N(omega)-nitro-L-arginine inhibits inducible HSP-70 via Ca(2+), PKC, and PKA in human intestinal epithelial T84 cells

The nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45 degrees C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC(50) of 0.0471 +/- 0.0007 microM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca(2+) dependent, we measured [Ca(2+)](i) after treating cells with L-NNA. L-NNA at 100 microM significantly decreased resting [Ca(2+)](i). Likewise, treatment with 1 microM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca(2+)](i) and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca(2+)](i) and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca(2+)](i) and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.     

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.     

Neuroprotective drug riluzole amplifies the heat shock factor 1 (HSF1)- and glutamate transporter 1 (GLT1)-dependent cytoprotective mechanisms for neuronal survival

Heat shock factor 1 (HSF1) mediates the cellular response to stress to increase the production of heat shock protein (HSP) chaperones for proper protein folding, trafficking, and degradation; failure of this homeostatic mechanism likely contributes to neurodegeneration. We show that the neuroprotective drug riluzole increased the amount of HSF1 in NG108-15 neuroprogenitor cells by slowing the specific turnover of HSF1 and supporting a more robust and sustained activation of HSF1. Using Hsp70-luciferase as a functional readout of the activity of HSF1, we show that riluzole amplified the heat shock induction of the reporter gene with an optimal increase at 1 μM. Immunocytochemical staining and Western blot quantitation of HSP70 in NG108-15 neuroprogenitor cells and embryonic spinal cord neurons provided corroborative evidence that riluzole amplified the HSF1-dependent regulation of HSP70 expression. Parallel studies on the GLT1 glutamate transporter showed that riluzole increased GLT1-reporter and GLT1 protein expression and that the increase was enhanced by heat shock and coincident with the increased expression of HSP70 and HSP90. This result is consistent with the anti-glutamatergic profile of riluzole and the presence of multiple heat shock elements on the GLT1 gene promoter, suggesting that riluzole may modulate GLT1 expression through HSF1. The increased HSP chaperones and GLT1 transporter blunted glutamate-induced and N-methyl D-aspartate receptor-mediated excitotoxic death. In summary, we show that riluzole increased the amount and activity of HSF1 to boost the expression of HSPs and GLT1 for neuroprotection under stress.