The febrile response is a complex physiological reaction to disease, including a cytokine-mediated increase in body temperature and the activation of inflammatory systems. Fever has beneficial roles in terms of disease prognosis, partly by suppressing the expression of inflammatory cytokines. However, the molecular mechanisms underlining the fever-mediated suppression of inflammatory gene expression have not been clarified. In this study, we showed that heat shock suppresses LPS-induced expression of IL-6, a major pyrogenic cytokine, in mouse embryonic fibroblasts and macrophages. Heat shock transcription factor 1 (HSF1) activated by heat shock induced the expression of activating transcription factor (ATF) 3, a negative regulator of IL-6, and ATF3 was necessary for heat-mediated suppression of IL-6, indicating a fever-mediated feedback loop consisting of HSF1 and ATF3. A comprehensive analysis of inflammatory gene expression revealed that heat pretreatment suppresses LPS-induced expression of most genes (86%), in part (67%) via ATF3. When HSF1-null and ATF3-null mice were injected with LPS, they expressed much higher levels of IL-6 than wild-type mice, resulting in an exaggerated febrile response. These results demonstrate a novel inhibitory pathway for inflammatory cytokines.

Heat shock transcription factor 1 (HSF1) not only regulates expression of heat shock genes in response to elevated temperature, but is also involved in developmental processes by regulating genes such as cytokine genes. However, we did not know how HSF1 regulates non-heat shock genes. Here, we show that constitutive HSF1 binding to the interleukin (IL)-6 promoter is necessary for its maximal induction by lipopolysaccharide (LPS) stimulation in mouse embryo fibroblasts and peritoneal macrophages. Lack of HSF1 inhibited LPS-induced in vivo binding of an activator NF-B and a repressor ATF3 to IL-6 promoter. Neither NF-B nor ATF3 binds to the IL-6 promoter in unstimulated HSF1-null cells even if they were overexpressed. Treatment with histone deacetylase inhibitor or a DNA methylation inhibitor restored LPS-induced IL-6 expression in HSF1-null cells, and histone modification enzymes were recruited on the IL-6 promoter in the presence of HSF1. Consistently, chromatin structure of the IL-6 promoter in the presence of HSF1 was more open than that in its absence. These results indicate that HSF1 partially opens the chromatin structure of the IL-6 promoter for an activator or a repressor to bind to it, and provides a novel mechanism of gene regulation by HSF1.One of major adaptive responses to high temperature stress in all living organisms is to induce heat shock proteins, which assist protein folding and inhibit protein denaturation (1). This response is regulated mainly at the level of transcription by heat shock transcription factor 1 (HSF1), 2 which can sense an increase in temperature (2, 3). The HSF family consists of four members (HSF1-4) in vertebrates, all of which bind to heat shock elements (HSE) (3). In addition to protecting cells from exposure to extreme temperature by inducing Hsp (4, 5), HSFs play critical functions in developmental processes such as gamategenesis and neurogenesis (6 -9), in maintenance of the sensory organs (10 -13), and in immune response (14, 15), partly by regulating expression of development-related genes such as FGF, LIF, and IL-6 cytokine genes and the p35 gene, an activator of cyclin-dependent kinase 5 as well as Hsp genes (9,11,13,14). Furthermore, HSF1 inhibits expression of tumor necrosis factor-␣ and IL-1␤ by binding directly to the tumor necrosis factor-␣ promoter (16), or by physically interacting with NF-IL6, an activator for the IL-1␤ gene (17). However, it is still unclear how HSF1 regulates expression of non-heat shock genes.To understand molecular mechanisms of the regulation of cytokine expression by HSF1, we further examined expression of IL-6. Although the effect of HSF1 on IL-6 expression is moderate, HSF1-mediated IL-6 expression may be involved in various aspects of inflammatory and immune response such as antibody production (14). Here, we found a novel function of HSF1 that partially opens the chromatin structure of the IL-6 promoter for an activator or a repressor to bind to it in unstressed conditions. MATERIALS AND METHODS Cell Cultures and Treatments-Primar...

The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers.

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Ischemic preconditioning (IPC) has protective effects against ischemia-perfusion injury of organs. In the present study, we investigated the associated mechanisms after performing remote IPC (rIPC) of lower limbs by clamping abdominal aorta in mice. Subsequent experiments showed decreased damage and paralysis of lower limbs following spinal cord injury (SCI). Concomitantly, plasma vascular endothelial growth factor (VEGF) levels were increased 24 h after rIPC compared with those in sham-operated animals. In subsequent microRNA analyses, thirteen microRNAs were downregulated in exosomes 24 h after rIPC. Further studies of femoral CD34-positive bone marrow (BM) cells confirmed downregulation of these seven microRNAs 24 h after rIPC compared with those in sham-operated controls. Subsequent algorithm-based database searches suggested that two of the seven microRNAs bind to the 3′ UTR of VEGF mRNA, and following transfection into CD34-positive BM cells, anti-miR-762, and anti-miR-3072-5p inhibitors led to increased VEGF concentrations. The present data suggest that rIPC transiently increases plasma VEGF levels by downregulating miR-762 and miR-3072-5p in CD34-positive BM cells, leading to protection against organ ischemia.