Smurf1 inhibits osteoblast differentiation and bone formation in vitro and in vivo

Abstract

Bone morphogenetic proteins (BMPs) are required for normal postnatal bone formation and osteoblast differentiation. There is evidence from recent studies that BMP signaling in osteoblasts is controlled by an ubiquitin-proteasome regulatory mechanism involving a cascade of enzymatic reactions. The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes. We have examined the role of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1), a member of the Hect domain family of E3 ubiquitin ligases in osteoblast function. Smurf1 has been found to interact with BMP-activated Smad1 and -5 and to mediate degradation of these Smad proteins. Recently we have found that Smurf1 mediates the protein degradation of the osteoblast-specific transcription factor Runx2/Cbfa1. To determine the role of Smurf1 in osteoblast differentiation, in the present studies we transfected a Smurf1 expression plasmid into 2T3 osteoblast precursor cells and found that Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation. To further investigate the role of Smurf1 in bone formation in vivo, we generated transgenic mice in which expression of the epitope-tagged Smurf1 transgene was targeted to osteoblasts using the murine 2.3-kb osteoblast-specific type I collagen promoter. In these transgenic mice, bone formation was significantly reduced during postnatal life. Our results demonstrate for the first time that Smurf1 plays a specific role in osteoblast differentiation and bone formation in vivo.

Fig.1. Smurf1 inhibits osteoblast differentiation in 2T3 cells

a and b, mRNA and protein expression of Smurf1 in 2T3 cells. FLAG-Smurf1 expression plasmid was transfected into 2T3 cells, and four stably transfected clones were selected and characterized. Expression of transfected FLAG-tagged Smurf1 mRNA was detected by reverse transcriptase-PCR using primers encoding FLAG sequence (upper primer) and Smurf1 cDNA (lower primer) (a). Expression of FLAG-Smurf1 protein was detected by Western blot analysis using an anti-FLAG monoclonal M2 antibody (b). c, Smurf1 inhibits ALP activity. ALP activity from four clones of 2T3/vector and four clones of 2T3/Smurf1 was measured. Smurf1 overexpression inhibited ALP activity. d, Smurf1 inhibits expression of osteoblast-specific genes. mRNA expression of osterix (Osx), Runx2, type I collagen (Col-I), and osteocalcin (OC) was analyzed by Northern blot analysis. Following phosphorimaging, the expression levels of these osteoblast-specific genes were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Smurf1 overexpression inhibited mRNA expression of Osx, Runx2, Col-I, and OC in 2T3 cells. e, Smurf1 inhibits mineralized bone nodule formation. 2T3/vector and 2T3/Smurf1 cells were cultured for 10 days in the absence or presence of BMP-2 (50 ng/ml). Transfection of Smurf1 inhibited BMP-2-induced mineralized bone nodule formation.

Fig.2. Smurf1 inhibits BMP signaling in 2T3 cells

a and b, reductions in Smad1 and Runx2 protein levels in 2T3/Smurf1 cells. Expression of Smad1 (a) and Runx2 (b) protein was detected by Western blot analysis. In 2T3/Smurf1 cells, Smad1 and Runx2 protein levels were decreased. c, inhibition of BMP signaling in 2T3/Smurf1 cells. 2T3/vector and 2T3/Smurf1 cells were transfected with BMP signaling reporter construct, 12×SBE-OC-Luc, and treated with 50 ng/ml BMP-2. Expression of Smurf1 inhibited BMP-2-stimulated luciferase activity. Transfection of Smad6 further inhibited BMP-2-stimulated luciferase activity. *, p < 0.05; #, p < 0.05, unpaired t test, compared with the same treatments in 2T3/vector group. d, the effect of Smurf1 on TGF-β signaling in 2T3 cells. 2T3/vector and 2T3/Smurf1 cells were transfected with TGF-β signaling reporter construct, p3TP-Lux, and treated with 2 ng/ml TGF-β. Transfection of Smurf1 had no significant effect on TGF-β signaling in 2T3 cells.

Fig.3. Smurf1 inhibits bone formation in Col1a1-Smurf1 transgenic mice

a, expression of the FLAG-tagged Smurf1 transgene in Col1a1-Smurf1 transgenic mice. Primary osteoblasts were isolated from calvariae of transgenic mice and their wild-type littermates. Expression of FLAG-Smurf1 protein in osteoblasts of Smurf1 transgenic mice was detected by Western blot analysis using the anti-FLAG M2 antibody. b and c, trabecular bone volume (BV) is decreased in Col1a1-Smurf1 transgenic mice. Bone volume was analyzed in two separate lines of transgenic mice in a defined area in proximal tibiae of 3-month-old Smurf1 transgenic mice and wild-type littermates (n = 10) (b). The bone volume was normalized to tissue volume (TV), and in Smurf1 transgenic mice, a 33% decrease in bone volume was observed compared with their wild-type littermates (b, c). *, p < 0.05, unpaired t test. d and e, BFR are decreased in Col1a1-Smurf1 transgenic mice. Bone formation rates were measured and calculated in the same area as bone volume was measured using the Osteomeasure system (Osteometrics Inc., Atlanta, GA). BFR was significantly decreased in Smurf1 transgenic mice compared with littermate control mice. *, p < 0.05, unpaired t test. f, ALP activity is reduced in bones of Col1a1-Smurf1 transgenic mice. Bone samples from Smurf1 transgenic mice and wild-type littermates were fixed in 70% ethanol and embedded in methylmethacrylate without prior decalcification. ALP activity was reduced in transgenic mice compared with their wild-type littermates.

Fig.4. Smurf1 inhibits osteoblast proliferation and differentiation in Col1a1-Smurf1 transgenic mice

a, osteoblast numbers are decreased in Col1a1-Smurf1 transgenic mice. Osteoblast numbers were counted in the same area as bone volume and bone formation rates were measured. A significant decrease in osteoblast numbers was found in Smurf1 transgenic mice. *, p < 0.05, unpaired t test. b and c, osteoblast proliferation was reduced in Col1a1-Smurf1 transgenic mice. BrdUrd labeling was performed using a BrdUrd assay kit (Zymed Laboratories Inc.). BrdUrd-positive cells on the periosteal surface of calvariae were counted and normalized to the total periosteal cell numbers. A significant decrease in BrdUrd-positive cells was found in Smurf1 transgenic mice. *, p < 0.05, unpaired t test. d, ALP activity is decreased in primary osteoblasts isolated from calvariae of Col1a1-Smurf1 transgenic mice. ALP activity in primary osteoblasts isolated from Smurf1 transgenic mice and their wild-type littermates was measured. In osteoblasts from Smurf1 transgenic mice, ALP activity was significantly decreased. *, p < 0.05, unpaired t test. e, mineralized bone nodule formation was inhibited in primary osteoblasts of Col1a1-Smurf1 transgenic mice. Primary osteoblasts isolated from Col1a1-Smurf1 transgenic mice and their wild-type littermates were cultured for 10 days in the absence or presence of BMP-2 (50 ng/ml). BMP-2-induced mineralized bone nodule formation was inhibited in osteoblasts isolated from Smurf1 transgenic mice.