Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation

Abstract

Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity.

Figure 1. The effect of actin-perturbing drugs on cell toxicity and F-actin organization.

(A) MTT analysis of BMSCs after treatment with Cyt B or Jasp for 48 h. For controls, BMSCs were cultured in medium containing 0.1% DMSO. (n = 3). (B) Cell apoptosis was determined by Annexin V/PI staining. BMSCs were treated with Cyt B or Jasp for 48 h. (n = 3). (C) Fluorescence images of the actin cytoskeleton (green) cultured for 48 h in osteogenic differentiation medium containing drugs at the indicated concentrations. (D) Fractal dimension analysis of morphological changes in F-actin cytoskeleton after 48 h of treatment with Cyt B or Jasp at the indicated concentrations (total 50 cells, n = 3). For each group, values are mean ± SD. *p < 0.05 vs. the DMSO group.

Figure 2. The effect of Cyt B and Jasp on TAZ nuclear aggregation and osteogenesis in BMSCs.

(A) ALP activity analysis of BMSCs after treatment with Cyt B or Jasp for 7 days. (n = 3). (B) AR-S of BMSCs after treatment with Cyt B or Jasp for 14 days. (n = 3). (C) Western blot analysis for the expression of tubulin and Laminin B in the cytoplasmic (Cy) and nuclear (Nu) fractions. (n = 3). (D) Western blot analysis of the relative nuclear translocation of TAZ after exposure to actin-perturbing drugs for 48 h. Lamin B was used as loading control for nuclear extracts. (n = 3). For each group, values are mean ± SD. *p < 0.05, **p < 0.01 vs. the DMSO group.

Figure 3. Depolymerized F-actin inhibited TAZ activation and osteogenesis in BMSCs independently of LATS.

(A) Western blot analysis of TAZ nuclear aggregation and total LATS1 expression in BMSCs transfected with siCON or siLATS1 with or without Cyt B after 48 h. Lamin B and GAPDH were used as loading controls for nuclear extracts and cell extracts, respectively. (n = 3). (B) AR-S of BMSCs transfected with siCON or siLATS1 after 14 days. (n = 3). (C) ALP activity analysis for BMSCs transfected with siCON or siLATS1 at day 7. (n = 3). For each group, values are mean ± SD. *p < 0.05 vs. the DMSO + siCON group, #p < 0.05 vs. the DMSO + siLATS1 group.

Figure 4. SMG inhibited osteogenic differentiation of BMSCs.

(A) ALP activity analysis of BMSCs after exposure to SMG for 48 h cultured in osteogenic medium. (n = 3). (B) Real-time PCR of relative Runx2 mRNA levels in BMSCs after exposure to SMG for 48 h. (n = 3). (C) Fluorescence images of the actin cytoskeleton (green) and nucleus (blue) of BMSCs cultured for 48 h in SMG. (D) Fractal analysis of morphological changes in F-actin cytoskeleton (total of 50 cells, n = 4). (E) Western blot analysis of TAZ nuclear aggregation in BMSCs. Lamin B was used as a loading control for nuclear extracts. (n = 4). For each group, values are mean ± SD. *p < 0.05, **p < 0.01 vs. the Control group.

Figure 5. Jasp recovered osteogenic differentiation of BMSCs in SMG.

(A) Western blot analysis for the nuclear aggregation of TAZ and total LATS1 expression in BMSCs transfected with siCON or siLATS1 and cultured in osteogenic medium with or without Jasp (10 nM) after 48 h. Lamin B and GAPDH were used as loading controls for nuclear extracts and cellular extracts, respectively. (n = 3). (B) ALP activity analysis of BMSCs after exposure to SMG for 48 h. (n = 3). (C) Real-time RT-PCR of relative Runx2 mRNA levels in BMSCs after exposure to SMG for 48 h. (n = 3). For each group, values are mean ± SD. #p < 0.05 vs. siLATS1 + SMG group.

Figure 6. LPA rescued osteogenic differentiation of BMSCs through the TAZ pathway.

(A) Fluorescence images of the actin cytoskeleton (green) and nucleus (blue) of BMSCs cultured for 48 h in SMG with or without LPA (2 μM). (n = 4). (B) Western blot analysis of TAZ nuclear aggregation in BMSCs after exposure to SMG for 48 h. Lamin B was used as a loading control for nuclear extracts. (n = 3). (C) Western blot analysis of TAZ nuclear aggregation in BMSCs transfected with siCON or siTAZ after exposure to SMG for 48 h cultured in osteogenic medium. (n = 3). (D) ALP activity analysis of BMSCs after exposure to SMG for 48 h. (n = 3). (E) Real-time RT-PCR of relative Runx2 mRNA levels in BMSCs after exposure to SMG for 48 h. (n = 3). For each group, values are mean ± SD. *p < 0.05, **p < 0.01.

Figure 7. LPA recovered osteogenic differentiation of BMSCs in SMG via F-actin.

(A) Fluorescence images of the actin cytoskeleton (green) and nucleus (blue) of BMSCs cultured for 48 h in SMG with or without Cyt B (1 μM). (n = 3). (B) Western blot analysis of TAZ nuclear aggregation in BMSCs after exposure to SMG for 48 h. Lamin B was used as a loading control for nuclear extracts. (n = 3). (C) ALP activity analysis of BMSCs after exposure to SMG for 48 h. (n = 3). (D) Real-time RT-PCR of relative Runx2 mRNA levels in BMSC after exposure to SMG for 48 h. (n = 3). For each group, values are mean ± SD. *p < 0.05, **p < 0.01 vs. the SMG + LPA group.