acids released from adipocytes with reduced DBC1 expression. In addition, DBC1-knockdown adipocytes had increased Glut4 expression. In summary, DBC1 can associate with STAT5A in adipocyte nucleus, but it does not appear to impact regulation of STAT5A target genes. Loss of adipocyte DBC1 modestly increases Glut4 gene expression and reduces TNFα-induced lipolysis. These observations are consistent with in vivo observations that show loss of DBC1 promotes metabolic health in mice.
