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Induction of plasminogen activator inhibitor 1 gene expression in adipocytes by thiazolidinediones ¹

HAYATO IHARA^*,, TETSUMEI URANO, AKIKAZU TAKADA and DAVID J. LOSKUTOFF^*2

^* Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA; and
Department of Physiology, Hamamatsu University School of Medicine, Hamamatsu, 431-3192 Japan

²Correspondence: Department of Vascular Biology, VB-3, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA. E-mail: loskutof{at}scripps.edu

SPECIFIC AIM

We used cultured 3T3-L1 cells and thiozolidinediones (TZDs) to test the hypothesis that peroxisome proliferator-activated receptor (PPAR-) induces plasminogen activator inhibitor (PAI-1) gene expression in adipocytes during adipogenesis. This transcription factor plays a prominent role in adipogenesis, and the increase in PAI-1 may be important for the adhesion and migration of adipocytes and for adipose tissue stability.

PRINCIPAL FINDINGS

1. TZDs accelerate 3T3-L1 adipocyte differentiation and induce PAI-1 gene expression
To determine whether activated PPAR- is involved in the regulation of PAI-1 gene expression during 3T3-L1 cell differentiation, 3T3-L1 cells were treated with 20 µM pioglitazone (a potent TZD) in the presence or absence of 5 µg/ml insulin. These treatments shortened the differentiation time significantly. For example, 9–10 days were required for these cells to fully differentiate when cultured in control differentiation medium (i.e., dexamethasone plus insulin). However, treatment with both pioglitazone and insulin promoted obvious differentiation in most of the cells by 3 days. Treatment with pioglitazone or insulin alone induced differentiation in some of the cells at 3 days, but the effects were modest compared to those induced by both agents.

Figure 1 shows the changes in PAI-1 mRNA after the various treatments as determined by quantitative RT-PCR. After an initial 24 h lag period, there was a rapid induction in PAI-1 mRNA in cells treated with pioglitazone plus insulin, with maximal induction (9.5-fold) occurring at 3 days. Treatment of these cells with pioglitazone alone or with insulin alone did not induce PAI-1 gene expression during this interval. However, a small but reproducible increase in PAI-1 mRNA was observed in the insulin-treated cultures at later times (e.g., 6–9 days). Induction of PAI-1 mRNA by pioglitazone/insulin at 3 days occurred in a dose-dependent manner and resulted in a 12-fold increase in the amount of PAI-1 antigen secreted into the medium.

View larger version (20K): [in this window] [in a new window]   Figure 1. Kinetics of PAI-1 mRNA induction during adipocyte cell differentiation. Confluent 3T3-L1 cells were treated with insulin and dexamethazone for 48 h to initiate differentiation, then washed with PBS. The cells were then treated for 9 days as indicated. At various times, total RNA was extracted from the cells and the relative levels of PAI-1 mRNA were determined by quantitative RT-PCR. The data points represent the values for control (), pioglitazone (), insulin (), and pioglitazone plus insulin (•). Results shown are the means ± SD of results from independent triplicate experiments.

2. PAI-1 gene expression is induced by other TZDs in the presence of insulin
The relative effect of pioglitazone/insulin on the induction of PAI-1 mRNA was compared with that of other members of the TZD family (e.g., troglitazone, ciglitazone, and clofibrate). Although pioglitazone/insulin and troglitazone/insulin stimulated PAI-1 mRNA similarly, the effect of ciglitazone/insulin was much smaller and there was no induction by clofibrate in the presence or absence of insulin. The magnitude of induction of PAI-1 mRNA was related to the relative potencies of the TZDs as activators of PPAR-.

3. TZDs/insulin induce PAI-1 by increasing the rate of transcription of the PAI-1 gene
The marked increase in PAI-1 mRNA levels after treatment of 3T3-L1 cells with pioglitazone and insulin could result from an increase in the stability of PAI-1 mRNA, from an increase in the rate of transcription of the PAI-1 gene, or from both of these processes. To address these possibilities, we determined the effect of pioglitazone/insulin on the half-life of PAI-1 mRNA and on the promoter activity of the PAI-1 gene. Actinomycin D chase experiments revealed that in untreated control cells, PAI-1 mRNA decayed with an estimated half-life of approximately 300 min and that this half-life was unchanged when the cells were treated with pioglitazone/insulin. In contrast, transient transfection experiments using a fragment from the 5'-flanking region of murine PAI-1 gene (i.e., nucleotides -1147/158) linked to the luciferase reporter gene demonstrated that pioglitazone in the presence of insulin stimulated the PAI-1 promoter by 10-fold over the untreated control (Fig. 2 ). Although pioglitazone and insulin alone also stimulated luciferase activity in these experiments, the presence of both agents was required for maximal induction. In any case, these experiments clearly demonstrate that the induction of PAI-1 mRNA and protein by TZDs/insulin results from an increase in the rate of transcription of the PAI-1 gene and not from stabilization of PAI-1 mRNA.

View larger version (13K): [in this window] [in a new window]   Figure 2. Effect of pioglitazone on the transcriptional activity of murine PAI-1/luciferase constructs transfected into 3T3-L1 cells. Confluent 3T3-L1 cells were treated with insulin and dexamethasone for 48 h to initiate differentiation, then washed with PBS. The cells were then treated with pioglitazone in the presence or absence of insulin for 24 h, washed twice with serum-free medium (D-MEM), and incubated with serum-free medium containing lipofectAMINE (5 µl/well) and DNA constructs (p-1147 PAI/Luc and pSVß-Gal; 1 µg each/well) for 5 h. After lipofection, the cells were incubated an additional 48 h with 20 µM of pioglitazone with or without 5 µg/ml of insulin. Cell lysates were prepared and assayed for luciferase activity. The data are presented as the fold increase in luciferase activity after treatment from three independent transfections.

CONCLUSIONS

We have shown that TZDs in the presence of insulin not only accelerate the rate of differentiation of 3T3-L1 cells into lipid-laden adipocytes, but also induce PAI-1 gene expression in these cells (Fig. 3 ). The induction of PAI-1 mRNA levels occurred in a dose-dependent manner and was not specific for pioglitazone/insulin, since other TZDs also induced PAI-1 mRNA in the presence of insulin. Although the half-life of PAI-1 mRNA was not altered by TZDs/insulin, this treatment induced PAI-1 promoter activity by more than 10-fold (Fig. 2) . These results demonstrate that PAI-1 gene expression during adipogenesis can be regulated by TZDs in the presence of insulin, possibly through interactions with PPAR- (Fig. 3) . The data also indicate that the induction of PAI-1 mRNA results primarily from an increase in the rate of transcription of the PAI-1 gene and not from stabilization of PAI-1 mRNA (Fig. 3) .

View larger version (14K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the potential contribution of TZD-activated PPAR- and insulin to the induction of adipocyte differentiation and PAI-1 gene expression. Activation of PPAR- in the presence of insulin not only enhances adipocyte differentiation, but also up-regulates PAI-1 gene transcription. The increase in PAI-1 mRNA leads to increases in PAI-1 synthesis by and secretion from the adipocyte.

The molecular basis of the effects of pioglitazone/insulin on PAI-1 promoter activity remains to be defined. The transfection studies (Fig. 2) indicate that the 1147 bp DNA fragment from the 5'-flanking region of the murine PAI-1 gene contains a responsive element(s) for pioglitazone. However, this fragment does not contain a completely matched PPAR-responsive element (PPRE). There is, however, an atypical PPRE around -185 bp; (i.e., -192, TCCCCC A TGCCCT, -178), and this sequence is similar to a nonconsensus PPRE recently demonstrated in the regulatory region of the lipoprotein lipase (LPL) gene. PPAR-RXR heterodimers can bind to this atypical PPRE-like sequence in human and mouse LPL promoters. These results raise the possibility that the induction of PAI-1 gene transcription by TZDs/insulin may be mediated by the binding of PPAR- with this atypical PPRE sequence. It is also possible that the pioglitazone-activated PPAR- could induce the expression of other transcription factor(s) (e.g., C/EBPs, STATs, ADD/SREBP, etc.) and that these transcription factors may in turn induce PAI-1 gene transcription. This possibility is supported by a number of considerations and preliminary observations. For example, the kinetics of induction of PAI-1 mRNA were slow (i.e., not immediate), with maximal induction observed only after 72 h of treatment (Fig. 1) . This relatively late induction profile is consistent with the possibility that de novo protein synthesis [e.g., for new transcription factor(s)] may be required for the induction of PAI-1 mRNA. The 5' regulatory region of the PAI-1 gene contains responsive elements for C/EBPs, STATs, and ADD/SREBP-1. All of these transcription factors are induced during adipogenesis, although they are not expressed in an adipose tissue-specific manner in vivo. It is possible that transcription factor(s) induced by PPAR- may activate PAI-1 gene transcription through these responsive elements; they may also act cooperatively with PPAR-. Indeed, PPAR- induces C/EBP expression in adipocytes and a strongly cooperative or synergistic effect is observed when both of these factors are expressed in the same cells. Finally, preliminary studies demonstrate that transfection of preadipocytes (e.g., undifferentiated 3T3-L1 cells that do not express endogenous C/EBP or PPAR-) with a PPAR- cDNA does not induce either endogenous PAI-1 mRNA or the luciferase activity from cotransfected reporter constructs. These findings suggest that PPAR-, together with additional but unknown transcription factor(s), is involved in the induction of PAI-1 gene expression by pioglitazone and insulin.

That both pioglitazone and insulin are required to accelerate the rate of adipogenesis and induce PAI-1 and that the increase in lipid accumulation and PAI-1 gene expression occur in parallel suggest that the induction of PAI-1 is part of the differentiation program. This hypothesis is consistent with previous studies showing that fully differentiated adipocytes contain significantly higher levels of PAI-1mRNA and antigen than their undifferentiated counterparts. Whether the increase in PAI-1 is required for, or is a consequence of, cell differentiation remains to be determined.

Our observations not only provide fundamental insights into the PAI-1 gene and its regulation, but also reveal clues about the mechanisms responsible for elevated PAI-1 in fully differentiated adipocytes. Nevertheless, the physiological role of PAI-1, if any, in adipocyte biology remains to be determined. It is possible that PAI-1 protects extracellular matrix components from proteolysis by limiting plasmin generation. Newly synthesized PAI-1 is deposited into the extracellular matrix of many cell types, and this PAI-1 could conceivably function to preserve the integrity of the loose connective tissue component that holds adipocytes together. This function may be more important in obesity, since adipocytes from obese individuals are much larger and tend to be more fragile then those from lean individuals. It also should be noted that PAI-1 binds very specifically to the adhesive glycoprotein vitronectin and regulates the adhesion and migration of a variety of cells on this extracellular matrix component. Vitronectin was recently detected in the adipose tissue, raising the possibility that PAI-1 may regulate preadipocyte migration and cell cluster formation during adipogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0570fje ; to cite this article, use FASEB J. (March 5, 2001) 10.1096/fj.00-0570fje

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