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Molecular mechanisms of tumor necrosis factor--mediated plasminogen activator inhibitor-1 expression in adipocytes

Manjula Pandey, David J. Loskutoff and Fahumiya Samad^*,1

^* La Jolla Institute for Molecular Medicine, Division of Vascular Biology, San Diego, California, USA;
University of California, Department of Medicine, La Jolla, California, USA; and
The Scripps Research Institute, Department of Cell Biology, La Jolla, California, USA

¹Correspondence: The La Jolla Institute for Molecular Medicine, Division of Vascular Biology, 4570 Executive Drive, Suite 100, San Diego, CA 92121, USA. E-mail:fsamad{at}ljimm.org

SPECIFIC AIMS

Increased expression of plasminogen activator inhibitor -1 (PAI-1) in adipose tissues is thought to contribute to both the cardiovascular and metabolic complications associated with obesity. Tumor necrosis factor (TNF-) is chronically elevated in adipose tissues of obese rodents and humans and has been directly implicated to induce PAI-1 in adipocytes. The aim of the present study was to evaluate the potential molecular mechanisms by which TNF- increases PAI-1 in the adipocyte.

PRINCIPAL FINDINGS

1. Chronic exposure of 3T3-L1 adipocytes to TNF- resulted in a more dramatic induction of PAI-1 mRNA than acute exposure; the magnitude of induction of PAI-1 by TNF- was directly correlated with the degree of TNF--mediated metabolic insulin resistance
3T3-L1 adipocytes were treated either for 3 (acute) or 24 (chronic) hours with recombinant mouse TNF- (8 ng/mL). Total RNA was extracted from the adipocyte monolayers and PAI-1 and ß-actin mRNA expression was determined using real-time RT-PCR and PAI-1 antigen in the conditioned medium was determined by an ELISA assay. Acute treatment of 3T3-L1 adipocytes for 3 h with TNF- resulted in a 6-fold increase in PAI-1 mRNA expression while no detectable increase was observed in the level of PAI-1 antigen in the conditioned medium. Chronic treatment of adipocytes with TNF- resulted in a 12-fold increase in PAI-1 mRNA and a 5-fold increase in PAI-1 antigen secreted into the medium.

Both elevated TNF- and PAI-1 have been consistently associated with insulin resistance. In our study, adipocytes treated for 24 h with TNF- showed a higher induction of PAI-1 when compared with adipocytes treated for only 3 h with TNF-. We therefore hypothesized that 24 h treatment with TNF- was sufficient to render these adipocytes metabolically insulin resistant and that this was closely correlated to the higher induction of PAI-1 observed in these cells. Indeed, insulin-mediated glucose uptake assays indicate that 24 h of TNF- treatment causes a significant decrease (P<0.0001) in insulin-mediated glucose transport with a concurrent increase in PAI-1 mRNA and protein expression.

2. In 3T3-L1 adipocytes TNF- induced PAI-1 mRNA by increasing the rate of transcription and not by increasing the stability of PAI-1 mRNA; de novo protein synthesis was not required for this process
To determine mechanisms by which TNF- induces PAI-1 mRNA in adipocytes, we first tested mRNA stability and degradation. 3T3-L1 adipocytes were pretreated with TNF- for 1 h, then incubated with the transcription inhibitor actinomycin D for 0, 1, 2, and 3 h. PAI-1 mRNA expression in cells treated with TNF- and actinomycin D was significantly lower than in cells treated with TNF- alone, suggesting that TNF- does not increase the stability of the PAI-1 message in adipocytes. The half-life of PAI-1 mRNA in untreated cells was 50 min and 49 min in cells treated with TNF-. Similarly, there was no difference in the rate of PAI-1 mRNA degradation between normal adipocytes (i.e., adipocytes treated acutely for 3 h with TNF-) or insulin-resistant adipocytes (i.e., adipocytes treated chronically for 24 h with TNF-). Thus, differences in PAI-1 mRNA stability do not contribute to TNF-mediated increase in PAI-1 expression in either normal or in insulin-resistant adipocytes.

To determine whether TNF--mediated increase in PAI-1 mRNA expression in adipocytes reflected an increase in the rate of transcription of the PAI-1 gene, heteronuclear (hn) PAI-1 RNA was measured from adipocytes treated for either 3 or 24 h with TNF- using intron-specific PAI-1 primer sequences. PAI-1 hnRNA expression was induced in adipocytes that were treated for either 3 or 24 h with TNF-, indicating that TNF- up-regulated PAI-1 gene transcription in these cells. Thus, increased steady-state PAI-1 mRNA levels observed after TNF- treatment of adipocytes are a result of increased transcription of the PAI-1 gene.

We next determined whether de novo protein synthesis was required for the induction of PAI-1 mRNA by TNF- in adipocytes. TNF--induced PAI-1 mRNA expression was not blocked in the presence of cycloheximide (2 µg/mL), indicating that de novo protein synthesis is not required for the induction of PAI-1 by TNF-. Instead, PAI-1 mRNA accumulation was further induced (i.e., superinduced) by cycloheximide; this superinduction appeared to be mediated by cycloheximide-induced stabilization of PAI-1 mRNA in adipocytes.

3. PAI-1 mRNA expression in response to acute short-term exposure to TNF- was mediated primarily via the p44/42 and PKC signaling pathways, while PAI-1 mRNA expression due to chronic TNF- exposure was mediated via these as well as additional, signaling molecules including the p38, PI-3kinase, tyrosine kinase, and NF-B pathways
TNF- activates several signaling molecules including the MAP kinases (i.e., the p44/42, p38, and JNK/SAPK), PI-3 kinase, tyrosine kinases, PKC, and the nuclear factor NF-B. Specific inhibitors of these distinct TNF- signaling pathways were used to begin to dissect the molecular mechanisms by which TNF- induces PAI-1 in adipocytes.

3T3-L1 adipocytes were either left untreated or were preincubated for an hour with each of the specific inhibitors at the indicated concentrations (Fig. 1 ) and PAI-1 mRNA expression was determined either 3 (Fig. 1A ) or 24 h (Fig. 1B ) after subsequent treatment with TNF-. Preincubation of cells with the p44/42 inhibitor PD98059 inhibited PAI-1 mRNA induction both in response to acute (3 h, P<0.02) and chronic (24 h, P<0.02) treatment with TNF-, while preincubation with the p38 inhibitor SB203580, inhibited PAI-1 induction only in response to chronic TNF- treatment (P<0.03). Inhibition of the MAP kinases p44/42 and p38 did not inhibit TNF- stimulated PAI-1 mRNA to basal levels in adipocytes treated chronically with TNF- (insulin-resistant adipocytes), suggesting that in these adipocytes additional signaling cascades may participate in this response.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of inhibitors of TNF- signaling on PAI-1 mRNA expression in adipocytes. 3T3-L1 adipocytes were grown and differentiated in 6-well culture plates as described in Materials and Methods. Adipocytes were pretreated for 1 h with DMSO, or with 50 µM PD98059, 10 µM SB 203580, 100 nM wortmannin, 50 µM Genistein, 20 µM SN50, and 10 µMGF 109203X. The cells were then treated with 8 ng/mL mouse recombinant TNF- for 3 or 24 h and PAI-1 mRNA expression (A, B) was determined using real-time RT-PCR and PAI-1 antigen (C) in the medium by an ELISA assay A–C). n = 6 ± SD.

TNF- also activates PI-3 kinase, various tyrosine kinases, and the protein kinase C (PKC) signaling pathways in adipocytes. Wortmannin, a specific inhibitor of PI-3 kinase, significantly inhibited PAI-1 expression in adipocytes treated chronically (24 h) with TNF- (P<0.017) with no significant effect on PAI-1 levels 3 h after TNF treatment (P<0.26). Similarly, inhibition of tyrosine kinase by genestein reduced PAI-1 mRNA in adiopcytes chronically treated with TNF- to a larger extent (P<0.002) than in adipocytes acutely treated with TNF- (P<0.2). A specific inhibitor to the transcription factor NF-B (SN50) reduced PAI-1 mRNA only in cells treated with TNF- for 24 h (P<0.05) and not in adipocytes acutely treated for 3 h with TNF-.

To investigate the role of PKC in TNF- induced PAI-1 synthesis in adipocytes, we examined the effect of GF 109203X, a highly potent and specific inhibitor of PKC on PAI-1 expression after acute and chronic exposure of adipocytes to TNF-. Pretreating adipocytes with GF109203X almost totally inhibited PAI-1 mRNA induction in response to both acute (P<0.019), and chronic (P<0.001) exposure of adipocytes to TNF-.

Figure 1C shows the effect of various signaling inhibitors on PAI-1 antigen secreted into the medium 24 h after TNF- treatment. Inhibiting the PKC pathway almost completely inhibited the TNF-mediated secretion of PAI-1 antigen (P<0.001). Inhibiting the PI3-kinase pathway inhibited TNF-mediated PAI-1 antigen levels (P<0.013) to a much smaller extent.

4. PAI-1 mRNA induction in adipocytes in response to insulin and transforming growth factor-ß (TGF-ß) was also mediated by the PKC signaling pathway
Hyperinsulinemia and elevated levels of TGF-ß are associated with obesity, and these molecules are potent inducers of PAI-1 in adipose tissues and in the adipocyte. Since the PKC pathway appears to be central to the induction of PAI-1 by TNF- in adipocytes, we questioned whether this pathway was involved in the regulation of PAI-1 by insulin and/or TGF-ß. Pretreating adipocytes with the PKC inhibitor GF109203X restored both insulin and TGF-ß-mediated PAI-1 mRNA to almost basal levels.

CONCLUSIONS AND SIGNIFICANCE

Although increased TNF- associated with obesity may be an important contributor to elevated plasma and adipose tissue expression of the procoagulant gene PAI-1, little is known regarding the molecular mechanisms that mediate the regulation of PAI-1 in the adipocyte. In the present study we demonstrated that both acute (3 h) and chronic (24 h) exposure of 3T3-L1 adipocytes to TNF- induces PAI-1 mRNA by increasing the rate of transcription of the PAI-1 gene, and that de novo protein synthesis is not required for this process. Moreover, the more dramatic increase in PAI-1 observed after chronic exposure of adipocytes to TNF- was intimately correlated with metabolic insulin resistance.

We demonstrated that TNF- regulates PAI-1mRNA expression through different pathways in metabolically insulin-sensitive (adipocytes treated for 3 h with TNF-) and in metabolically insulin-resistant (adipocytes treated for 24 h with TNF-) cells (Fig. 1) . Adipocytes treated for 3 h with TNF-, appear to require the activation of p44/42 and PKC for PAI-1mRNA induction. The more dramatic induction of PAI-1 observed in adipocytes treated for 24 h with TNF- was more complex. For example, besides the p44/42 MAPK and PKC pathways, these cells appeared to utilize the p38 MAP kinase pathway, tyrosine kinases and a signaling cascade that required the activation of PI3-kinase and the transcription factor NF-B. Our results indicate that the PKC pathway may be central in the signaling cascade that leads to the induction of PAI-1 by TNF- in adipocytes.

In addition to TNF-, levels of insulin and TGF-ß are increased in obesity, and these molecules have been shown to induce PAI-1 biosynthesis in adipocytes. Our studies demonstrate that the PKC pathway also is used by adipocytes in the insulin and TGF-ß-mediated expression of PAI-1, suggesting that the PKC pathway may be a common signaling pathway involved in the induction of PAI-1 not only by TNF- but also by insulin and TGF-ß, all of which are elevated in obesity and induce PAI-1 in the adipocyte.

These studies provide new information regarding the molecular mechanisms in the adipocyte that leads to the induction of PAI-1, a gene that is consistently elevated in obesity; it is not only an established risk factor for cardiovascular disease but may contribute to the metabolic risk associated with obesity. Our results further suggest that PKC inhibitors may provide a means for down-regulating PAI-1 expression in adipocytes.

View larger version (25K): [in this window] [in a new window]   Figure 2. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3459fje;

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This Article Full Text (PDF) All Versions of this Article: 19/10/1317 04-3459fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pandey, M. Articles by Samad, F. Search for Related Content PubMed PubMed Citation Articles by Pandey, M. Articles by Samad, F.