We examined the influence of sepsis, induced by cecal ligation and puncture in rats, on the protein and gene expression and hormone binding activity of the glucocorticoid receptor (GR) in skeletal muscle. Sepsis resulted in increased GR mRNA and protein levels and upregulated hormone binding activity in extensor digitorum longus and soleus muscles. Scatchard analysis suggested that the increased GR hormone binding activity reflected an increased number of hormone binding sites, whereas receptor affinity for glucocorticoids was unchanged. The GR antagonist RU-38486 blocked the sepsis-induced increase in GR expression and hormone binding activity, implicating a positive regulatory effect of glucocorticoids on GR expression and binding activity under the present experimental conditions. The results suggest that glucocorticoid-dependent metabolic changes in skeletal muscle during sepsis may reflect not only high circulating glucocorticoid levels but increased amounts and hormone binding activity of the GR as well.

In previous studies, the heat shock response, induced by hyperthermia or sodium arsenite, increased interleukin (IL)-6 production in intestinal mucosa and cultured human enterocytes. A novel way to induce the heat shock response, documented in other cell types, is treatment with proteasome inhibitors. It is not known if proteasome inhibition induces heat shock in enterocytes or influences IL-6 production. Here we tested the hypothesis that treatment of cultured Caco-2 cells, a human intestinal epithelial cell line, with proteasome inhibitors induces the heat shock response and stimulates IL-6 production. Treatment of Caco-2 cells with one of the proteasome inhibitors MG-132 or lactacystin activated the transcription factor heat shock factors (HSF)-1 and -2 and upregulated cellular levels of the 72-kDa heat shock protein HSP-72. The same treatment resulted in increased gene and protein expression of IL-6, a response that was blocked by quercetin. Additional experiments revealed that the IL-6 gene promoter contains a HSF-responsive element and that the IL-6 gene may be regulated by the heat shock response. The present results suggest that proteasome inhibition induces heat shock response and IL-6 production in enterocytes and that IL-6 may be a heat shock-responsive gene, at least under certain circumstances. The observations are important considering the multiple biological roles of IL-6, both locally in the gut mucosa and systemically, and considering recent proposals in the literature to use proteasome inhibitors in the clinical setting to induce the heat shock response.

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Author ContributionsThe manuscript was written through contributions of all authors. NDT, LCP, MRK1 (Karver), and MDS designed TF-targeted peptide sequences. MKK performed all conventional TEM. MKK conceived and designed the rat experiments and performed the surgeries. HAK assisted with the rat hemorrhage model. MKK and RHL conceived and designed the mouse experiments, with laser injury performed by RHL. DCG and BRD provided assistance with tissue processing and handling. MKK conceived, designed, and performed the TEG experiments. DCG provided additional assistance with TEGs. MDS and TDC performed CD spectroscopy. TDC performed cryo-TEM, SAXS, and WAXS. MRK1 synthesized all PAs. WB, EBP, LCP, NDT, TAP, SIS, MRK2 (Kibbe) helped guide the research. MKK, NDT, MRK2 interpreted all results. EBP assisted with statistical analysis. EBP and JRR performed CAC measurements. MKK prepared the initial draft of the manuscript. NDT and MRK2 critically revised the manuscript. MRK2, SIS, and BG provided oversight and funding of the entire project. All authors have given approval to the final version of the manuscript. Supporting InformationA table of the three letter codes, amino acid sequences, and corresponding Factor VII residues for the peptides incorporated into PA molecules; crystal structure models of the putative interaction sites of the targeting peptides on TF; HPLC-MS traces showing purity of synthesized PAs; cryogenic TEM of 25% SFE and 75% SBC-2 PA nanofibers; WAXS analysis of backbone, 25% SFE, and 75% SBC-2 PA nanofibers; CD spectroscopy of FKD and TQD PA nanofibers; fluorescent quantification of tested ratios of SFE and SBC-2 PA nanofibers; the critical aggregation concentration determinations for the 75% SBC-2, 25% SFE, and backbone PAs; and real-time localization of 75% SBC-2 in a mouse laser injury model. This material is available free of charge via the internet at http:// pubs.acs.org.