Angiopoietin-like 4 confers resistance to hypoxia/serum deprivation-induced apoptosis through PI3K/Akt and ERK1/2 signaling pathways in mesenchymal stem cells

Abstract

Angiopoietin-like 4 (ANGPTL4) is a potential anti-apoptotic agent for various cells. We examined the protective effect of ANGPTL4 on hypoxia/serum deprivation (SD)-induced apoptosis of MSCs, as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured in vitro. Apoptosis was induced by hypoxia/SD for up to 24 hr, and assessed by flow cytometry and TUNEL assay. Expression levels of Akt, ERK1/2, focal adhesion kinase (FAK), Src, Bcl-2, Bax, cytochrome C and cleaved caspase-3 were detected by Western blotting. Integrin β1 mRNA was detected by qRT-PCR. Mitochondrial membrane potential was assayed using a membrane-permeable dye. Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat ANGPTL4 in a concentration dependent manner. Moreover, ANGPTL4 decreased the hypoxia/SD-induced caspase-3 cleavage and the cytochrome C release, but increased the Bcl-2/Bax ratio and the mitochondrial membrane potential. Decreased expression of integrin β1, the ANGPTL4 receptor was observed during hypoxia/SD conditions, however, such decrease was reversed by ANGPTL4. In addition, ANGPTL4 induced integrin β1-associated FAK and Src phosphorylation, which was blocked by anti-integrin β1 antibody. ANGPTL4 also reversed the hypoxia/SD-induced decrease of Akt and ERK 1/2 phosphorylation, and the effect of ANGPTL4 was abolished by inhibitors of either integrins, ERK1/2, or phosphatidylinositol 3-kinase (PI3K). Blocking integrinβ1, Akt or ERK largely attenuated anti-apoptotic effect of ANGPTL4. ANGPTL4 protects MSCs from hypoxia/SD-induced apoptosis by interacting with integrins to stimulate FAK complex, leading to downstream ERK1/2 and PI3K/Akt signaling pathways and mimicking the pathway in which MSCs contact with the extracellular matrix.

Figure 1. MSCs produced ANGPTL4 upon exposure to hypoxia/SD.

MSCs were cultured under hypoxia/SD or normal conditions for 24 hours. (A) mRNA levels of ANGPTL4 molecules were analyzed by qRT-PCR analysis as described in the methods. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control). (B ) release of ANGPTL4 was analyzed by ELISA(Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control).

Figure 2. ANGPTL4 protected MSCs from hypoxia/SD-induced apoptosis.

Apoptosis was measured by flow cytometry and TUNEL assay in MSCs pre-incubated with ANGPTL4 (100 ng/mL) for 1 hr, and then either kept (ANGPTL4 Pre-1 hr +24 hr) or removed (ANGPTL4 Pre-1 hr) during a 24 hr hypoxia/SD condition. Cells were exposed to ANGPTL4 when were subjected to hypoxia/SD with no prior-incubations (ANGPTL4 24 hr). MSCs cultured in complete medium were used as controls. (A and B) Survival and apoptosis were measured using Annexin V/PI staining and flow cytometry. Annexin V^−/PI⁻ cells (Q3) were considered as intact or alive cells, Annexin V⁺/PI⁻ (Q4) were considered as early apoptotic cells, Annexin V⁺/PI⁺ cells (Q2) were considered as late apoptotic cells, and Annexin V^−/PI⁺ cells (Q1) were considered as necrotic cells. One representative experiment out of three is shown in A. Quantitative analysis of apoptotic index (fold increase relative to control) by FACS is shown in B. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. hypoxia/SD; ▴P<0.05 vs. ANGPTL4 Pre-1 hr +24 hr). (C and D) TUNEL staining on MSCs. The number of TUNEL⁺ cells is shown in C. Quantitative analysis of TUNEL⁺ cells (fold increase relative to control) is shown in D. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. hypoxia/SD; ▴P<0.05 vs. ANGPTL4 Pre-1 hr +24 hr).

Figure 3. ANGPTL4 protected MSCs against hypoxia/SD-induced apoptosis.

ANGPTL4 (1–1000 ng/mL) was pre-incubated with the cells for 1 hr in complete medium before exposure to hypoxia/SD conditions, and then was maintained in the incubation medium throughout the hypoxia/SD treatment period. (A) Representative image of flow cytometric dot plots analyses of apoptotic cells after Annexin V/PI staining. (B) Image showing TUNEL⁺ cells in each group. (C and D) Composite values of apoptotic index and percentage of TUNEL⁺ cells. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. hypoxia/SD). (E) Representative image of Western blots of procaspase-3 cleavage induced by hypoxia/SD and its inhibition by ANGPTL4 using anticaspase-3 antibody. (The blots shown are representative of three independent experiments). (F) Composite values of the ratio of cleaved caspase-3 and procaspase-3 relative to β-actin. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. hypoxia/SD).

Figure 4. Effects of ANGPTL4 on cell viability and proliferation.

ANGPTL4 was added into the culture media alone in order to test its effect on MSCs viability and proliferation. (A and B) Trypan blue assessment indicated that ANGPTL4 alone even up to 1000 ng/mL was not toxic to cultured MSCs. (Each column represents the mean ± SD of three independent experiments; P>0.05). (C and D) Proliferation growth curves of MSCs were obtained by the CCK-8 assay. In the presence of 100 ng/mL ANGPTL4, the proliferation rate did not show significant changes during 3-days ANGPTL4 treatment in the normal culture (C), and 24-hr hypoxia/SD culture (D). (Each data point represents the mean ± SD of three independent experiments; P>0.05).

Figure 5. Effects of ANGPTL4 on cell survival under hypoxia/SD.

MSCs were transfected with a siRNA against the ANGPTL4 transcript. The cells were also transfected witn a non-targeting siRNA(siRNA-NT) as control. The siRNA-mediated transfection efficiency was demonstrated by western blot (A and B) and ELISA(C). (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs.siRNA-ANGPTL4). Then MSCs were treated with hypoxia/SD for 24 hr. Cells under normal culture were used as control. Then the apoptosis was measured by FACS(D and E).(Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control; ▴P<0.05 vs. hypoxia/SD+siRNA-ANGPTL4).

Figure 6. ANGPTL4 influenced expression of integrin β1 in MSCs.

(A to C) mRNA and protein levels of integrin β1 molecules were analyzed by qRT-PCR and western blot analysis as described in the methods. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control; ▴P<0.05 vs. hypoxia/SD). (D) Expression levels of integrin β1 on the MSCs surfacewere analyzed by immunofluorescence assay.

Figure 7. ANGPTL4 protected MSCs from apoptosis through an ANGPTL4-integrin β1 related molecules pathway.

(A to D) Expression levels of p-FAK (A and C) and p-Src (B and D) were analyzed by Western blot assay. (Fold changes compared to FAK or Src. Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control; ▴P<0.05 vs. hypoxia/SD+ANGPTL4 ). (E to H) MSCs were transfected with a siRNA against the integrin β1 transcript. The cells were also transfected witn a non-targeting siRNA(siRNA-NT) as control. The siRNA-mediated transfection efficiency was demonstrated by western blot (E and F). (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs.siRNA-integrinβ1) Then MSCs were treated with hypoxia/SD for 24 hr. In parallel experiments, cells were pretreated with ANGPTL4 (100 ng/mL) 1 hr prior to exposure to hypoxia/SD. The drug was maintained in the incubation medium throughout the hypoxia/SD treatment period. Then the apoptosis was measured by FACS(G and H).(Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control; ▴P<0.05 vs. hypoxia/SD, & P<0.05 vs. hypoxia/SD +siRNA-integrinβ1).

Figure 8. ANGPTL4 induced ERK1/2 and PI3K/Akt pathways activated.

(A–C) MSCs were stimulated with ANGPTL4 (100 ng/mL) for the indicated times, and then expressions of Akt and Phospho-Akt (S473) (A), Akt and Phospho-Akt (T308) (B), and ERK1/2 and Phospho-ERK1/2 (T202/Y204) (C) were analyzed by Western blotting. Fold changes were compared to Akt or ERK1/2. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. ANGPTL4 −60 min). (D–F) Representative image of Western blots of Akt and Phospho-Akt (S473) (D), Akt and Phospho-Akt (T308) (E), and ERK1/2 and Phospho-ERK1/2 (T202/Y204) (F). Under hypoxia/SD, cells that were pretreated with ANGPTL4 (100 ng/mL) and incubated in hypoxia/SD conditions for 24 hr. Compounds on (D) and (E) were pretreated with LY294002 (25 µM), or integrin β1 antibody. (F) was pretreated with U0126 or integrin β1 antibody. Fold changes were compared to Akt or ERK1/2. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control; ▴P<0.05 vs. hypoxia/SD+ANGPTL4 −100 ng/mL).

Figure 9. ANGPTL4 protected MSCs from Hypoxia/SD-induced apoptosis through integrin-sensitive ERK1/2 and PI3K/Akt pathways.

To determine the role of respective kinase pathways in anti-apoptotic actions of ANGPTL4, MSCs were transfected with siRNA against Akt or ERK genes, as well as the scrambled siRNA. The siRNA-mediated transfection efficiency was demonstrated by Western blotting (A to D).(Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. siRNA-Akt; *P<0.05 vs. siRNA-ERK). MSCs were then treated with hypoxia/SD for 24 hr. In parallel experiments, cells were pretreated with ANGPTL4 (100 ng/mL) 1 hr prior to exposure to hypoxia/SD, and was maintained in the incubation medium throughout the hypoxia/SD treatment period. The apoptosis rate was analysed by flow cytometry (E and F).(Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control; ▴P<0.05 vs. hypoxia/SD+ANGPTL4 −100 ng/mL; &P<0.05 vs. hypoxia/SD +siRNA-NT).

Figure 10. ANGPTL4 exerted anti-apoptotic effects via inhibition of mitochondrial dysfunction.

MSCs were treated with hypoxia/SD for 24 hr. In parallel experiments, cells were pretreated with either integrin β1 antibody (20 µM) or LY294002 (25 µM) or U0126 (20 µM) for 90 min before exposure to hypoxia/SD. When present, ANGPTL4 (100 ng/mL) was added in the presence of each drug for 1 hr prior to exposure to hypoxia/SD. All drugs were maintained in the incubation medium throughout the hypoxia/SD treatment period. Bcl-2/Bax ratio (A and B), cytochrome C (C and D), and caspase-3 (E and F) were detected by Western blotting. (Each example shown is representative of three experiments). Fold changes were compared to control. (G) MMP with JC-1 staining. (Each column represents the mean ± SD of three independent experiments; *P<0.05 vs. control; ▴P<0.05 vs. hypoxia/SD+ANGPTL4 −100 ng/mL).