Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2 h and recovered for 4 h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.     

Specific induction of heat shock protein 27 by glucocorticoid in osteoblasts

It is generally recognized that osteoporosis is a common complication of patients with glucocorticoid excess and that glucocorticoid receptor is associated with heat shock protein (HSP) 70 and HSP90 in a heterocomplex. In the present study, we investigated whether glucocorticoid induces HSP27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Dexamethasone time-dependently increased the levels of HSP27, while having no effect on the levels of HSP70 or HSP90. The effect of dexamethasone was dose-dependent in the range between 0.1 nM and 0.1 microM. Dexamethasone induced an increase of the levels of mRNA for HSP27. Dexamethasone induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by dexamethasone. In addition, SB203580 reduced the dexamethasone-stimulated increase of the mRNA levels for HSP27. The dexamethasone-induced phosphorylation of p38 MAP kinase was reduced by SB203580. These results strongly suggest that glucocorticoid stimulates the induction of neither HSP70 nor HSP90, but HSP27 in osteoblasts, and that p38 MAP kinase is involved in the induction of HSP27.     

Characterization of cold-induced heat shock protein expression in neonatal rat cardiomyocytes

Cardiac surgery is usually performed under conditions of cardioplegicischemic arrest. To protect the heart during the ischemic period, themyocardium is exposed to varying degrees of hypothermia. Althoughhyperthermia is known to induce the heat shock response, the moleculareffects of hypothermia on the myocardium have not been investigated. We havestudied the effect of hypothermia on the induction of heat shock proteins inprimary cultures of neonatal cardiomyocytes. Cold stress in cardiomyocytesinduced a 6 fold increase in the heat shock protein HSP70 as compared tocontrol. Increased HSP70 protein levels correlated with induction of HSP70mRNAs. Maximal levels of HSP70 protein appeared 4-6 h following recoveryfrom cold shock, indicating the transient nature of the response. Inductionof HSP25 mRNA was also observed in cold-shocked cardiomyocytes, even thoughincreased HSP25 protein levels were not detected. Our results indicate thathypothermia is capable of inducing the heat shock response in neonatalcardiomyocytes.     

Heat shock factor 1 is a key regulator of the stress response in Chlamydomonas

We report here on the characterization of heat shock factor 1 (HSF1), encoded by one of two HSF genes identified in the genome of Chlamydomonas reinhardtii. Chlamydomonas HSF1 shares features characteristic of class A HSFs of higher plants. HSF1 is weakly expressed under non-stress conditions and rapidly induced by heat shock. Heat shock also resulted in hyperphosphorylation of HSF1, and the extent of phosphorylation correlated with the degree of induction of heat shock genes, suggesting a role for phosphorylation in HSF1 activation. HSF1, like HSFs in yeasts, forms high-molecular-weight complexes, presumably trimers, under non-stress, stress and recovery conditions. Immunoprecipitation of HSF1 under these conditions led to the identification of cytosolic HSP70A as a protein constitutively interacting with HSF1. Strains in which HSF1 was strongly under-expressed by RNAi were highly sensitive to heat stress. 14C-labelling of nuclear-encoded proteins under heat stress revealed that synthesis of members of the HSP100, HSP90, HSP70, HSP60 and small HSP families in the HSF1-RNAi strains was dramatically reduced or completely abolished. This correlated with a complete loss of HSP gene induction at the RNA level. These data suggest that HSF1 is a key regulator of the stress response in Chlamydomonas.     

Mechanism of simvastatin on induction of heat shock protein in osteoblasts

It has recently been reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) stimulate bone formation. However, the mechanism of stimulation of bone metabolism by statins is not precisely clarified. In this study, we investigated whether simvastatin induces heat shock protein (HSP) 27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Simvastatin increased the levels of HSP27 while having little effect on the levels of HSP70 or HSP90. The effect of simvastatin on HSP27 accumulation was dose dependent. Cycloheximide reduced the accumulation. Simvastatin induced an increase in the levels of mRNA for HSP27. Actinomycin D suppressed the mRNA levels. Simvastatin induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase among the MAP kinase superfamily. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by simvastatin while SB202474, a negative control of p38 MAP kinase inhibitor, had no effect. SB203580 reduced the simvastatin-increased mRNA levels for HSP27. Lovastatin, another statin, also induced the HSP27 accumulation and SB203580 suppressed the HSP27 accumulation. These results strongly suggest that statins such as simvastatin do not stimulate the induction of HSP70 and HSP90, but do stimulate the induction of HSP27 in osteoblasts and that p38 MAP kinase plays a role in this induction.     

Methotrexate enhances prostaglandin D2-stimulated heat shock protein 27 induction in osteoblasts

As for the pathogenesis of rheumatoid arthritis (RA), prostaglandins (PGs) act as important mediators of inflammation and joint destruction. Among them, PGD2 is well recognized as a potent regulator of osteoblastic functions. We previously showed that PGD2 stimulates the induction of heat shock protein 27 (HSP27) via protein kinase C (PKC)-dependent p38 mitogen-activated protein (MAP) kinase and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Therefore, it is a current topic to clarify how HSP27 plays a role for regulating osteoblastic functions in the lesion of RA. On the other hand, methotrexate (MTX) is one of the most effective medicines for the treatment of RA. Here, we examined the effect of MTX on PGD2-stimulated HSP27 induction in MC3T3-E1 cells. The cells were pretreated with various doses of MTX including therapeutic dosage for RA, and then stimulated by PGD2. MTX significantly enhanced the PGD2- increased levels of HSP27 in a dose-dependent manner, although MTX alone had no effect on the levels of HSP27. In addition, MTX amplified the PGD2-increased levels of HSP27 mRNA. On the contrary, MTX had little effect on PGD2-induced formation of inositol phosphates, PKC activation and phosphorylations of MAP kinases. Our results strongly suggest that MTX enhances PGD2-stimulated HSP27 induction at a point downstream from MAP kinases in osteoblasts.     

Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation

The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.