Insulin activates CCAAT/enhancer binding proteins and proinflammatory gene expression through the phosphatidylinositol 3-kinase pathway in vascular smooth muscle cells

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-kappaB 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-beta and C/EBP-delta but not C/EBP-alpha 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-beta and C/EBP-delta in VSMCs. These results clearly indicate that the activation of PI3K induced proinflammatory gene expression through activating C/EBP-beta and C/EBP-delta but not NF-kappaB, which may explain the proinflammatory effect of insulin in the insulin-resistant state.     

Effects of hyperthermia on p53 protein expression and activity

Although p53 responses after DNA damage have been investigated extensively, p53 responses after heat shock, which exerts cytotoxic action by mechanisms other than direct induction of DNA damage, are less well characterized. We investigated, therefore, the effect of hyperthermic exposures on the levels and DNA-binding activity of p53. Experiments were carried out with U2OS and ML-1 cells, known to express wild-type p53 protein. Although heating at 41 degrees C for up to 6 h had only a small effect on p53 levels or DNA binding activity, exposure to temperatures between 42.5 and 45.5 degrees C caused an immediate decrease in protein levels that was associated with a reduction in DNA binding activity. This observation is compatible with a high lability of p53 to heat shock, or heat sensitivity of the pathway regulating p53 levels in non-stressed cells. When cells were heated to 42.5 degrees C and returned to normal temperatures, a strong p53 response associated with an increase in protein levels and DNA binding activity was observed, suggesting the production of p53-inducing cellular damage. At higher temperatures, however, this response was compromised in an exposure-time-dependent manner. The increase in DNA binding activity was more heat sensitive than the increase in p53 levels and was inhibited at lower temperatures and shorter exposure times. Thus, the pathway of p53 activation is itself heat sensitive and compromised at high levels of exposure. Compared to p53 activation after exposure to ionizing radiation, heat-induced activation is rapid and short lived. When cells were exposed to combined heat and radiation, the response observed approximated that of cells exposed to heat alone. Wortmannin at 10 microM inhibited p53 activation for up to 2 h after heat shock suggesting the involvement of wortmannin-sensitive kinases, such as DNA-PK and ATM. Heat shock causes phosphorylation of p53 at Serine-15, but there is no correlation between phosphorylation at this site and activation of the protein. The results in aggregate indicate p53 activation in the absence of DNA damage by a heat-sensitive mechanism operating with faster kinetics than radiation-induced p53 activation. The former response may induce pathways preventing other stimuli from activating p53, as heat-induced activation of p53 is dominant over activation of p53 by DNA damage in combined-treatment experiments. These observations suggest means for abrogating p53 induction after DNA damage with the purpose of potentiating response and enhancing cell killing.     

Differential regulation of macrophage CCAAT-enhancer binding protein isoforms by lipopolysaccharide and cytokines

The regulation of the C/EBP family in macrophages by LPS and cytokines is of potentially crucial importance in several pathophysiological conditions. The action of LPS and three cytokines on the expression of C/EBP mRNA, protein and functional DNA binding activity in the murine J774.2 cell line was therefore studied. Exposure of the cells to LPS, IL-1, IFN-gamma and TNF-alpha produced a reduction of C/EBP alpha mRNA levels and a corresponding increase in the expression of C/EBP beta and C/EBP delta. EMSA showed time-dependent changes in the DNA binding activity of individual C/EBP isoforms and demonstrated the participation of heterodimers between the different members in DNA-protein interactions. Additionally, mediator-specific changes in the kinetics and magnitude of C/EBP mRNA expression pattern and profile of DNA-protein interactions were observed. These studies provide novel insights into the potential mechanisms that may be responsible for the mediator-specific regulation of macrophage gene expression through the C/EBP family.     

Arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1 beta-stimulated Caco-2 cells independent of the heat shock response

Recent studies suggest that sodium arsenite downregulates NF-kappaB activity by inhibiting phosphorylation and subsequent degradation of IkappaBalpha. Many effects of sodium arsenite are secondary to induction of heat shock proteins. The role of the heat shock response in arsenite-induced inhibition of NF-kappaB, however, is not known. We examined the involvement of the heat shock response in arsenite-induced inhibition of NF-kappaB activity in IL-1beta-stimulated Caco-2 cells, a human colorectal adenocarcinoma cell line with enterocytic properties. Treatment of the cells with IL-1beta resulted in increased IkappaB kinase activity, reduced levels of IkappaBalpha and increased NF-kappaB DNA binding activity. Sodium arsenite blocked all of these responses to IL-1beta without inducing changes in heat shock factor activity or heat shock protein levels. Results from additional experiments showed that the protective effect of sodium arsenite on IkappaBalpha was not influenced by the oxygen radical scavenger catalase or by inhibitors of the MAP-kinase signaling pathway. The present results suggest that sodium arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1beta-stimulated Caco-2 cells independent of the heat shock response. In addition, stabilization of IkappaBalpha by sodium arsenite does not require oxygen radical formation or activation of the MAP kinase signaling pathway.     

Interleukin-6 induces thermotolerance in cultured Caco-2 cells independent of the heat shock response

In recent studies, induction of the heat shock response increased IL-6 production in gut mucosa in vivo and in cultured Caco-2 cells in vitro. The heat shock response is associated with increased survival of cells exposed to otherwise lethal hyperthermia, so called thermotolerance, but the role of IL-6 in the induction of thermotolerance is not known. We tested the hypothesis that treatment of cultured Caco-2 cells with IL-6 results in the development of thermotolerance. Cells were treated with human recombinant IL-6 for 1h followed by 3 h recovery in cytokine-free medium whereafter cells were exposed to heat stress (48 degrees C for 2 h). In untreated cells, the heat stress resulted in an approximately 80% cell death. In cells treated with IL-6, cell viability after heat stress was significantly improved and was doubled at an IL-6 concentration of 20 ng/ml. Treatment of the cells with other cytokines (IL-4, IL-10, IL-1beta, or TNFalpha) did not induce thermotolerance, suggesting that the effect of IL-6 may be specific for this cytokine. The induction of thermotolerance by IL-6 was blocked by an IL-6 receptor antibody, suggesting that the development of thermotolerance was receptor-mediated. Treatment of cells with IL-6 did not induce an heat shock response as suggested by unaltered heat shock protein 70 and 90 levels and unaffected heat shock factor DNA binding activity. In addition, the IL-6-induced thermotolerance was not inhibited by quercetin. The present study provides the first evidence of IL-6-induced thermotolerance and suggests that this effect of IL-6 is independent of the heat shock response.     

Proteasome inhibition drastically but reversibly impairs murine lymphocyte development

The proteasome inhibitor bortezomib, which induces cell death in various cancer cell lines including lymphatic neoplasias, has recently been approved for the treatment of relapsed multiple myeloma. Important mechanisms of proteasome inhibitor-mediated tumor cell death are the inhibition of NF-kappaB activation and induction of the terminal unfolded protein response (UPR). However, little is known about effects of bortezomib on developing and mature lymphocytes. Therefore, Balb/C mice were injected with bortezomib and lymphocyte subsets were analyzed. This treatment resulted in dramatically decreased numbers of T and B lymphocyte precursors, while mature lymphocytes were only partially affected. Thymocytes were almost depleted 3 days after a single bortezomib injection, pro-B and pre-B cells already after 2 days. Thymocytes and B cell precursors recovered within 2 weeks. The decreased numbers of developing lymphocytes were due to apoptotic cell death accompanied by strongly increased caspase 3/7 activity. Within 8 h after bortezomib injection, there was a strong induction of heat shock protein 70 and C/EBP homologous protein in bone marrow B cells, indicating endoplasmic reticulum stress and activation of the terminal UPR, respectively. Hence, induction of apoptosis by proteasome inhibition can dramatically affect lymphocyte development, a fact which has important implications for the clinical use of bortezomib, especially in situations with ongoing lymphopoiesis.