Estradiol‑enhanced osteogenesis of rat bone marrow stromal cells is associated with the JNK pathway

Abstract

Bone marrow stromal cells (BMSCs) can differentiate into osteoblasts. The present study investigated the osteogenic effects of estradiol, as well as the role of the c‑Jun N‑terminal kinase (JNK) signaling pathway in promoting estradiol‑enhanced osteogenesis of rat (r)BMSCs. rBMSCs were treated for 7 days with or without estradiol and further treated with or without the JNK‑specific inhibitor SP600125. The role of estrogen during rBMSC osteogenesis was evaluated by alkaline phosphatase activity and mineralized nodule formation using the Gomori method and Alizarin red S staining, respectively. Subsequently, the mRNA expression levels of transforming growth factor-β1 (TGF‑β1) and core‑binding factor α1 (Cbfα1) were evaluated by reverse transcription‑quantitative polymerase chain reaction, and TGF‑β1, Cbfα1 and phosphorylated (p)‑JNK protein expression was detected by western blotting. All groups treated with SP600125 expressed low levels of TGF‑β1 and Cbfα1 mRNA and protein, and low p‑JNK protein expression. Compared with the control cells, rBMSCs cultured with estradiol exhibited a significant upregulation in the expression levels of osteogenic genes and proteins. The present study demonstrated that estradiol enhanced osteogenic differentiation of rBMSCs and that the JNK signaling pathway was involved in this process, providing insights into the molecular mechanisms involved in rBMSC osteogenesis upon estradiol stimulation.

Figure 1.

Morphology and cell surface markers of rBMSCs. Morphology of primary rBMSCs at (A) 5 days and (B) passage 4 rBMSCs at 6 days (magnification, ×100). Flow cytometric analysis of rBMSCs for (C) CD29, (D) CD90.1 and (E) CD45. rBMSCs, rat bone marrow stromal cells.

Figure 2.

ALP activity of rBMSCs treated with various concentrations of E2. The optimal E2 concentration and incubation time were determined as 10⁻⁸ mol and 7 days, respectively (**P<0.01). E2, estradiol; IND, induction medium; pNPP, p-nitrophenyl phosphate; rBMSCs, rat bone marrow stromal cells.

Figure 3.

ALP staining and mineral nodule formation of rat bone marrow stromal cells. (A and B) ALP staining was performed using Gomori's method. (C and D) Mineral nodule formation was determined by Alizarin red S staining. *P<0.05 and **P<0.01. ALP, alkaline phosphatase; E2, estradiol; IND, induction medium; SP, SP600125.

Figure 4.

Western blot analysis of JNK and p-JNK protein in rat bone marrow stromal cells. Significant differences among each group are noted by *P<0.05 and **P<0.01. E2, estradiol; IND, induction medium; JNK, c-Jun N-terminal kinase; p, phosphorylated; SP, SP600125.

Figure 5.

Cbfα1 mRNA and protein expression levels in rat bone marrow stromal cells. Reverse transcription-quantitative polymerase chain reaction and western blot analysis of Cbfα1 mRNA and protein expression, respectively. GAPDH blot is the same as in Fig. 6, as Cbfα1, TGF-β1 and GAPDH were all probed for on the same membrane. *P<0.05 and **P<0.01. Cbfα1, core-binding factor α1; E2, estradiol-treated group; IND, induction medium group; SP, SP600125.

Figure 6.

TGF-β1 mRNA and protein expression levels in rat bone marrow stromal cells. Reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to detect TGF-β1 mRNA and protein levels, respectively. GAPDH blot is the same as in Fig. 5, as Cbfα1, TGF-β1 and GAPDH were all probed for on the same membrane. *P<0.05 and **P<0.01. E2, estradiol group; IND, induction medium; SP, SP600125; TGF-β1, transforming growth factor-β1.