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Link to original content: https://pubmed.ncbi.nlm.nih.gov/16931573/
Leptin increases tissue inhibitor of metalloproteinase I (TIMP-1) gene expression by a specificity protein 1/signal transducer and activator of transcription 3 mechanism - PubMed Skip to main page content
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. 2006 Dec;20(12):3376-88.
doi: 10.1210/me.2006-0177. Epub 2006 Aug 24.

Leptin increases tissue inhibitor of metalloproteinase I (TIMP-1) gene expression by a specificity protein 1/signal transducer and activator of transcription 3 mechanism

Songbai Lin  1 , Neeraj K Saxena, Xiaokun Ding, Lance L Stein, Frank A Anania
Affiliations

Leptin increases tissue inhibitor of metalloproteinase I (TIMP-1) gene expression by a specificity protein 1/signal transducer and activator of transcription 3 mechanism

Songbai Lin et al. Mol Endocrinol. 2006 Dec.

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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Figures

Fig. 1
Fig. 1. TIMP-1 Gene Expression Is Elevated by Leptin Both in Vivo and in Vitro
A, Total RNA extracted from whole livers from the four treatment groups was used to determine mRNA expression of TIMP-1 as described in Materials and Methods by RT-qPCR. Level of mRNA expression, observed in sham-operated lean littermates was set at 100% of control. Results are means ± se and represent 12 reactions for each treatment condition. *, P < 0.01 compared with lean sham-operated values. B, Subconfluent activated rat HSCs were serum starved for 16 h with 0.1% FBS in DMEM and then exposed to increasing concentrations of leptin for 24 h and analyzed for TIMP-1 mRNA expression by RT-qPCR. Values are means ± se, expressed as fold change relative to the control (no leptin). *, P < 0.01. The results are representative of three independent experiments. C, Activated rat HSCs were treated with leptin (100 ng/ml) for the times indicated and analyzed for TIMP-1 mRNA expression by RT-qPCR. The results, representative of three independent experiments, are compared with respect to control (no leptin) at the respective time points). *, P < 0.01. D, Activated HSCs were grown in DMEM with 0.1% FBS for 16 h pretreated with leptin (0 or 100 ng/ml) for 4 h and subsequently treated with actinomycin D [5 µg/ml]. Cells were harvested for total RNA at time points indicated after addition of actinomycin D. TIMP-1 mRNA was quantitated using RT-qPCR and normalized to 18 S rRNA. Results are expressed as fold change relative to the samples at time zero. The entire experiment was repeated three separate times in triplicate. TIMP-1 mRNA stability was evaluated by computing the best fit linear curve on a linear plot of the mRNA fold change vs. time for leptin and nonleptin treatment groups. SF, Serum free.
Fig. 2
Fig. 2. Localization of a Human TIMP-1 Promoter Region (−1482 to +88) Mediating the Effect of Leptin on TIMP-1 Transcription in Activated Rat HSCs
A, Various lengths of human TIMP-1 promoter sequences were cloned upstream from luciferase reporter gene as described in Materials and Methods. The schematic diagram represents six deletion mutants: TIMP-1–1482, TIMP-1–989, TIMP-1–582, TIMP-1–369, TIMP-1–229, and TIMP-1–108 used for promoter analysis. B, Transient transfection of activated HSCs with respective promoter activities by firefly luciferase assay. The assay was repeated three times in triplicate and represent the mean ± se; *, P < 0.01 with respect to TIMP-1–229. SF, Serum free.
Fig. 3
Fig. 3. The Effect of Leptin on the Binding of HSC Nuclear Extracts to Oligonucleotides Spanning the 229-bp TIMP-1 Promoter
A, Schematic diagram of six oligonucleotides spanning the 229 bp TIMP-1 promoter were synthesized, annealed and radiolabeled as probes for EMSA. B, Nuclear extracts (5 µg) were prepared from activated primary rat HSCs, serum starved for 16 h, and exposed to leptin (100 ng/ml) for 24 h. Leptin enhanced binding of nuclear extracts prepared from treated HSCs to probes 1 and 3 when compared with binding activities of untreated extracts. C, To identify complexes formed for probes 1 and 3, competition and antisera reactions were performed as described in Materials and Methods. FP, Free probe; SF, serum-free, untreated control; L, leptin treatment; Cold 50×, competition assay from leptin-treated nuclear extracts in the presence of 50-fold excess unlabeled probe 3 (P3). Sp1 and Sp3 antisera were added to reaction mixtures for supershift assay in lanes 5 and 6, respectively, and supershift complexes were formed (long arrow). The assay is representative of three independent experiments.
Fig. 4
Fig. 4. Mutation of the Sp1 Sites Abolishes Effect of Leptin on TIMP-1 Promoter Activity
A, Four substitution mutation constructs for the TIMP-1–229 deletion mutant were synthesized as described in Materials and Methods. B, Subconfluent primary activated HSCs were transiently transfected with these constructs to determine the role of Sp1 in mediating leptin-enhanced TIMP-1 promoter activity as assessed by firefly luciferase activity. Relative promoter activity from three independent experiments performed in triplicate expressed as mean ± se. *, P < 0.01 with respect to basal TIMP-1–229 promoter activity. SF, Serum free.
Fig. 5
Fig. 5. Leptin Increases Phosphorylation, But Not Protein Level, of Sp1 and Sp3
A, Subconfluent primary activated HSCs were serum starved for 16 h, and then cultured in the presence of leptin (100 ng/ml) or vehicle for the times indicated before cellular proteins were harvested. The Western blots were performed as described in Materials and Methods in triplicate using antibody to pSTAT3. B, Primary activated HSCs were grown and starved as described above, and then cultured in the presence of leptin (100 ng/ml) or vehicle for the times indicated. Sp1 and Sp3 proteins were immunoprecipitated (IP) and the immunoprecipitates were subjected to SDS-PAGE. The membranes were probed with antiphosphoserine antibody to evaluate the phosphorylation status of Sp1 and Sp3. The membranes were also probed with anti-Sp1 and -Sp3 antibody to monitor the quantity of Sp1 or Sp3. The gels shown were representative of three independent experiments. Maximal phosphorylation occurs for Sp1 at 4 h with minimal phosphorylation of Sp3. IB, Immunoblot.
Fig. 6
Fig. 6. ChIP Assay Reveals that Leptin Enhances the Binding of Sp1, But Not Sp3 or pSTAT3, to the Rat TIMP-1 Promoter
A, Subconfluent primary activated HSCs were serum starved for 16 h, and then cultured in the presence of leptin (100 ng/ml) or vehicle for 24 h before the cells were lysed and sonicated. Formaldehyde cross-linked chromatin from HSCs was incubated with Sp1, Sp3, pSTAT3, or control antisera. Immunoprecipitated DNA was analyzed by PCR with primer to proximal promoter of rat TIMP-1 gene or glyceraldehyde phosphate dehydrogenase (GAPDH) gene. Total input DNA at a 1:100 dilution was used as a positive control of the PCRs. DNA prepared from chromatin that was immunoprecipitated with nonimmune rabbit sera (+IgG) was used as a negative control for assays. The lanes are designated either according to the antisera used for the ChIP assay or by “Input,” which indicates the preimmunoprecipitated chromatin used in the PCR. The gel shown is representative of three independent experiments. B, Protein-DNA complexes immunoprecipitated were measured by real-time PCR. Results, expressed as fold change differences of DNA (amplified promoter) between leptin and vehicle samples were calculated relative to GAPDH as described in Materials and Methods. *, P < 0.01, leptin treatment vs. untreated control. Results are means ± se and represent three independent studies. SF, Serum free.
Fig. 7
Fig. 7. AG490 Inhibited Leptin-Stimulated STAT3 and Sp1 Phosphorylation and Abolished the Leptin-Enhanced TIMP-1 Promoter Activity
A, Subconfluent primary activated HSCs were serum starved for 16 h, and then cultured with leptin (100 ng/ml) or vehicle with or without AG490 (10 µm) for 4 h before cellular proteins were harvested. IL6 (10 ng/ml) was used as positive control for STAT3 activation. pSTAT3 and STAT3 were assayed by immunoblot (IB) analysis as shown. Equal loading of proteins in each lane was confirmed by probing membranes for β-actin. B, Identical experimental design to A was conducted to probe to determine whether AG490 blocked phosphorylation of Sp1 after immunoprecipitation and probed with anti-phosphoserine antibody to evaluate the phosphorylation status of Sp1. The membranes were also probed with anti-Sp1 to monitor the quantity of Sp1. The present study is representative of two independent experiments. C, Subconfluent primary activated HSCs were transfected with TIMP-1–229, serum starved for 16 h, and then cultured as described above for 24 h before cell lysates were harvested for measurement of luciferase activity. Relative promoter activity from three independent experiments performed in triplicate are expressed as means ± se; *, P < 0.01. D, 90% confluent primary activated HSCs were transfected with FLAG-SOCS-3 expression vector or corresponding empty vector as described elsewhere. The cells were serum starved for 16 h, and then cultured with leptin (100 ng/ml), IL-6 (10 ng/ml) or vehicle for 4 h before cellular proteins were harvested. The lysates were immunoprecipitated using Sp1 antibody or control anti-sera. Total input protein was assessed to show the successful transfection of FLAG-SOCS-3 expression vector using flag antibody.
Fig. 8
Fig. 8. Sp1 and STAT3 Complex Was Detected and STAT3 Antibodies Disrupt the Leptin-Induced Binding of Sp1 to the TIMP-1 Promoter
A, Primary activated HSCs were transfected with 24 µg of Sp1 expression vector (pN3-Sp1FL-complete), serum starved for 16 h, and treated with or without leptin for 24 h. Nuclear protein was extracted and 200 µg of protein were subjected to immunoprecipitation (IP) using an antibody against Sp1 or antirabbit IgG (nonimmune sera). The immunoprecipitated proteins were separated and subjected to immunoblot analysis with either anti-pSTAT3, or anti-phosphoserine. Twenty micrograms of nuclear protein were input for detecting Sp1 expression. Immunoblot (IB) with either anti-pSTAT3 or anti-phosphoserine antibodies of leptin-treated primary activated HSC nuclear protein lysate immunoprecipitated with anti-Sp1 antibody revealed pSTAT3-Sp1 complexes. COIP, Coimmunoprecipitation. B, To identify a physical mechanism whereby STAT3 would complex with Sp1, EMSA was carried out using 5 µg nuclear extracts prepared from leptin-treated HSCs for all lanes. Lane 1, Free probe; lane 2, serum free (SF) exposed HSC nuclear extracts; lane 3, leptin-treated primary activated HSC nuclear extract with probe 3 (cf. Fig. 3) only (Leptin); lane 4, supershift assay with nonimmune serum IgG; lane 5, antibody to Sp1 resulting in supershift of Sp1-olignonucleotide complex (Sp1); lane 6, STAT3 antisera disrupted Sp1 or Sp3 binding to the oligonucleotide, but STAT1 antisera (lane 7) did not result in nuclear protein-oligonucleotide disruption. This EMSA is representative of three independent experiments.
Fig. 9
Fig. 9. Potential Mechanism for Leptin Activation of STAT3 and Sp1 in the Regulation of the TIMP-1 Promoter
Based upon the data presented leptin activates STAT3 phosphorylation via Jak2. This process also results in the phosphorylation of Sp1 as well as nuclear translocation and binding of phosphorylated Sp1 to the TIMP-1 promoter. Based on ChIP analysis (Fig. 5) and coimmunoprecipitation and EMSAs (Fig. 8), we propose that STAT3 nuclear translocation results in the physical association of pSTAT3 and phosphorylated Sp1.
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