Heat shock protein 90 protects rat mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the PI3K/Akt and ERK1/2 pathways

Abstract

Mesenchymal stem cell (MSC) transplantation has shown a therapeutic potential to repair the ischemic and infracted myocardium, but the effects are limited by the apoptosis and loss of donor cells in host cardiac microenvironment. The aim of this study is to explore the cytoprotection of heat shock protein 90 (Hsp90) against hypoxia and serum deprivation-induced apoptosis and the possible mechanisms in rat MSCs. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was assessed by Hoechst 33258 nuclear staining and flow cytometric analysis with annexin V/PI staining. The gene expression of Toll-like receptor-4 (TLR-4) and V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ErbB2) was detected by real-time polymerase chain reaction (PCR). The protein levels of cleaved caspase-3, Bcl-2, Bcl-xL, Bax, total-ERK, phospho-ERK, total-Akt, phospho-Akt, and Hsp90 were detected by Western blot. The production of nitric oxide was measured by spectrophotometric assay. Hsp90 improves MSC viability and protects MSCs against apoptosis induced by serum deprivation and hypoxia. The protective role of Hsp90 not only elevates Bcl-2/Bax and Bcl-xL/Bax expression and attenuates cleaved caspase-3 expression via down-regulating membrane TLR-4 and ErbB2 receptors and then activating their downstream PI3K/Akt and ERK1/2 pathways, but also enhances the paracrine effect of MSCs. These findings demonstrated a novel and effective treatment strategy against MSC apoptosis in cell transplantation.

Fig. 1

Protection of MSCs against hypoxia and serum deprivation-induced apoptosis by preconditioning with rhHsp90α Rat MSC viability was evaluated by MTT assay. Cell viability was calculated as the percentage of viable cell number of each sample divided by that of the normoxia and serum-contained control. Cell viability of MSCs preconditioned with rhHsp90α at the dosage of 0.1–10 μmol/L was significantly increased compared to that of control (^* P<0.05)

Fig. 2

Attenuated apoptosis of MSCs exposed to hypoxia and serum deprivation by exogenous rhHsp90α preconditioning (a) Annexin V/PI binding by flow cytometry was performed to detect the early apoptosis of MSCs under hypoxia and serum deprivation (SD) conditions or normoxia and serum-contained conditions (SC) as control. Annexin V⁺ and PI⁺ cells were counted as the apoptotic cells (right top), and the percentage of apoptosis cells was calculated. The apoptosis percentage of MSCs preconditioned with rhHsp90α (0.01–10 μmol/L) decreased compared to those without rhHsp90α preconditioning, as exposed to hypoxia and serum deprivation (^* P<0.05 for 0.1–1 μmol/L, ^** P<0.01 for 10 μmol/L); (b) Hoechst 33258 staining was introduced to detect nuclear apoptotic bodies as the late characteristics of apoptosis. Representative immunofluorescence staining of MSCs depicted the nuclear apoptotic bodies (arrows) in the left panel. Cells with nuclear apoptotic bodies were counted as apoptosis cells, and the percentage of apoptosis cells was calculated (right panel). Compared to those without rhHsp90α preconditioning, the apoptosis percentage in MSCs preconditioned with rhHsp90α decreased as exposed to hypoxia and SD (0.01–10 μmol/L) (^** P<0.01). Cells under normoxia and SC as control group

Fig. 2

Attenuated apoptosis of MSCs exposed to hypoxia and serum deprivation by exogenous rhHsp90α preconditioning (a) Annexin V/PI binding by flow cytometry was performed to detect the early apoptosis of MSCs under hypoxia and serum deprivation (SD) conditions or normoxia and serum-contained conditions (SC) as control. Annexin V⁺ and PI⁺ cells were counted as the apoptotic cells (right top), and the percentage of apoptosis cells was calculated. The apoptosis percentage of MSCs preconditioned with rhHsp90α (0.01–10 μmol/L) decreased compared to those without rhHsp90α preconditioning, as exposed to hypoxia and serum deprivation (^* P<0.05 for 0.1–1 μmol/L, ^** P<0.01 for 10 μmol/L); (b) Hoechst 33258 staining was introduced to detect nuclear apoptotic bodies as the late characteristics of apoptosis. Representative immunofluorescence staining of MSCs depicted the nuclear apoptotic bodies (arrows) in the left panel. Cells with nuclear apoptotic bodies were counted as apoptosis cells, and the percentage of apoptosis cells was calculated (right panel). Compared to those without rhHsp90α preconditioning, the apoptosis percentage in MSCs preconditioned with rhHsp90α decreased as exposed to hypoxia and SD (0.01–10 μmol/L) (^** P<0.01). Cells under normoxia and SC as control group

Fig. 3

Expression of endogenous Hsp90 and membrane receptors of ErbB2 and TLR-4 (a) Western blot was run to detect the expression of endogenous Hsp90 on MSCs under normoxia and serum-contained (SC) conditions (Lane 1), hypoxia and serum deprivation (SD) (Lane 2), and preconditioning with exogenous rhHsp90α (10 μmol/L) (Lane 3). Representative bands were shown in the top panel. Hypoxia and SD induced endogenous Hsp90 expression in MSCs compared to normoxia and SC (^** P<0.01). Exogenous rhHsp90α preconditioning did not change the Hsp90 expression compared to MSCs without preconditioning (^* P>0.05, bottom panel); (b) Cell ErbB2 and TLR-4 receptors were analyzed by quantitative RT-PCR. Hypoxia and SD led to increased expression of ErbB2 and TLR-4 mRNA on MSCs, compared to normoxia and SC (^** P<0.01). Expression of ErbB2 and TLR-4 mRNA decreased on MSCs preconditioned with exogenous rhHsp90α, compared to MSCs without preconditioning (^## P<0.01)

Fig. 3

Expression of endogenous Hsp90 and membrane receptors of ErbB2 and TLR-4 (a) Western blot was run to detect the expression of endogenous Hsp90 on MSCs under normoxia and serum-contained (SC) conditions (Lane 1), hypoxia and serum deprivation (SD) (Lane 2), and preconditioning with exogenous rhHsp90α (10 μmol/L) (Lane 3). Representative bands were shown in the top panel. Hypoxia and SD induced endogenous Hsp90 expression in MSCs compared to normoxia and SC (^** P<0.01). Exogenous rhHsp90α preconditioning did not change the Hsp90 expression compared to MSCs without preconditioning (^* P>0.05, bottom panel); (b) Cell ErbB2 and TLR-4 receptors were analyzed by quantitative RT-PCR. Hypoxia and SD led to increased expression of ErbB2 and TLR-4 mRNA on MSCs, compared to normoxia and SC (^** P<0.01). Expression of ErbB2 and TLR-4 mRNA decreased on MSCs preconditioned with exogenous rhHsp90α, compared to MSCs without preconditioning (^## P<0.01)

Fig. 4

Effect of exogenous rhHsp90α preconditioning on PI3K/Akt and ERK1/2 pathways Representative bands of the interested proteins are shown on the top panel. Compared to normoxia and serum-contained (SC) conditions (Lane 1), hypoxia and serum-deprivation (SD) (Lane 2) resulted in a 3.0-fold decrease of p-Akt/Akt and p-ERK/ERK expression on MSCs, which was reversed by exogenous rhHsp90α preconditioning (^** P<0.01, respectively). MSCs incubated with 17-AAG (40 nmol/L) (Lane 4), an Hsp90 inhibitor, reduced p-Akt/Akt and p-ERK/ERK expression of MSCs preconditioning with exogenous rhHsp90α (10 μmol/L) (Lane 3) (^# P<0.05; ^## P<0.01)

Fig. 5

Effect of exogenous rhHsp90α preconditioning on the apoptotic proteins Representative bands of the interested proteins are shown on the top pannel. Compared to normoxia and serum-contained (SC) (Lane 1), hypoxia and serum deprivation (SD) (Lane 2) decreased the expression of Bcl-2 and Bcl-xL proteins, and increased the expression of Bax and cleaved caspase-3 proteins in MSCs (^** P<0.01, respectively). Preconditioning MSCs with exogenous rhHsp90α (10 μmol/L) (Lane 3) elevated the expression of Bcl-2 and Bcl-xL proteins, and alleviated the expression of Bax and cleaved caspase-3 proteins (^## P<0.01, respectively), which were reversed by 17-AAG (an Hsp90 inhibitor, 40 nmol/L) (Lane 4), wortmannin (a PI3K inhibitor, 0.2 μmol/L) (Lane 5) and U0126 (an ERK1/2 inhibitor, 10 μmol/L) (Lane 6); ^** P<0.01 respectively. β-actin as the internal standard

Fig. 6

Exogenous rhHsp90α preconditioning induced NO synthesis in MSCs exposed to hypoxia and serum deprivation (SD) NO production in the supernatant of each MSC culture was quantitated by colorimetric analysis. NO levels were significantly elevated in MSCs preconditioned with exogenous rhHsp90α at the dosage of 1–10 μmol/L (^* P<0.05, ^** P<0.01), compared to 0 μmol/L rhHsp90α group