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

Abstract

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 2h and recovered for 4h 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.

Fig. 1

Expression of individual HSPs under uninjured condition (Control) and following recovery for 4 h after 2 h of injury (Injury) in Vector control (VC cells) and HSP27 transfected cells (27OE cells). Shown are the Western blots of cell lysates stained with antibodies directed against transfected human HSP27 (Panel A), endogenous HSP25 (Panel B), endogenous HSP70 (Panel C) and actin (Panel D).

Fig. 2

Effect of HSF decoy on HSP expression, with and without injury by ATP depletion, in 27OE cells. Panel A shows representative Western blots of 27OE cell lysate stained with antibodies directed against HSP27, HSP25, HSP70 and actin respectively. Panel B is a graph of densitometry (mean ± SEM) of Western blots probed for endogenous HSP70 and HSP25 and transfected HSP27, expressed as change from uninjured control (n = 5 for all conditions). * represents p<0.05 between groups.

Fig. 3

Western blot of 27OE cells stained with antibody directed against HSP70, HSP25, HSP27, and actin. Cells were from uninjured (C) and injured (I) conditions treated either with vehicle or with siRNA against HSP70 (I + siRNA-70).

Fig. 4

Na,K-ATPase detachment in injured VC cells and 27OE cells injured in the presence or absence of HSF decoy or of siRNA-70. The graph is a summary of detergent extractable, soluble Na,K-ATPase that is detached from the cytoskeleton (mean ± SEM). Each experimental group is expressed as percent change from parallel, uninjured control cells. Below the graph is a representative Western blot of soluble Na,K-ATPase in each of the experimental conditions. n = 5 for all conditions. * represents p<0.05 between groups.

Fig. 5

Immunoprecipitation of HSP27 and co-precipitation of actin in 27OE cells following no injury (Lane 1), injury for 2 h (Lane 2) and injury followed by recovery for 4 h (Lane 3). Incubation of HSP27 antibody alone with Protein G (Lane 4) was used to clarify target proteins from immunoglobulin chains which have molecular weights near target protein. Lanes 5 in both A and B are positive controls. Parallel immunoprecipitation with non-specific normal mouse IgG was performed as a negative control in all three conditions (Lanes 6, 7, and 8). Shown are Western blots stained for HSP27 (Panel A) and Actin (Panel B). These experiments were repeated three times to confirm consistency.

Fig. 6

Immunoprecipitation of actin and co-precipitation of HSP27 in 27OE cells following no injury (Lane 1), injury for 2 h (Lane 2) and injury followed by recovery for 4 h (Lane 3). Incubation of actin antibody alone with Protein G (Lane 4) was used to clarify target proteins from immunoglobulin chains which have molecular weights near target protein. Lanes 5 in both A and B are positive controls. Parallel immunoprecipitation with non-specific normal mouse IgG was performed as a negative control in all three conditions (Lanes 6, 7, and 8). Shown are Western blots stained for Actin (Panel A) and HSP27 (Panel B). These experiments were repeated three times to confirm consistency.

Fig. 7

Immunoprecipitation of HSP27 and co-precipitation of actin in 27OE cells following no injury (Lane 1), injury for 2 h (Lane 2) and injury followed by recovery (Lane 3) in the presence of HSF decoy (Panel A) or siRNA-70 (Panel B). Shown are Western blots stained for actin. These experiments were repeated three times to confirm consistency.