Defects within the insulin signaling pathways may lead to insulin resistance in peripheral tissues. The prevalence of obesity generally increases with age and is associated with insulin resistance characterized by an elevation in hepatic glucose production, impaired glucose tolerance and defects in the ability of the insulin receptor to transduce insulin signals. Cross-sectional studies have associated obesity and other components of the so-called metabolic syndrome with low-grade inflammation. A strong positive correlation has been reported between adipose tissue levels of adipocytokines (e.g., interleukin (IL)-6, tumor necrosis factor (TNF)) and the increased secretion of acute phase proteins by the liver of obese subjects. Hence, better understanding of the complex interplay between proinflammatory cytokines and insulin signal transduction could lead to the development of effective strategies to reduce or delay the progression of complications associated with insulin resistance states, such as Type 2 diabetes. ? ? We have completed a study whereby the effect of the thiol antioxidant pyrrolidine dithiocarbamate (PDTC) on IL-6 signaling has been elucidated. As noted earlier, IL-6 is a proinflammatory cytokine that has been implicated in the expression of acute phase plasma proteins (APPs) and hepatic insulin resistance through activation of the JAK/STAT3 pathway. Although previous studies have demonstrated that PDTC exerts protection against inflammatory responses, its role in the regulation of IL-6 receptor signaling remains unclear. Here we show that treatment of cultured HepG2 hepatoma cells with PDTC inhibits IL-6-stimulated tyrosine phosphorylation and subsequent nuclear translocation of STAT3 in a dose- and time-dependent fashion. No inhibition of JAK-1 activity was observed. To provide insight into PDTC signaling, we constructed a conditionally active STAT3 by fusing it with the ligand-binding domain of the estrogen receptor (STAT3-ER). In the presence of 4-hydroxytamoxifen, STAT3-ER was translocated in the nucleus of HepG2 cells in a phosphorylation-independent manner, and treatment with PDTC mitigated the response. While STAT3 coprecipitated with the chaperone heat-shock protein 90 (Hsp90) in control cells, coprecipitation of the two proteins was greatly reduced after PDTC treatment or following exposure to geldanamycin, a selective Hsp90 inhibitor. As a result, there was a decrease in IL-6-induced association of STAT3 with the transcriptional coactivators, FOXO1a and C/EBPbeta, together with significant reduction in the expression of SOCS-3 protein and that of two major APPs (alpha2-macroglobulin and haptoglobin). Importantly, treatment of HepG2 cells and a primary culture of rat hepatocytes with PDTC restored insulin responsiveness that was abrogated by IL-6. These studies are consistent with the ability of PDTC to down-regulate IL-6-induced STAT3 activation by altering the stability of STAT3-Hsp90 complex. It remains unknown whether PDTC can inhibit the development of insulin resistance in obesity and in aging, two chronic in vivo models of low grade inflammation. Therefore, we are planning an in vivo study to explore the role of PDTC in the liver in animal models of obesity and insulin resistance. High fat diet and diabetes increase oxidative injury including a proinflammatory response and lipid peroxidation leading to the development of non-alcoholic steatohepatitis (NASH). We will assess whether diabetes-prone mice maintained on high fatty diet and subjected to PDTC treatment will develop a proinflammatory response (e.g., acute-phase protein secretion) and ultimately lipid peroxidation in the liver will be evaluated. Similarly, it will be important to determine if this lipid dysregulation is STAT3-driven. A number of studies have indicated that PDTC is well tolerated in vivo. It is our hope that PDTC will be a useful tool and provide therapeutic/functional benefits by delaying onset and/or reducing duration of hepatic insulin resistance.? ? In a second project, we investigated the role of the actin-binding protein filamin A (FLNa) in the modulation of proinflammatory cytokine signaling. FLNa interacts not only with actin, but also with plasma membrane proteins, enzymes and adapter molecules. This scaffolding property of FLNa allows functional interaction between intracellular signaling proteins with the actin cytoskeleton and with cell membrane receptors. In our previous study, FLNa was found to associate with the insulin receptor and attenuate insulin-induced activation of the Ras/MAPK pathway. Using the human melanoma cell line M2, which does not express FLNa, and its subclone M2A7, transfected with human FLNa cDNA, we observed that TNFalpha activation of NF-kappaB was greatly reduced in filamin-lacking cells, while insulin stimulation of the Ras/ERK cascade was significantly higher in these M2 cells. The coordinate induction of insulin signaling and repression of TNFalpha-mediated responses provides a molecular rationale for the antagonism between insulin and proinflammatory cytokines, and highlights the need to further investigate the role of FLNa as regulator of cytokine and insulin signaling. ? ? Before initiating the study of the role of FLNa in cytokine signaling, we examined the effect of FLNa on the regulation of signaling pathways that control the expression of matrix metalloproteinases (MMPs), which have been linked with tumor invasion and metastasis. The lack of FLNa in human M2 melanoma cells was associated with constitutive expression and secretion of active MMP-9 in the absence of MMP-2 upregulation. M2 cells displayed greater invasiveness and stronger MMP-9 production than their M2A7 counterparts where FLNa had been stably reintroduced. Using an MMP-9 promoter construct (pMMP-9-Luc), in vitro kinase assays, and genetic and pharmacological approaches, we demonstrate that FLNa mediated transcriptional down-regulation of pMMP-9-Luc by suppressing the constitutive hyperactivity of the Ras/MAPK extracellular signal-regulated kinase (ERK) cascade. Experimental evidence indicated that this phenomenon is due to the destabilization of Ras-GRF1, a guanine nucleotide exchange factor that activates H-Ras by facilitating the release of GDP. Ectopic expression of Ras-GRF1 was accompanied by ERK activation and produced transcriptional upregulation of pMMP-9-Luc, whereas a catalytically inactive dominant negative Ras-GRF1, which prevented ERK activation, suppressed pMMP-9-Luc expression, indicating the functional requirement for Ras-GRF1 and active ERK in this process. Our results indicate that expression of FLNa regulates constitutive activation of the Ras/ERK pathway and melanoma cell invasion through a MMP-9 dependent mechanism. This work has been recently submitted for publication. On-going work involves the study of the role of FLNa-mediated downregulation of Ras-GRF1 in cytokine and insulin signaling in HepG2 hepatoma cells.
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| Zhu, Tie-Nian; He, Hua-Jun; Kole, Sutapa et al. (2007) Filamin A-mediated down-regulation of the exchange factor Ras-GRF1 correlates with decreased matrix metalloproteinase-9 expression in human melanoma cells. J Biol Chem 282:14816-26 |
| He, Hua-Jun; Zhu, Tie-Nian; Xie, Yi et al. (2006) Pyrrolidine dithiocarbamate inhibits interleukin-6 signaling through impaired STAT3 activation and association with transcriptional coactivators in hepatocytes. J Biol Chem 281:31369-79 |
