Down-regulation of tissue inhibitor of metalloproteinase-3 (TIMP-3) expression is necessary for adipocyte differentiation - PubMed (original) (raw)

Down-regulation of tissue inhibitor of metalloproteinase-3 (TIMP-3) expression is necessary for adipocyte differentiation

Denis Bernot et al. J Biol Chem. 2010.

Abstract

Matrix metalloproteinase activity is essential for proper extracellular matrix remodeling that takes place during adipose tissue formation. Four tissue inhibitors of matrix metalloproteinases (TIMPs) regulate their activity. However, the role of TIMPs in adipocyte differentiation is poorly understood. We found that the expression of all TIMPs was modified during adipocyte differentiation, but that of TIMP-3 was distinguished by its extreme down-regulation. TIMP-3 expression was closely linked to the differentiation process. Indeed, it remained low during the adipocyte differentiation but increased when cell differentiation was prevented. We identified the transcription factor Sp1 as being responsible for the regulation of TIMP-3 expression during adipocyte differentiation. Overexpression of TIMP-3 reduced adipocyte differentiation, underlining its active role in this process. TIMP-3 overexpression decreased the expression of the early and obligate key inductors of adipogenesis Krüppel-like factor 4 (Klf4), early growth response 2 (Egr2/Krox20), and CAAT/enhancer-binding protein beta (C/EBPbeta). Our results indicate that during preadipocyte differentiation, the Sp1-dependent decrease in TIMP-3 expression is required for the successful implementation of the adipocyte differentiation program.

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Figures

FIGURE 1.

FIGURE 1.

Expression of TIMPs during adipocyte differentiation. A and B, real time PCR analysis of mRNAs levels of adipocyte markers (SREBP-1c, PPARγ, and leptin), preadipocyte marker (Pref1) (A), and TIMP-1, -2, -3, -4 during adipocyte differentiation of 3T3-L1 cells (B). Inset, Western blot of TIMP-3 contained in the extracellular matrix. C, real time PCR analysis of TIMP-3 mRNA levels of mouse embryonic fibroblasts (MEF), human visceral (VIS) and subcutaneous (SC) preadipocytes before (d−2) and after 2 days (d0) of treatment with the induction mixture. Days of analysis are indicated below graphs. Error bars, S.D.

FIGURE 2.

FIGURE 2.

Adipocyte differentiation maintains low level of TIMP-3. 3T3-L1 cells cultured in the absence or presence of glucose were submitted to the adipogenic treatment. TIMP-3 (left) and Pref1 (right) mRNA levels were analyzed by real time PCR. Days of analysis are indicated below graphs. *, significant versus the situation with glucose (p < 0.007). Error bars, S.D.

FIGURE 3.

FIGURE 3.

hTIMP-3 overexpression impairs adipocyte differentiation. 24 h after the transfection with empty plasmid or hTIMP-3 expression vector, 3T3-L1 cells were untreated (Cont) or treated (IC) for 48 h with the induction mixture. Caspase activity (A) and cell number (B) were measured as described under “Experimental Procedures.” After 3 days of differentiation, TIMP-3 expression was analyzed by Western blotting (C, upper panel), and intracellular lipid accumulation was visualized by oil red O staining (C, lower panel). D, mRNAs levels of adipocyte markers (SREBP-1c, PPARγ, and leptin), preadipocyte marker (Pref1), and TIMP-3 were analyzed by real time PCR after 3 days of differentiation. Error bars, S.D.

FIGURE 4.

FIGURE 4.

TIMP-3 expression is regulated by transcriptional events. A, 3T3-L1 cells were stimulated for 24 h with Mix, Dex, and Mix+Dex, then TIMPs and PPARγ mRNA levels were analyzed by real time PCR. B, Dex and Mix were added to 3T3-L1 cells 6 h after their transfection with TIMP-3 luciferase reporter gene, and luciferase activity was measured 24 h later. Cont, untreated. C, 12 h after the stimulation with Dex and Mix, TIMP-3 nuclear pre-mRNA levels were analyzed by real time PCR using intronic primers. Error bars, S.D.

FIGURE 5.

FIGURE 5.

Mode of action of Dex and Mix on TIMP-3 expression. A, 3T3-L1 cells were untreated (Cont) or treated for 24 h with Dex, 1 μ

m

Ru 486 (Ru) alone, or in association with Dex (Ru+Dex). B, same as A except that cells were treated with Mix, 1 μ

m

rolipram (Rol), 400 μ

m

dibutyryl-cAMP (cAMP), and then TIMP-3 mRNA levels were analyzed by real time PCR. Inset, Mix was added to 3T3-L1 cells 24 h after their transfection with pCRE-SEAP. SEAP protein accumulated in the medium was measured 12 h later. Error bars, S.D.

FIGURE 6.

FIGURE 6.

Sp1-driven TIMP-3 transcription is reduced by Mix and Dex. A, Western blot analysis of nuclear Sp1 from 3T3-L1 cells untreated (Cont) or treated for 24 h with Dex and Mix. B, same as Fig. 4_B_ except that cells were transfected with Sp1 luciferase reporter vector. C, cells were cotranfected with Sp1 or TIMP-3 luciferase reporter vector together with empty plasmid or Sp1 expression vector, and luciferase activity was measured 24 h later. D, cells were untreated (Cont) or treated for 24 h with Dex or Mix in the absence or presence of WP631 (100 n

m

), and TIMP-3 mRNA levels were analyzed by real time PCR. Error bars, S.D.

FIGURE 7.

FIGURE 7.

Adipocyte differentiation maintains a low Sp1 transcriptional activity. 24 h after the transfection with Sp1 luciferase gene reporter, 3T3-L1 cells were treated for 48 h with the induction mixture and then the cells were incubated for 24 h in a complete medium with insulin (Ins), without insulin (−Ins), or without glucose (−Glu), and luciferase activity was measured. Error bars, S.D.

FIGURE 8.

FIGURE 8.

hTIMP-3 overexpression inhibits Klf4, Egr2/Krox20, and C/EBPβ expression. A, real time PCR analysis of the mRNAs levels of Klf4, Egr2/Krox20, and C/EBPβ following the addition of the induction mixture. B, 24 h after transfection of 3T3-L1 cells with an empty plasmid or with hTIMP-3 expression vector, cells were left untreated or treated for 30 min with the induction mixture, and then mRNAs levels of Klf4, Egr2/Krox20, and C/EBPβ were analyzed by real time PCR. C, Western blot analysis of the expression of C/EBPβ following the addition of the induction mixture. D, 24 h after transfection of 3T3-L1 cells with an empty plasmid or with hTIMP-3 expression vector cells were left untreated or treated for 4 h with the induction mixture then C/EBPβ expression was analyzed by Western blotting. Error bars, S.D. *, significant versus respective controls.

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