BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells

Abstract

The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway.

Fig 1. Effects of BST2 knockdown by siRNA on BST2 expression in hAD-BMSCs.

RT-PCR and immunoblotting analyses (A) as well as qRT-PCR (B) demonstrated that the expression of BST2 was lower in cells treated for 5 days with OS and siBST2 #1 or #2 than in cells treated with OS and non-targeting siRNA (siCont). (C) Expression of BST2 in the human bone marrow. Representative immunostaining of BST2 in the human bone marrow was observed under light microscope (magnification ×400). Brown staining (marked with a red arrow) indicates the expression of BST2 in bone marrow. Values are expressed as means ± SEM (standardized mean difference) **, P<0.01 compared withthe expression level in cells treated with the control siRNA (siCont) (n = 4).

Fig 2. Effects of BST2 knockdown on osteoblast differentiation in hAD-BMSCs.

ALP staining (A) and ALP activity (n = 4) (B) were reduced after BST2 knockdown. At 21 days of cultivation, BST2 knockdown by siRNA resulted in a significant reduction in AR-S staining for calcium accumulation (C). Values are expressed as means ± SEM. **, P<0.01 compared with the control siRNA-treated group (siRNA Cont).

Fig 3. Expression of osteoblast differentiation markers following BST2 knockdown.

hAD-BMSCs transfected with siRNA were cultured for 5 days in the presence or absence of OS, and qRT-PCR was performed to measure the expression of ALP, COL1α1, BSP, OCN, and OSX, with GAPDH as the internal control. Relative expression levels were normalized to the expression of the untreated negative control sample. Compared with the untreated control, increased expression levels of ALP, OSX, COL1α1, BSP, and OCN were apparent in cells treated with OS and the control siRNA; however, the expression of these markers was decreased after BST2 knockdown by siRNA. Values are expressed as means ± SEM. *, P<0.05; **, P<0.01, when compared with cells treated with OS and the control siRNA.

Fig 4. Effects of BST2 on RUNX2 expression.

hAD-BMSCs transfected with siRNA were cultured for 5 days in the presence or absence of OS, and RT-PCR (A), qRT-PCR (B), and immunoblot analyses were performed. GAPDH was used as an internal standard, and the relative expression of RUNX2 was normalized to the expression levels in of cells treated with OS alone. qRT-PCR data showed that RUNX2 expression was reduced following BST2 knockdown with siBST2 #1 and #2. To determine protein expression, the cells were lysed and the proteins were analyzed by SDS-PAGE and immunoblotting (C). The band densities for RUNX2 were normalized to those for β-actin. RUNX2 mRNA and protein expression were significantly reduced following BST2 knockdown by siRNA. Data are expressed as means ± SEM (n = 4, **P<0.01vs. OS-treated control).

Fig 5. BMP2 signaling pathway was influenced by BST2.

hAD-BMSCs transfected with siRNA were cultured for 5 days in the presence or absence of OS, and an RT-PCR analysis was performed. GAPDH was used as an internal standard, and the relative expression levelsof BMP2, BMPR1A, BMPR1B, and BMPR2 were normalized to the expression levels of control cells treated with OS alone. The expression levels of BMP2 and BMPR1A were significantly reduced following BST2 knockdown by siRNA (A). The cells were lysed and the proteins were analyzed by SDS-PAGE and immunoblot analyses. The band densities for SMADs 1, 4, and p-SMAD1/5/8 were normalized to those of β-actin and T-SMAD1/5/8. SMAD1, SMAD4 and p-SMAD1/5/8 expression levels were significantly reduced following BST2 knockdown by siRNA (B). Data are expressed as means ± SEM (n = 4, **P < 0.01 vs. OS-treated controls).