Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1.

Heat shock factor 1 (HSF1) is a serine-rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA-binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild-type HSF1 when expressed in hsf1(-/-) cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.

Primary chondrocytes resist hydrostatic pressure-induced stress while primary synovial cells and fibroblasts show modified Hsp70 response.

OBJECTIVE: During joint loading, chondrocytes in the articular cartilage are subjected to gradients of high compressive hydrostatic pressure (HP). In response to diverse chemical or physical stresses, heat shock genes are induced to express heat shock proteins (Hsps). This study sought to examine the role of Hsps in baroresistance in primary bovine chondrocytes and synovial cells, as well as in primary human fibroblasts. METHODS: Northern blotting was used to analyze the steady-state levels of hsp70 mRNA in the primary cells exposed to HP or heat stress. Hsp70 protein accumulation was analyzed by Western blotting, and the DNA-binding activity was examined by gel mobility shift assay. RESULTS: Primary bovine chondrocytes which have been adapted to live under pressurized conditions showed negligible Hsp70 response upon HP loading, whereas primary bovine synovial cells and human fibroblasts accumulated hsp70 mRNA and protein when subjected to HP. The response was initiated without activation of the heat shock transcription factor 1. Interestingly, pre-conditioning of the barosensitive fibroblasts with HP or heat shock reduced the Hsp70 response, indicating induction of baroresistance. CONCLUSION: This study suggests that Hsp70 can play an important role in the early stages of adaptation of cells to HP. Thus, the Hsp70 gene expression upon HP loading may serve as one indicator of the chondrocytic phenotype of the cells. This can be of use in the treatment of cartilage lesions.

Formation of nuclear HSF1 granules varies depending on stress stimuli.

In concert with the stress-induced activation of human heat shock factor 1 (HSF1), the factor becomes inducibly phosphorylated and accumulates into nuclear granules. To date, these processes are not fully understood. Here, we show that although stress caused by the proteasome inhibitors MG132 and clasto-lactacystine beta-lactone induces the expression of Hsp70, the formation of HSF1 granules is affected differently in comparison to heat shock. Furthermore, proteasome inhibition increases serine phosphorylation on HSF1, but to a lesser extent than heat stress. Our results suggest that, depending on the type of stress stimulus, the multiple events associated with HSF1 activation might be affected differently.

Protein synthesis is required for stabilization of hsp70 mRNA upon exposure to both hydrostatic pressurization and elevated temperature.

We have recently described that in chondrocytic cells high hydrostatic pressure (HP) causes a heat shock response via mRNA stabilization without a transcriptional activation of the hsp70 gene. In this study, we investigated whether this exceptional regulatory mechanism occurs more generally in different types of cells. Indeed, hsp70 mRNA and protein accumulated in HeLa, HaCat and MG-63 cells under 30 MPa HP, without DNA-binding of heat shock transcription factor 1 (HSF1) to the heat shock element of the hsp70 gene or formation of nuclear HSF1 granules, revealing a lack of transcriptional activation. Moreover, we observed that protein synthesis is needed for mRNA stabilization. Thus, high HP offers a model to study the mechanisms of hsp70 mRNA stabilization without HSF1-mediated induction of the heat shock gene response.

Thermotolerance and cell death are distinct cellular responses to stress: dependence on heat shock proteins.

We tested the hypothesis that heat shock protein (Hsp) induction and cell death are mutually exclusive responses to stress. Despite activation of heat shock transcription factor 1 at temperatures ranging from 40 to 46 degrees C, Hsp72 and Hsp27 were not induced above 42 degrees C. Moreover, cells underwent apoptosis at 44 degrees C and necrosis at 46 degrees C, with mitochondrial cytochrome c release at both temperatures. However, only apoptosis was associated with caspase activation. Treatment of cells with z-VAD-fmk prior to heat shock at 44 degrees C failed to restore Hsp induction despite inhibition of heat-induced apoptosis. Furthermore, accumulation of Hsps after incubation at 42 degrees C rendered the cells resistant to apoptosis. These results suggest that lack of Hsp induction is the cause rather than the consequence of cell death.

Conventional and novel PKC isoenzymes modify the heat-induced stress response but are not activated by heat shock.

In mammalian cells, the heat-induced stress response is mediated by the constitutively expressed heat shock transcription factor 1 (HSF1). Upon exposure to elevated temperatures, HSF1 undergoes several post-translational modifications, including inducible phosphorylation or hyperphosphorylation. To date, neither the role of HSF1 hyperphosphorylation in regulation of the transcriptional activity of HSF1 nor the signaling pathways involved have been characterized. We have previously shown that the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), markedly enhances the heat-induced stress response, and in the present study we elucidate the mechanism by which PKC activation affects the heat shock response in human cells. Our results show that several conventional and novel PKC isoenzymes are activated during the TPA-mediated enhancement of the heat shock response and that the enhancement can be inhibited by the specific PKC inhibitor bisindolylmaleimide I. Furthermore, the potentiating effect of TPA on the heat-induced stress response requires an intact heat shock element in the hsp70 promoter, indicating that PKC-responsive pathways are able to modulate the activity of HSF1. We also demonstrate that PKC is not activated by heat stress per se. These results reveal that PKC exhibits a significant modulatory role of the heat-induced stress response, but is not directly involved in regulation of the heat shock response.

Alpha2B-adrenoceptors couple to Ca2+ increase in both endogenous and recombinant expression systems.

The ability of cloned human alpha2B-adrenoceptors heterologously expressed in Sf9 cells and endogenous alpha2B-adrenoceptors in NG 108-15 neuroblastoma x glioma cells to couple to increase of intracellular Ca2+ was studied. Ca2+ increases in NG 108-15 cells were detectable but slight, whereas those in alpha2B-adrenoceptor-expressing Sf9 cells were greater. In the latter, the maximum Ca2+ increase correlated positively, and the EC50-value of noradrenaline negatively, with the receptor expression density. The order of potency of the agonists was D-medetomidine ([D]-4-[5]-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole) > noradrenaline approximately = clonidine > oxymetazoline, with clonidine and UK14,304 (5-bromo-N-[4,5-dihydro-1H-imidazole-2-yl]-6-quinoxalinamine) being weak partial agonists. In Sf9 cells Ca2+ increases consisted of concomitant mobilization from an intracellular store and influx of extracellular Ca2+. In these cells alpha2B-adrenoceptor stimulation also increased the inositol 1,4,5-trisphosphate mass. We conclude that alpha2B-adrenoceptors can couple to intracellular Ca2+ increases which may involve prior activation of phospholipase C.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate enhances the heat-induced stress response.

Induction of heat shock gene expression is mediated by specific heat shock transcription factors (HSFs), but the signaling pathways leading to activation of HSFs are poorly understood. To elucidate whether protein kinase C-responsive signaling pathways could be involved in the regulation of heat shock gene expression, we have examined the effects of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) on the heat-induced stress response in K562 cells. We demonstrate that TPA treatment markedly enhances heat shock gene expression during heat stress, although TPA alone does not induce the heat shock response. This TPA-mediated enhancement can initially be detected as an accelerated acquisition of DNA binding and transcriptional activity of HSF1 resulting in elevated Hsp70 protein concentrations. In the presence of TPA, the attenuation of HSF1 DNA binding activity during continuous exposure to heat shock occurs more rapidly and in concert with the appearance of newly synthesized Hsp70, which supports earlier studies on the autoregulatory role of Hsp70 in deactivation of HSF1. During heat stress, a correlation between the hyperphosphorylation of HSF1 and its transcriptional activity was observed, in both the presence and the absence of TPA. Our results show that the heat-induced stress response can be significantly modulated by activation of protein kinase C-responsive signaling pathways.