title: Identification of regulatory elements in the human adipose most abundant gene transcript-1 ( apM-1) promoter: role of SP1/SP3 and TNF-alpha as regulatory pathways.
abstract: OBJECTIVE
  The human adipocyte-specific apM-1 (adipose most abundant gene transcript-1) gene encodes for a secretory protein of the adipose tissue that seems to play a role in the pathogenesis of obesity-related insulin resistance and its expression is inhibited by TNF-alpha. Our aim was to characterize the tissue-specific regulation of the recently cloned apM-1 promoter and the mechanisms of TNF-alpha-induced downregulation of the apM-1 gene.
  
  
  METHODS
  We characterised the apM-1 gene by electrophoretic mobility shift assays (EMSA) and luciferase reporter gene assays (LRA).
  
  
  RESULTS
  Although several putative binding sites for transcription factors known to be involved in adipogenesis such as C/EBP and PPARgamma are present in the promoter, we could not detect any binding of these nuclear proteins from differentiated adipocytes. However, a proximal SP1 binding site specifically binds both, recombinant SP1 protein and SP1 derived from adipocyte nuclear extracts. Since the expression of SP1 during adipocyte differentiation has not yet been analysed, we could show by using EMSA, that binding activity of SP1 is increased during adipocyte differentiation. The stimulatory activity of SP1 was confirmed in LRA by cotransfection experiments in S2 Schneider cells lacking endogenous SP factors. An inhibitory activity of SP3 on the stimulatory effect of SP1 could be confirmed in LRA by contransfection experiments in adipocytes. Nuclear extracts from adipocytes incubated with TNF-alpha showed a reduced binding activity of SP1.
  
  
  CONCLUSIONS
  SP1 is expressed and its binding activity is enhanced during adipocyte differentiation. SP1 has stimulatory effects, SP3 has inhibitory effects on apM-1 promoter activity, mediated by a proximal SP1 binding site. The mechanism of TNF-alpha-induced inhibition of apM-1 gene expression is, at least in part, due to a decrease of transcriptional SP1 binding activity caused by TNF-alpha and thus provides a new mechanism of TNF-alpha-dependent signalling.
authors.affiliation: Department of Internal Medicine I, University of Regensburg, 93042 Regensburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Barth
authors.forename: T
authors.initials: T
authors.lastname: Langmann
authors.forename: J
authors.initials: J
authors.lastname: Schölmerich
authors.forename: G
authors.initials: G
authors.lastname: Schmitz
authors.forename: A
authors.initials: A
authors.lastname: Schäffler
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D052242:Adiponectin; D000818:Animals; D002454:Cell Differentiation; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000502:physiology*; D006706:Homeostasis; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008156:Luciferases / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D052242:Adiponectin; D004268:DNA-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051705:Sp3 Transcription Factor; D008156:Luciferases