Leptin Increases Tissue Inhibitor of Metalloproteinase I (TIMP-1) Gene Expression by a Specificity Protein 1/Signal Transducer and Activator of Transcription 3 Mechanism

Abstract Leptin has properties of a profibrogenic cytokine. In liver, the activated hepatic stellate cell (HSC) is responsible for a net production of extracellular matrix. A key molecule synthesized is the tissue inhibitor of metalloproteinase I (TIMP-1), which acts to inhibit the activity of matrix metalloproteinases. The purpose of the present study was to determine how leptin, a gp130 cytokine, orchestrates the regulation of TIMP-1 gene activation and expression. Transient transfection of primary HSCs revealed that leptin significantly increased luciferase activity of a 229-bp TIMP-1 promoter construct (TIMP-1–229). An EMSA revealed that leptin enhanced specificity protein 1 (Sp1) binding. Site-directed mutagenesis for Sp1 reduced the enhancing effect of leptin on TIMP-1 transcriptional activation, and this effect was dose dependent on the number of Sp1 sites mutated. Chromatin immunoprecipitation revealed that leptin enhanced binding of Sp1; however, inhibition of signal transducer and activator of transcription (STAT) 3 phosphorylation by AG490 also blocked Sp1 phosphorylation and significantly reduced leptin-associated TIMP-1–229 promoter activity, indicating that one mechanism for leptin-increased transcriptional activity is via phosphorylation of Sp1 and subsequent promoter binding. Finally, we demonstrate that leptin also results in intranuclear pSTAT3 binding to Sp1. We propose a novel mechanism whereby leptin-mediated TIMP-1 transcription employs a Sp1/pSTAT3-dependent mechanism, one of which is a noncanonical association between Sp1 and pSTAT3. These data provide a new molecular mechanism whereby the adipocytokine leptin plays a role in complications of the metabolic syndrome.

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Signal Transducer and Activator of Transcription 3α and Specificity Protein 1 Interact to Upregulate Intercellular Adhesion Molecule-1 in Ischemic–Reperfused Myocardium and Vascular Endothelium

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/01.atv.0000168428.96177.24 ◽

2005 ◽

Vol 25 (7) ◽

pp. 1395-1400 ◽

Cited By ~ 38

Author(s):

Xiao Ping Yang ◽

Kaikobad Irani ◽

Subhendra Mattagajasingh ◽

Anthony DiPaula ◽

Firdous Khanday ◽

...

Keyword(s):

Adhesion Molecule ◽

Vascular Endothelium ◽

Intercellular Adhesion ◽

Intercellular Adhesion Molecule ◽

Signal Transducer ◽

Intercellular Adhesion Molecule 1 ◽

Specificity Protein 1 ◽

Specificity Protein ◽

Reperfused Myocardium

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Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A ofBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.182.24.6975-6982.2000 ◽

2000 ◽

Vol 182 (24) ◽

pp. 6975-6982 ◽

Cited By ~ 4

Author(s):

Janet K. Hatt ◽

Philip Youngman

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Mutational Analysis ◽

Response Regulator ◽

Gene Activation ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Specific Gene ◽

Dna Binding Domain ◽

Conserved Residues

ABSTRACT The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-formingBacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

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Transcription factors YY1, Sp1 and Sp3 modulate dystrophin Dp71 gene expression in hepatic cells

Biochemical Journal ◽

10.1042/bcj20160163 ◽

2016 ◽

Vol 473 (13) ◽

pp. 1967-1976 ◽

Cited By ~ 2

Author(s):

Katia Peñuelas-Urquides ◽

Carolina Becerril-Esquivel ◽

Laura C. Mendoza-de-León ◽

Beatriz Silva-Ramírez ◽

José Dávila-Velderrain ◽

...

Keyword(s):

Transcription Factors ◽

Promoter Region ◽

Site Directed Mutagenesis ◽

Cellular Processes ◽

Specificity Protein 1 ◽

Hepatic Cells ◽

Dna Elements ◽

Yin And Yang ◽

Specificity Protein ◽

Flanking Regions

Dystrophin Dp71, the smallest product encoded by the Duchenne muscular dystrophy gene, is ubiquitously expressed in all non-muscle cells. Although Dp71 is involved in various cellular processes, the mechanisms underlying its expression have been little studied. In hepatic cells, Dp71 expression is down-regulated by the xenobiotic β-naphthoflavone. However, the effectors of this regulation remain unknown. In the present study we aimed at identifying DNA elements and transcription factors involved in Dp71 expression in hepatic cells. Relevant DNA elements on the Dp71 promoter were identified by comparing Dp71 5′-end flanking regions between species. The functionality of these elements was demonstrated by site-directed mutagenesis. Using EMSAs and ChIP, we showed that the Sp1 (specificity protein 1), Sp3 (specificity protein 3) and YY1 (Yin and Yang 1) transcription factors bind to the Dp71 promoter region. Knockdown of Sp1, Sp3 and YY1 in hepatic cells increased endogenous Dp71 expression, but reduced Dp71 promoter activity. In summary, Dp71 expression in hepatic cells is carried out, in part, by YY1-, Sp1- and Sp3-mediated transcription from the Dp71 promoter.

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