HSP27 regulates p53 transcriptional activity in doxorubicin-treated fibroblasts and cardiac H9c2 cells: p21 upregulation and G2/M phase cell cycle arrest.

Treatment of cancer patients with anthracyclin-based chemotherapeutic drugs induces congestive heart failure by a mechanism involving p53. However, it is not known how p53 aggravates doxorubicin (Dox)-induced toxicity in the heart. On the basis of in vitro acute toxicity assay using heat shock factor-1 (HSF-1) wild-type (HSF-1(+/+)) and HSF-1-knockout (HSF-1(-/-)) mouse embryonic fibroblasts and neonatal rat cardiomyocyte-derived H9c2 cells, we demonstrate a novel mechanism whereby heat shock protein 27 (HSP27) regulates transcriptional activity of p53 in Dox-treated cells. Inhibition of p53 by pifithrin-alpha (PFT-alpha) provided different levels of protection from Dox that correlate with HSP27 levels in these cells. In HSF-1(+/+) cells, PFT-alpha attenuated Dox-induced toxicity. However, in HSF-1(-/-) cells (which express a very low level of HSP27 compared with HSF-1(+/+) cells), there was no such attenuation, indicating an important role of HSP27 in p53-dependent cell death. On the other hand, immunoprecipitation of p53 was found to coimmunoprecipitate HSP27 and vice versa (confirmed by Western blotting and matrix-assisted laser desorption/ionization time of flight), demonstrating HSP27 binding to p53 in Dox-treated cells. Moreover, upregulation of p21 was observed in HSF-1(+/+) and H9c2 cells, indicating that HSP27 binding transactivates p53 and enhances transcription of p21 in response to Dox treatment. Further analysis with flow cytometry showed that increased expression of p21 results in G(2)/M phase cell cycle arrest in Dox-treated cells. Overall, HSP27 binding to p53 attenuated the cellular toxicity by upregulating p21 and prevented cell death.

Doxorubicin-induced cardiotoxicity: direct correlation of cardiac fibroblast and H9c2 cell survival and aconitase activity with heat shock protein 27.

The use of doxorubicin (Dox) and its derivatives as chemotherapeutic drugs to treat patients with cancer causes dilated cardiomyopathy and congestive heart failure due to Dox-induced cardiotoxicity. In this work, using heat shock factor-1 wild-type (HSF-1(+/+)) and HSF-1 knockout (HSF-1(-/-)) mouse fibroblasts and embryonic rat heart-derived cardiac H9c2 cells, we show that the magnitude of protection from Dox-induced toxicity directly correlates with the level of the heat shock protein 27 (HSP27). Western blot analysis of normal and heat-shocked cells showed the maximum expression of HSP27 in heat-shocked cardiac H9c2 cells and no HSP27 in HSF-1(-/-) cells (normal or heat-shocked). Correspondingly, the cell viability, measured [with (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] after treatment with various concentrations of Dox, was the highest in heat-shocked H9c2 cells and the lowest in HSF-1(-/-) cells. Depleting HSP27 in cardiac H9c2 cells by small interfering (si)RNA also reduced the viability against Dox, confirming that HSP27 does protect cardiac cells against the Dox-induced toxicity. The cells that have lower HSP27 levels such as HSF-1(-/-), were found to be more susceptible for aconitase inactivation. Based on these results we propose a novel mechanism that HSP27 plays an important role in protecting aconitase from Dox-generated O(2)*(-), by increasing SOD activity. Such a protection of aconitase by HSP27 eliminates the catalytic recycling of aconitase released Fe(II) and its deleterious effects in cardiac cells.

Stimulation of cysteinyl leukotriene production in mast cells by heat shock and acetylsalicylic acid.

Immunoglobulin (Ig) E-dependent activation of mast cells is central to the allergic response. The engagement of IgE-occupied receptors initiates a series of molecular events that causes the release of preformed, and de novo synthesis of, allergic mediators. Cysteinyl leukotrienes are able to contract airway smooth muscle and increase mucus secretion and vascular permeability and recruit eosinophils. Mast cells have also recently been recognized as active participants in innate immune responses. Heat stress can modulate innate immunity by inducing stress proteins such as heat-shock proteins (HSPs). We previously demonstrated that treatment of mast cells with heat shock or acetylsalicylic acid results in an increase of TNF-alpha and IL-6 release. This effect was paralleled by expression of HSP70. In the current study, we further investigated the effects of heat shock and acetylsalicylic acid on the activation of mast cells and the release of cysteinyl leukotrienes. In mouse mast cells, derived from a culture of bone marrow cells, responsiveness to heat shock, acetylsalicylic acid and exogenous or endogenous HSP70 was monitored by measuring leukotriene C4 release. We show that after heat shock treatment and exposure to acetylsalicylic acid leukotriene production was increased. Moreover, exogenous rHSP70 also induced leukotriene production. Because it has been reported that leukotriene production in mast cells may be mediated by Toll like receptor (TLR) activation, and HSP70 also activates TLRs signaling, we further explored these issues by using mast cells that are not able to produce HSP70, i.e. heat shock factor-1 (HSF-1) knockout cells. We found that in HSF-1 knockout bone marrow derived mast cells, heat shock and acetylsalicylic acid failed to induce release of leukotrienes. Moreover, in wild type cells the surface expression of TLR4 was attenuated, whereas the intracellular expression was up-regulated. We conclude that heat shock and acetylsalicylic acid induce the production and release of heat shock proteins from mast cells, which in turn stimulate leukotriene synthesis through activation of TLR4.

Induction of HSP70 is dispensable for anti-inflammatory action of heat shock or NSAIDs in mast cells.

OBJECTIVE: It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid, ibuprofen, and indomethacin, induce anti-inflammatory effects through inhibition of cyclooxygenase enzyme activity. However, it has also been established that a variety of their anti-inflammatory effects are independent of cyclooxygenase. In the search for alternative modes of action, it was found that NSAIDs share some cellular effects with heat shock treatment. This prompted us to investigate whether NSAIDs modulate production of proinflammatory cytokines by mast cells through the heat shock response. MATERIALS AND METHODS: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-) mice, responsiveness to heat shock and NSAIDs was monitored by measuring tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production and signaling pathways. RESULTS: In bone marrow-derived mast cells (BMMC), we found that heat shock and a number of NSAIDs induced heat shock protein 70 (HSP70), which was closely paralleled with inhibition of IL-6 and TNF-alpha production. Surprisingly, in BMMC from HSF-1(-/-)mice, heat shock and selected NSAIDs were still able to suppress cytokine production in the absence of HSP70 induction. CONCLUSION: In this article, we provide evidence that inhibition of release of proinflammatory cytokines by NSAIDs and heat shock may be attributed to inhibition of the inhibitory nuclear factor kappaB (NF-kappaB) kinase activity, extracellular signal-regulated kinases 1/2, and p38 pathways, resulting in decreased transcriptional activity of the NF-kappaB pathway.