Effect of GARP on osteogenic differentiation of bone marrow mesenchymal stem cells via the regulation of TGFβ1 in vitro

Abstract

Mesenchymal stem cells (MSCs), which have multipotential differentiation and self-renewal potential, are possible cells for tissue engineering. Transforming growth factor β1 (TGFβ1) can be produced by MSCs in an inactive form, and the activation of TGFβ1 functions as an important regulator of osteogenic differentiation in MSCs. Recently, studies showed that Glycoprotein A repetitions predominant (GARP) participated in the activation of latent TGFβ1, but the interaction between GARP and TGFβ1 is still undefined. In our study, we successfully isolated the MSCs from bone marrow of rats, and showed that GARP was detected in bone mesenchymal stem cells (BMSCs). During the osteogenic differentiation of BMSCs, GARP expression was increased over time. To elucidate the interaction between GARP and TGFβ1, we downregulated GARP expression in BMSCs to examine the level of active TGFβ1. We then verified that the downregulation of GARP decreased the secretion of active TGFβ1. Furthermore, osteogenic differentiation experiments, alkaline phosphatase (ALP) activity analyses and Alizarin Red S staining experiments were performed to evaluate the osteogenic capacity. After the downregulation of GARP, ALP activity and Alizarin Red S staining significantly declined and the osteogenic indicators, ALP, Runx2, and OPN, also decreased, both at the mRNA and protein levels. These results demonstrated that downregulated GARP expression resulted in the reduction of TGFβ1 and the attenuation of osteoblast differentiation of BMSCs in vitro.

Keywords: Bone marrow mesenchymal stem cell; Bone regeneration; GARP; TGFβ1.

Figure 1. BMSCs morphological characters observed under light microscope.

The images revealed that BMSCs showed a spindal shape and typical fibrablast appearance. BMSCs, bone marrow mesenchymal stem cells. (A) 40X. Bar = 500 µm (B) 100X. Bar = 100 µm (C) 200X. Bar = 50 µm (D) 400X. Bar = 20 µm.

Figure 2. Cytometry results of BMSCs.

Cytometry results demonstrated that BMSCs were positive for CD 44 (95.82%), CD 90 (99.85%), CD 105 (99.09%) and negative for CD 45 (5.53%). BMSCs, bone marrow mesenchymal stem cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin. (A) count; (B) Blank Control; (C) CD 44; (D) CD 90; (E) CD 105; (F) CD 45; (t test).

Figure 3. Immunofluorescent staining experiment.

For immunofluorescent staining experiment, we used GARP antibody to show the expression of GARP protein, homologous anti-IgG antibody as isotype control and DAPI to show the location of nucleus. GARP, Glycoprotein A repetitions predominant; DAPI, diamidino-phenyl-indole. (A) and (D) showed the merge image of two groups; (B) and (E) showed the FITC immunofluorescent staining; (C) and (F) showed the DAPI staining; 200X; Bar = 50 µm.

Figure 4. GARP expression during osteogenic differentiation.

According to the qRT-PCR and western blot experiments results, on days 7 and 14 after osteogenic differentiation, GARP mRNA and protein levels increased significantly, compared to the days 0. (A) qRT-PCR experiment of GARP mRNA expression; (B) western blot experiment of GARP protein expression; (t test, * P < 0.05).

Figure 5. qRT-PCR results of GARP expression.

qRT-PCR results showed that GARP-sh4 and GARP-sh5 shRNA could significantly down-regulate the mRNA expression of GARP in BMSCs.(t test, * P < 0.05).

Figure 6. Western blot experiments of GARP expression.

Western blot experiments showed that GARP total protein expression in GARP-sh group was decreased, compared to control group.

Figure 7. Cytometry results of GARP expression.

Cytometry results demonstrated that the GARP expression was 26.1% in NC group and 10.7% in GARP-sh group. (t test); (A) the count number of tested cells; (B) blank group; (C) NC group; (D) GARP-sh group.

Figure 8. Cell Counting Kit-8 (CCK-8) analysis of transfected BMSCs.

CCK-8 analysis result demonstrated that cells of GARP-sh group and NC group were in the slow growth period on the first two days after seeding in the plate. On the days 3, the growth pattern of cells began to display an exponential growth phase. On the days 6, the cells reached to the plateau phase. There was no significant differences between two groups (t test.).

Figure 9. ELISA experiments of mature TGFβ1 level in GARP-sh and NC groups.

ELISA experiments showed that mature TGFβ1 level was down-regulated in GARP-sh group.(t test, * P < 0.05).

Figure 10. Osteogenic differentiation related factors’ mRNA expression results.

Osteogenic differentiation results demonstrated ALP, Runx 2 and OPN mRNA expression was decreased in GARP-sh group at osteogenic differentiation 7 days and 14 days. (t test, * P < 0.05); (A) mRNA expression of ALP; (B) mRNA expression of Runx2; (C) mRNA expression of OPN.

Figure 11. Western blot analysis of osteogenic related factors’ protein expression.

Western blot analysis showed that ALP, Runx2 and OPN protein expressions were decreased in GARP-sh group.

Figure 12. ALP and Alizarin Red S staining analysis of osteogenic differentiation.

After 7 days osteogenic differentiation, ALP staining experiments showed that the staining of NC was much higher than the GARP-sh group. on 14 days after osteogenic differentiation, Alizarin Red S staining results showed that there was more mineralized nodule formation in the NC group, compared to the GARP-sh group. (A) ALP and Alizarin Red S staining with the gross appearance. (B) ALP and Alizarin Red S staining under the microscopic view (40X). Bar = 500 µm.