Increased COUP-TFII Expression Mediates the Differentiation Imbalance of Bone Marrow-Derived Mesenchymal Stem Cells in Femoral Head Osteonecrosis

Abstract

Objective: Bone marrow-derived mesenchymal stem cells (BMSCs) have multilineage differentiation potential, which allows them to progress to osteogenesis, adipogenesis, and chondrogenesis. An imbalance of differentiation between osteogenesis and adipogenesis will result in pathologic conditions inside the bone. This type of imbalance is also one of the pathological findings in osteonecrosis of the femoral head (ONFH). Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) was previously reported to mediate the differentiation of mesenchymal stem cells. This study investigated the expression of the osteogenesis regulator Runx2, osteocalcin, the adipogenesis regulator PPARγ, and COUP-TFII in the femoral head tissue harvested from ONFH patients, and characterized the effect of COUP-TFII on the differentiation of primary BMSCs.

Methods: Thirty patients with ONFH were recruited and separated into 3 groups: the trauma-, steroid- and alcohol-induced ONFH groups (10 patients each). Bone specimens were harvested from patients who underwent hip arthroplasty, and another 10 specimens were harvested from femoral neck fracture patients as the control group. Expression of the osteogenesis regulator Runx2, osteocalcin, the adipogenesis regulator PPARγ, C/EBP-α, and COUP-TFII was analyzed by Western blotting. Primary bone marrow mesenchymal cells were harvested from ONFH cells treated with COUP-TFII RNA interference to evaluate the effect of COUP-TFII on MSCs.

Results: ONFH patients had significantly increased expression of the adipogenesis regulator PPARγ and C/EBP-α and decreased expression of the osteogenesis regulator osteocalcin. ONFH bone tissue also revealed higher COUP-TFII expression. Immunohistochemical staining displayed strong COUP-TFII immunoreactivity adjacent to osteonecrotic trabecular bone. Increased COUP-TFII expression in the bone tissue correlated with increased PPARγ and decreased osteocalcin expression. Knockdown of COUP-TFII with siRNA in BMSCs reduced adipogenesis and increased osteogenesis in mesenchymal cells.

Conclusion: Increased COUP-TFII expression mediates the imbalance of BMSC differentiation and progression to ONFH in patients. This study might reveal a new target in the treatment of ONFH.

Figure 1

(a) The femoral head was extracted and split in half, and the necrotic bone around the center of the femoral head (circle) was harvested. (b) Compared with the control group, the trauma, steroid, and alcohol groups all showed increased numbers of adipocytes and decreased trabecular bone (black arrows) in the H&E sections (100x). (c) The BV/TV of femoral heads in the osteonecrotic groups all showed significant decreases compared with those in the control group. (Data are presented as mean ± SD; t test, ^∗P < 0.05, compared with the control group; BV/TV, bone volume/total volume.)

Figure 2

(a and b) In the osteonecrotic groups, the key factors (PPARγ and C/EBP-α) controlling adipogenesis were significantly decreased compared with the control group. (c and d) On the other hand, the osteogenesis marker (osteocalcin) was significantly decreased in all of the osteonecrotic groups. (Data are presented as mean ± SD; t test, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and the difference was statistically significant compared with the control group.)

Figure 3

(a) Immunohistochemical images (200x) of osteonecrotic tissue showing COUP-TFII expression in the bone specimens. In the osteonecrotic groups, osteonecrotic lesions displayed strong COUP-TFII expression compared with those in the control group. (b and c) Western blot analysis showed increased expression of COUP-TFII in all the osteonecrotic groups compared with the control group. (Data are means ± SD; t test, ^∗P < 0.05, ^∗∗P < 0.05, ^∗∗∗P < 0.001, and the difference was statistically significant compared with the control group.)

Figure 4

Morphological analyses of bone marrow mesenchymal stem cells (BMSCs). (a) Initial primary BMSCs from osteonecrotic patients demonstrated spindle-shaped morphology (magnification, 200x). (b) Following four generations (16 days), the cells presented with a similar spindle morphology (magnification, 200x). (c) Flow cytometry results of mesenchymal stem cells (MSC) showed that the cells were positive for the markers CD90, CD105, and CD73 and negative for CD45/CD34/CD11b/CD19/HLA-DR PE.

Figure 5

(a) COUP-TFII promotes the adipogenic differentiation of mesenchymal cells. COUP-TFII siRNA-treated BMSC cells and controlled BMSC cells underwent adipocyte differentiation, and cells were stained with oil red O and photographed on day 14. Decreasing adipogenesis was noted after COUP-TFII knockdown (magnification, 200x). (b and c) Impaired adipogenesis in COUP-TFII-depleted cells was confirmed by western blotting, with decreased expression of PPARγ and C/EBP-α. (AM: adipogenic medium; KD: knockdown; Data are presented as mean ± SD; t test, ^∗∗P < 0.01, ^∗∗∗P < 0.001.)

Figure 6

(a) Alizarin red staining of BMSCs that underwent osteogenesis for 28 days. When cultured in osteogenic medium, COUP-TFII knockdown BMSCs showed increased osteogenic differentiation compared with control BMSCs (magnification, 200x). (b and c) Increased osteogenesis in COUP-TFII-depleted cells was confirmed by western blotting with increased expression of Runx2. (OM: osteogenic medium; KD: knockdown; data are presented as the means ± SD; t test, ^∗∗P < 0.01.)