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Insulin Activates CCAAT/Enhancer Binding Proteins and Proinflammatory Gene Expression through the Phosphatidylinositol 3-Kinase Pathway in Vascular Smooth Muscle Cells*

Open AccessPublished:July 26, 2002DOI:https://doi.org/10.1074/jbc.M206266200
      Phosphatidylinositol 3-kinase (PI3K) is a key molecule mediating signals of insulin in vascular smooth muscle cells (VSMCs). To examine the effect of chronic activation of PI3K on the gene expression of VSMCs, membrane-targeted p110CAAX, a catalytic subunit of PI3K, was overexpressed in rat VSMCs by adenovirus-mediated gene transfer. Similar to insulin's effects, cells overexpressing p110CAAX exhibited a 10- to 15-fold increase in monocyte chemoattractant protein-1 (MCP-1) mRNA expression as compared with the control cells. Electrophoretic mobility shift assay analysis showed that the overexpression of p110CAAX activated neither the NF-κB binding nor the activator protein (AP-1) binding activities. We found that two CCAAT/enhancer binding protein (C/EBP) binding sites located between 2.6 and 3.6 kb upstream of the MCP-1 gene were responsible for the induction by p110CAAX. The overexpression of C/EBP-β and C/EBP-δ but not C/EBP-α caused 6- to 8-fold induction of MCP-1 promoter activity. Consistently, the overexpression of p110CAAX as well as insulin induced mRNA expression and nuclear expression of C/EBP-β and C/EBP-δ in VSMCs. These results clearly indicate that the activation of PI3K induced proinflammatory gene expression through activating C/EBP-β and C/EBP-δ but not NF-κB, which may explain the proinflammatory effect of insulin in the insulin-resistant state.
      MCP-1
      monocyte chemoattractant protein-1
      AP-1
      activator protein
      C/EBP
      CCAAT/enhancer binding protein
      DMEM
      Dulbecco's modified Eagle's medium
      DTT
      dithiothreitol
      EMSA
      electrophoretic mobility shift assay
      ERK
      extracellular signal-regulated kinase
      FCS
      fetal calf serum
      GSK3
      glycogen synthase kinase 3
      IκB
      inhibitor of kappa B
      JNK
      c-Jun N-terminal kinase
      MAPK
      mitogen-activated protein kinase
      PAI-1
      plasminogen activator inhibitor 1
      PI3K
      phosphatidylinositol 3-kinase
      PMA
      phorbol myristate acetate
      TNF-α
      tumor necrosis factor-α
      VSMCs
      vascular smooth muscle cells
      m.o.i.
      multiplicity of infection
      Ad
      adenovirus
      PBS
      phosphate-buffered saline
      IL
      interleukin
      Several lines of clinical evidence have clearly shown that hyperinsulinemia is an independent risk factor for the development of atherosclerosis (
      • Fontbonne A.
      • Tchobroutsky G.
      • Eschwege E.
      • Richards J.L.
      • Claude J.R.
      • Rosselin G.E.
      ,
      • Despres J.P.
      • Lamarche B.
      • Mauriege P.
      • Cantin B.
      • Dagenais G.R.
      • Moorjani S.
      • Lupien P.J.
      ,
      • Reaven G.M.
      ). It has been proposed that insulin may have an important role in the pathogenesis of atherosclerosis (
      • Stout R.W.
      ), although the data from in vitro experiments show that insulin acts as both an anti-atherogenic and atherogenic hormone (
      • Scherrer U.
      • Randin D.
      • Vollenweider P.
      • Vollenweider L.
      • Nicod P.
      ,
      • Stout R.W.
      • Bierman E.L.
      • Ross R.
      ). Previously, we observed that the hyperinsulinemic state caused by high fructose diet induced the production of superoxide anion and decreased nitric oxide production, resulting in the activation of the transcription factor NF-κB and expression of its target genes in the cardiovascular tissues of rats (
      • Shinozaki K.
      • Nishio Y.
      • Okamura T.
      • Yoshida Y.
      • Maegawa H.
      • Kojima H.
      • Masada M.
      • Toda N.
      • Kikkawa R.
      • Kashiwagi A.
      ). The activation of NF-κB is reported to stimulate the expression of inflammatory genes found in atheromatous lesions such as monocyte chemoattractant protein-1 (MCP-1)1 and vascular cell adhesion molecule-1 (
      • Landry D.B.
      • Couper L.L.
      • Bryant S.R.
      • Lindner V.
      ,
      • Bustos C.
      • Hernandez-Presa M.A.
      • Ortego M.
      • Tunon J.
      • Ortega L.
      • Perez F.
      • Diaz C.
      • Hernandez G.
      • Egido J.
      ). Therefore, the activation of NF-κB in the vascular tissues in the hyperinsulinemic state may affect the pathogenesis of atherosclerotic lesions. Although the increased production of reactive oxygen species is one possible mechanism, how the hyperinsulinemic state activates the NF-κB in cardiovascular tissues is still unknown. In addition, recent clinical studies have shown that levels of C-reactive protein as well as fibrinogen, in vivo signs of inflammation, are elevated in patients with hyperinsulinemia (
      • Wu T.
      • Dorn J.P.
      • Donahue R.P.
      • Sempos C.T.
      • Trevisan M.
      ), suggesting that inflammatory responses are also induced in the liver of hyperinsulinemic patients.
      Insulin activates both the Ras/mitogen-activated protein kinase (MAPK) pathway and phosphatidylinositol 3-kinase (PI3K) pathway in many insulin-sensitive tissues or cells (
      • White M.F.
      • Kahn C.R.
      ). Previously, we showed that, in vascular smooth muscle cells (VSMCs), insulin at 1–10 nm concentration preferentially activates the PI3K pathway rather than the MAPK pathway resulting in stimulated amino acid uptake but not thymidine incorporation into DNA (
      • Takagi Y.
      • Kashiwagi A.
      • Tanaka Y.
      • Maegawa H.
      • Shigeta Y.
      ,
      • Obata T.
      • Kashiwagi A.
      • Maegawa H.
      • Nishio Y.
      • Ugi S.
      • Hidaka H.
      • Kikkawa R.
      ). These results indicate that the activation of PI3K does not directly stimulate cell proliferation at these insulin concentrations. Recently, it was reported that the activation of Akt, a serine/threonine kinase, downstream of the PI3K pathway stimulates the transcription factor NF-κB through the activation of inhibitor of kappa B (IκB) kinase in U293 cells (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ,
      • Romashkova J.A.
      • Makarov S.S.
      ). Thus, insulin may stimulate NF-κB through activating PI3K and Akt and may play a role in the induction of proinflammatory gene expression and promotion of the inflammatory process in vascular tissues. Among the inflammatory processes found in an atheromatous lesion, the initial event is recruitment of monocytes to the lesion (
      • Ross R.
      ). Monocyte chemoattractant protein-1 (MCP-1) plays a key role in the recruitment of monocytes (
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ). In fact, strong expression of MCP-1 protein is observed in the human atherosclerotic lesion (
      • Takeya M.
      • Yoshimura T.
      • Leonard E.J.
      • Takahashi K.
      ) and the selective knock-out of CCR2, the receptor of MCP-1, prevents the initiation of the atherosclerotic process in vivo (
      • Boring L.
      • Gosling J.
      • Cleary M.
      • Charo I.F.
      ). Similar to other proinflammatory genes, the expression of MCP-1 is also regulated transcriptionally under the control of NF-κB in human and rat cells (
      • Ueda A.
      • Ishigatsubo Y.
      • Okubo T.
      • Yoshimura T.
      ,
      • Wang Y.
      • Rangan G.K.
      • Goodwin B.
      • Tay Y.C.
      • Harris D.C.
      ).
      In the present study, to elucidate the role of the activation of PI3K in the inflammatory gene expression elicited through possible activation of NF-κB in VSMCs, we continuously increased the PI3K activity in cultured VSMCs using the recombinant adenovirus expressing membrane-targeted PI3K (p110CAAX). As previously reported (
      • Egawa K.
      • Sharma P.M.
      • Nakashima N.
      • Huang Y.
      • Huver E.
      • Boss G.R.
      • Olefsky J.M.
      ), the specific activation of PI3K mimics insulin activation. This technique allows us to study the direct effect of the activation of PI3K on the gene expression in VSMCs and the mechanisms by which it regulates proinflammatory gene expression in VSMCs. Unexpectedly, our study showed that the continuous activation of PI3K by the overexpression of p110CAAX increased the MCP-1 gene expression without activating NF-κB in VSMCs. Here, we report the molecular mechanisms regulating MCP-1 gene expression in VSMCs by insulin or a direct activation of PI3K through activating CCAAT/enhancer binding proteins (C/EBPs) but not NF-κB.

      DISCUSSION

      In the present study, we found that the overexpression of p110CAAX in VSMCs activated the downstream of the PI3K signal pathway, such as Akt and GSK3, but not MAPKs. The specific and chronic activation of PI3K by the overexpression of p110CAAX as well as the stimulation with a near physiological dose of insulin induced gene expression of a proinflammatory cytokine, MCP-1, in VSMCs through increasing the nuclear expression of C/EBP-β and C/EBP-δ. These observations may provide a new concept for the pathophysiological role of PI3K activation for the enhancement of proinflammatory gene expression observed in the development of atheromatous lesions.
      Although atherosclerosis has been considered to be the accumulation of lipids within the arterial wall, it has recently been emphasized that a series of inflammatory processes is involved in the development of the atherosclerotic lesion and the onset of acute coronary syndromes (
      • Libby P.
      ). The initial step of the process is recruitment of monocytes to the lesion. In this process, the cytokine MCP-1 is thought to play a major role. Actually, in human atheromatous lesions, strong expression of MCP-1 is observed (
      • Takeya M.
      • Yoshimura T.
      • Leonard E.J.
      • Takahashi K.
      ). The induction of MCP-1 is one of the inflammatory responses regulated by many factors, including proinflammatory cytokines, hormones, lipopolysaccharide, and modified lipids (
      • Chen X.L.
      • Tummala P.E.
      • Olbrych M.T.
      • Alexander R.W.
      • Medford R.M.
      ). The present study reveals that insulin is also a factor evoking the initial step of the inflammatory response in VSMCs. Recently, several lines of clinical evidence have supported the role of the hyperinsulinemic state in the development of atherosclerosis and the association of the insulin-resistant state with increased level of a marker of inflammation (
      • Festa A.
      • D'Agostino Jr., R.
      • Howard G.
      • Mykkanen L.
      • Tracy R.P.
      • Haffner S.M.
      ). Our finding may suggest one of the molecular mechanisms by which hyperinsulinemia induces inflammatory process in the artery.
      Most previous reports have shown that the induction of MCP-1 is regulated by transcription factor NF-κB (
      • Hernandez-Presa M.
      • Bustos C.
      • Ortego M.
      • Tunon J.
      • Renedo G.
      • Ruiz-Ortega M.
      • Egido J.
      ). Wang et al.(
      • Wang Y.
      • Rangan G.K.
      • Goodwin B.
      • Tay Y.C.
      • Harris D.C.
      ) have shown that the activation of NF-κB plays a key role in the induction of rat MCP-1 gene expression through its binding to the NF-κB response elements located at −2280 bp and −2261 bp from the starting point of MCP-1 transcription. Interestingly, we found that insulin or activation of PI3K induced the MCP-1 gene expression in VSMCs without the activation of NF-κB. We confirmed this finding in three experiments. First, in contrast to the effect of TNF-α stimulation, the activation of PI3K by overexpression of p110CAAX did not enhance the binding activity of NF-κB based on the assessment of EMSA and the degradation of IκB-α. Furthermore, the activation of PI3K did not increase the luciferase activity of pGL3 plasmid carrying the 2.6-kb upstream region of MCP-1 gene, which contained NF-κB response elements, and finally, the specific mutation of NF-κB response elements at −2280 bp and −2261 bp did not affect the p110CAAX-induced luciferase activity of pGL3 plasmid carrying the 3.6-kb upstream region of MCP-1 gene. These observations clearly indicate that the activation of NF-κB is not involved in the induction of promoter activity of MCP-1 by the overexpression of p110CAAX. We observed that the mutation of NF-κB response elements decreased the promoter activities of MCP-1 in both cells with overexpression of p110CAAX and control plasmid, indicating that the NF-κB response elements have a role in the basal promoter activity of the MCP-1 in VSMCs. Like NF-κB, AP-1 and sequence-specific transcription factor (Sp1) were not involved in the induction of MCP-1 gene expression by the activation of PI3K as assessed by EMSA and luciferase assay (data not shown). We found that two C/EBP binding elements located at −2579 to −2591 bp and −3107 to −3118 bp from the starting point of MCP-1 transcription had a role in the gene expression of MCP-1 induced by the overexpression of p110CAAX. The involvement of the C/EBP binding elements was confirmed in our experiments showing that specific mutation of two C/EBP binding elements abolishes the enhancement of promoter activity of MCP-1 by the overexpression of p110CAAX. In addition, the result that the overexpression of C/EBP-β and C/EBP-δ but not C/EBP-α increased the MCP-1 promoter activity also supports the involvement of C/EBP-β and C/EBP-δ in the p110CAAX-induced MCP-1 promoter activity. A similar involvement of C/EBP in the induction of IL-6 and MCP-1 gene expression by lipopolysaccharide was reported in a lymphoblastic cell line (
      • Hu H.M.
      • Tian Q.
      • Baer M.
      • Spooner C.J.
      • Williams S.C.
      • Johnson P.F.
      • Schwartz R.C.
      ).
      We observed that the overexpression of p110CAAX increased the nuclear expression of C/EBP-β and C/EBP-δ using EMSA, Western blot analysis, and immunostaining of the cells with antibodies against C/EBPs. These results clearly indicate that the overexpression of p110CAAX facilitates the translocation of C/EBP-β from the perinuclear region to the nuclei while the mRNA contents of C/EBP-β and C/EBP-δ were elevated by possible de novosynthesis. In addition, we found a marked increase in the mRNA expression of C/EBP-δ but only a modest increase in the mRNA expression of C/EBP-β in the cells overexpressed with p110CAAX. In fact, the enhancement of the binding activity of C/EBP-β by a phosphorylation or dephosphorylation mechanism was reported (
      • Wegner M.
      • Cao Z.
      • Rosenfeld M.G.
      ,
      • Trautwein C.
      • Caelles C.
      • van der Geer P.
      • Hunter T.
      • Karin M.
      • Chojkier M.
      ). Hanlon et al. (
      • Hanlon M.
      • Sturgill T.W.
      • Sealy L.
      ) reported that phosphorylation of C/EBP-β by ERK2 increased the interaction with serum response factor resulting in elevated c-fos promoter activity, although downstream effectors of PI3K such as Rac, Cdc42, and Akt did not have a role in regulating the interaction of C/EBP-β and serum response factor (
      • Hanlon M.
      • Sturgill T.W.
      • Sealy L.
      ). On the other hand, it is reported that growth hormone decreases the phosphorylation of C/EBP-β and enhances its activity through increasing the phosphorylation of Akt and GSK3 in 3T3 cells (
      • Piwien-Pilipuk G.
      • Van Mater D.
      • Ross S.E.
      • MacDougald O.A.
      • Schwartz J.
      ). Although we did not measure the amount of phosphorylation of C/EBP-β in this study, considering that the overexpression of p110CAAX increased the phosphorylation of Akt and GSK3 while phosphorylation of MAPKs was not increased, the activation of PI3K by overexpression of p110CAAX may enhance the activity of C/EBP-β by modulating the Akt and GSK3 activities in VSMCs. This increase in the C/EBP-β activity might possibly induce the C/EBP-δ expression (
      • Poli V.
      ).
      We found that insulin at 1–10 nm also increased the mRNA expression of MCP-1 as well as C/EBP-β and C/EBP-δ expression in VSMCs. These effects of insulin were completely blocked in the presence of wortmannin, suggesting that the activation of PI3K played a key role in the gene regulation of MCP-1 and C/EBP-β and C/EBP-δ by insulin. In addition, we found that the phosphorylation of Akt by insulin was not blocked by insulin-like growth factor-1 receptor antibody (data not shown), indicating that insulin activated the PI3K through the insulin receptor-insulin receptor substrates pathway in VSMCs. Because our findings, that insulin and activation of PI3K both regulate proinflammatory gene expressions, were obtained in cultured VSMCs, they do not necessarily indicate that PI3K and C/EBPs are activated in the hyperinsulinemic state in vivo. Jianget al. (
      • Jiang Z.Y.
      • Lin Y.W.
      • Clemont A.
      • Feener E.P.
      • Hein K.D.
      • Igarashi M.
      • Yamauchi T.
      • White M.F.
      • King G.L.
      ) have shown that selective resistance of the PI3K pathway to insulin resulted in relative activation of MAPKs in the vascular tissues of genetic hyperinsulinemic Zucker fatty rats. We found, however, an elevation of phosphorylation of Akt in the vascular tissues from the Zucker fatty rats in the fasting state (data not shown). These results suggest that PI3K in the vascular tissues may be activated continuously by the chronic hyperinsulinemia found in the insulin-resistant state, although the acute response to insulin is blunted. Thus, the hyperinsulinemia in the insulin-resistant state may have a role in the development of atherosclerosis through activating the PI3K in VSMCs, although the role of insulin in atherogenesis has been a matter of debate for years (
      • Stout R.W.
      ,
      • Stout R.W.
      • Bierman E.L.
      • Ross R.
      ,
      • Kuboki K.
      • Jiang Z.Y.
      • Takahara N., Ha, S.W.
      • Igarashi M.
      • Yamauchi T.
      • Feener E.P.
      • Herbert T.P.
      • Rhodes C.J.
      • King G.L.
      ). We should, however, note that our present investigation was mainly done under culture condition where VSMCs were present alone. In the vascular tissues, VSMCs are always accompanied by endothelial cells. Because PI3K activates the endothelial nitric-oxide synthase (
      • Montagnani M.
      • Chen H.
      • Barr V.A.
      • Quon M.J.
      ), insulin activates PI3K in VSMCs and increases NO production in endothelial cells at the same time in vascular tissue. It is reported that NO has anti-atherogenic and anti-inflammatory actions (
      • Garg U.C.
      • Hassid A.
      ). In fact, inhibition of endothelial nitric-oxide synthase induced MCP-1 production and inflammatory response in cardiovascular tissues (
      • Tomita H.
      • Egashira K.
      • Kubo-Inoue M.
      • Usui M.
      • Koyanagi M.
      • Shimokawa H.
      • Takeya M.
      • Yoshimura T.
      • Takeshita A.
      ). Interestingly, NO production was decreased in the insulin-resistant state in humans (
      • Steinberg H.O.
      • Chaker H.
      • Leaming R.
      • Johnson A.
      • Brechtel G.
      • Baron A.D.
      ) and rat models (
      • Shinozaki K.
      • Kashiwagi A.
      • Nishio Y.
      • Okamura T.
      • Yoshida Y.
      • Masada M.
      • Toda N.
      • Kikkawa R.
      ) even in the presence of hyperinsulinemia. Thus, to clarify the role of PI3K activation in proinflammatory gene expression underin vivo condition, one should always evaluate NO production in endothelial cells, because it might affect proinflammatory gene expression profoundly in VSMCs.
      In conclusion, the chronic activation of PI3K with the use of adenovirus-mediated overexpression of p110CAAX and stimulation with 1–10 nm insulin in VSMCs induced nuclear expression of C/EBP-β and C/EBP-δ, resulting in induction of MCP-1 gene expression. The involvement of C/EBP-β and C/EBP-δ in the gene expression of various inflammatory cytokines other than MCP-1, such as IL-1β, IL-6, IL-8, IL-12, and TNF-α (
      • Poli V.
      ,
      • Merola M.
      • Blanchard B.
      • Tovey M.G.
      ), has been reported. Thus, our findings might indicate that activation of insulin signals in VSMCs promotes the inflammatory reaction leading to the development of atherosclerosis, although we need further investigation in vivo to explore the role of hyperinsulinemia in the induction of inflammatory response in various tissues, including vascular tissues.

      Acknowledgments

      We thank Dr. Jerrold M. Olefsky for providing Ad5-p110CAAX and Dr. Steven L. McKnight for giving expression vectors for C/EBP-α, C/EBP-β, and C/EBP-δ.

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