Carnosic acid prevents heat stress-induced oxidative damage by regulating heat-shock proteins and apoptotic proteins in mouse testis.

We previously showed that activation of the A1 adenosine receptor protected the kidney against ischemia-reperfusion injury by induction and phosphorylation of heat shock protein 27 (HSP27). Here, we used mice that overexpress human HSP27 (huHSP27) to determine if kidneys from these mice were protected against injury. Proximal tubule cells cultured from the transgenic mice had increased resistance to peroxide-induced necrosis compared to cells from wild-type mice. However, after renal ischemic injury, HSP27 transgenic mice had decreased renal function compared to wild-type mice, along with increased renal expression of mRNAs of pro-inflammatory cytokines (TNF-alpha, ICAM-1, MCP-1) and increased plasma and kidney keratinocyte-derived cytokine. Following ischemic injury, neutrophils infiltrated the kidneys earlier in the transgenic mice. Flow cytometric analysis of lymphocyte subsets showed that those isolated from the kidneys of transgenic mice had increased CD3(+), CD4(+), CD8(+), and NK1.1(+) cells 3 h after injury. When splenocytes or NK1.1(+) cells were isolated from transgenic mice and adoptively transferred into wild-type mice there was increased renal injury. Further, depletion of lymphocytes by splenectomy or neutralization of NK1.1(+) cells resulted in improved renal function in the transgenic mice following reperfusion. Our study shows that induction of HSP27 in renal tubular cells protects against necrosis in vitro, but its systemic increase counteracts this protection by exacerbating renal and systemic inflammation in vivo.

Skeletal muscle atrophy occurs under a variety of conditions and can result from alterations in both protein synthesis and protein degradation. The muscle-specific E3 ubiquitin ligases, MuRF1 and MAFbx, are excellent markers of muscle atrophy and increase under divergent atrophy-inducing conditions such as denervation and glucocorticoid treatment. While deletion of MuRF1 or MAFbx has been reported to spare muscle mass following 14 days of denervation, their role in other atrophy-inducing conditions is unclear. The goal of this study was to determine whether deletion of MuRF1 or MAFbx attenuates muscle atrophy after 2 weeks of treatment with the synthetic glucocorticoid dexamethasone (DEX). The response of the triceps surae (TS) and tibialis anterior (TA) muscles to 14 days of DEX treatment (3 mg kg(-1) day(-1)) was examined in 4 month-old male and female wild type (WT) and MuRF1 or MAFbx knock out (KO) mice. Following 14 days of DEX treatment, muscle wet weight was significantly decreased in the TS and TA of WT mice. Comparison of WT and KO mice following DEX treatment revealed significant sparing of mass in both sexes of the MuRF1 KO mice, but no muscle sparing in MAFbx KO mice. Further analysis of the MuRF1 KO mice showed significant sparing of fibre cross-sectional area and tension output in the gastrocnemius (GA) after DEX treatment. Muscle sparing in the MuRF1 KO mice was related to maintenance of protein synthesis, with no observed increases in protein degradation in either WT or MuRF1 KO mice. These results demonstrate that MuRF1 and MAFbx do not function similarly under all atrophy models, and that the primary role of MuRF1 may extend beyond controlling protein degradation via the ubiquitin proteasome system.

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.

] In fact, recent studies have demonstrated experimentally that increasing the burden of misfolded proteins in the heart can contribute to the development of cardiac dysfunction. In this review, we discuss the role of heat shock proteins (HSPs) in common cardiac diseases, including cardiac hypertrophy, heart failure, and ischemia/reperfusion injury. Furthermore, we delineate the many specific mechanisms by which these chaperones, cochaperones, and heat shock factor (HSF) transcription factors have been found to be cardioprotective in experimental models. Lastly, we review recent studies involving drugs that are being developed (and currently used) to increase the expression (and presumably function) of chaperone/cochaperone systems that may be applicable to the treatment of common cardiac diseases and familial cardiac diseases with a pathogenesis that includes a major component of misfolded proteins (eg, desminopathies).

Heat shock protein 25 (HSP25) interferes negatively with apoptosis through several pathways that involve its direct interaction with cytochrome c or Akt. Here we show that HSP25 inhibits protein kinase C (PKC) delta-mediated cell death through direct interaction. HSP25 binds to kinase-active PKCdelta to inhibit its kinase activity and translocation to the membrane, which results in reduced cell death. Deletion constructs of HSP25 and PKCdelta identified amino acids 90-103 of HSP25 and the C-terminal V5 region of PKCdelta as binding sites. In addition, the interaction between HSP25 and PKCdelta induced HSP25 phosphorylation at Ser-15 and Ser-86, and these phosphorylations permitted HSP25 release from PKCdelta. Based on these observations, we propose that after PKCdelta activation, HSP25 binds to the exposed V5 region of PKCdelta. This novel function of HSP25 accounts for its cytoprotective properties via the inhibition of PKCdelta and the enhancement of HSP25 phosphorylation.

Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease.

Plants are capable of responding to various environmental stresses by initiating the expression of genes that encode proteins involved in plant growth, fruit ripening, maintaining protein homeostasis, and combating heat stress (HS) by activating heat tolerance systems. The mechanism of resisting against HS is very intricate, and the molecular basis and involvement of the related gene network in Capsicum annuum L. are not fully understood. There are five different heat shock proteins (HSPs) reported in the literature, namely, small HSPs (sHSPs), CaHSP60s, CaHSP70s, CaHSP90s, and CaHSP100s, which play a pivotal role in heat stress response (HSR) in C. annuum. Heat shock factors (HSFs) and heat stress elements (HSEs) govern the transcriptional modifications and control the relative expression of heat shock proteins (HSPs). The heat stress response is the reprogramming of the molecular cascades involving the cell stress responses against the HSR, which is characterized by the increased production of molecular chaperones, which help the plants to counter the negative physiological impacts on proteins, induced by heat and other abiotic stresses. Therefore, understanding the detailed molecular mechanisms of C. annuum in response to extreme temperatures is critical for exploring how they will be affected by climate change and how they behave to cope with these varied climate extremes. This study is focused on providing a complete understanding of the molecular cascades in C. annuum L.’s response to HS, which starts with the sensation of HS signals and activation of the relative molecular cascades that are responsible for the activation of HSFs and initiate their primary targets, e.g., HSPs. Overall, this review provides deep insights into all the cellular responses during HS with a special focus on categorization and physiological aspects of HSPs and HSFs.

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.

Murine heat-shock protein 70 (HSP70) protein, which is produced from 2 genes, hsp70.1 and hsp70.3, is known to protect the brain against ischemic injury. However, little information is available on the antiapoptotic mechanism of HSP70.1 protein after cerebral ischemia. To evaluate the role of HSP70.1 protein in ischemia, we analyzed the mitochondrial apoptotic pathway using hsp70.1 knockout (KO) mice and their wild-type (WT) mice. hsp70.1 KO and WT mice underwent focal ischemia for 120 minutes. DNA fragmentation was evaluated by TUNEL staining. Cytochrome c release and the activation of caspase-3 were analyzed by Western blotting and immunohistochemistry. hsp70.1 mRNA was not detected in hsp70.1 KO mice after ischemia, and HSP70 protein expression was markedly suppressed versus WT mice. KO mice showed a significantly greater infarction volume and DNA fragmentation in the cortex than WT mice at 24 hours after ischemia. At 8 hours, cytochrome c release into the cytoplasm was markedly higher in KO mice than in WT mice. Caspase-3 activation was also significantly enhanced in KO mice versus WT mice, as evidenced by higher levels of activated caspase-3 and cleaved gelsolin. These findings suggest that the deletion of the hsp70.1 gene increases cytochrome c release into the cytoplasm and subsequent caspase-3 activation, thereby exacerbating apoptosis after focal cerebral ischemia.

Cell Type–Dependent Pro- and Anti-Inflammatory Role of Signal Transducer and Activator of Transcription 3 in Alcoholic Liver Injury

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, is gastrointestinal complications. Both NSAID-dependent cyclooxygenase inhibition and gastric mucosal apoptosis are involved in NSAID-produced gastric lesions, and this apoptosis is mediated by the endoplasmic reticulum stress response and resulting activation of Bax. Heat shock proteins (HSPs) have been suggested to protect gastric mucosa from NSAID-induced lesions; here we have tested this idea genetically. The severity of gastric lesions produced by indomethacin was worse in mice lacking heat shock factor 1 (HSF1), a transcription factor for hsp genes, than in control mice. Indomethacin administration up-regulated the expression of gastric mucosal HSP70. Indomethacin-induced gastric lesions were ameliorated in transgenic mice expressing HSP70. After indomethacin administration, fewer apoptotic cells were observed in the gastric mucosa of transgenic mice expressing HSP70 than in wild-type mice, whereas the gastric levels of prostaglandin E(2) for the two were indistinguishable. This suggests that expression of HSP70 ameliorates indomethacin-induced gastric lesions by affecting mucosal apoptosis. Suppression of HSP70 expression in vitro stimulated indomethacin-induced apoptosis and activation of Bax but not the endoplasmic reticulum stress response. Geranylgeranylacetone induced HSP70 at gastric mucosa in an HSF1-dependent manner and suppressed the formation of indomethacin-induced gastric lesions in wild-type mice but not in HSF1-null mice. The results of this study provide direct genetic evidence that expression of HSP70 confers gastric protection against indomethacin-induced lesions by inhibiting the activation of Bax. The HSP inducing activity of geranylgeranylacetone seems to contribute to its gastroprotective activity against indomethacin.

Newborn animals are particularly sensitive to hypoxic stress. Oxygen is spared for sensitive tissues, including brain and heart. Scarce information is available concerning the molecular effects of hypoxia in the gastrointestinal tract (GIT). Moreover, stress protein expressions, including heat shock proteins (HSP), are still poorly documented in the GIT. Our objective was to determine the possible effect of hypoxia on HSP expression at birth. After western blotting, alphaB crystallin, HSP 27, and HSP 70 expressions were determined in newborn controls and piglets exposed to 1 or 4 h hypoxia (5% O2, 95% N2) allowed to recover from 1 to 68 h. Cytosol and nuclei were also separated and the extracts were tested for HSF1 and alphaB crystallin expressions. Surprisingly, alphaB crystallin was overexpressed in the stomach and colon in animals submitted to hypoxia, whereas HSP 27 and HSP 70 expression remained stable. Increases and return to basal levels in HSF1 and alphaB crystallin were simultaneously observed in the unique nuclear compartment. To our knowledge, the present study is the first to demonstrate the oxygen dependency of an HSP in the GIT, particularly in the colon in newborn piglets. The kinetics of alphaB crystallin overexpression after hypoxia parallels the activation of HSF1. This observation possibly indicates a correlation between this factor and alphaB crystallin after hypoxia. Taken together, the present results open the field of wide investigation about the specific response of this low-molecular-weight HSP and its possible involvement in pathological states in the GIT such as stomach and colon.

Gastric Inflammation, Metaplasia, and Tumor Development in Gastrin–Deficient Mice

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, senescent decline and ageing is broadly influenced by genetic and extrinsic factors. Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Here, we survey the molecular mechanisms that link ageing to main stress response pathways, and mediate age-related changes in the effectiveness of the response to stress. We also discuss how each pathway contributes to modulate the ageing process. A better understanding of the dynamics and reciprocal interplay between stress responses and ageing is critical for the development of novel therapeutic strategies that exploit endogenous stress combat pathways against age-associated pathologies.

Adiponectin Deficiency Protects Mice From Chemically Induced Colonic Inflammation