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Our laboratory previously showed that osteoactivin (OA) is a novel, osteoblast-related glycoprotein that plays a role in osteoblast differentiation and function. The purpose of this study was to examine the regulation of OA expression by BMP-2 and the role OA plays as a downstream mediator of BMP-2 effects in osteoblast function. Using primary osteoblast cultures, we tested different doses of BMP-2 on the regulation of OA expression during osteoblast development. To test whether Smad-1 signaling is responsible for BMP-2 regulation of OA expression, osteoblast cultures were transfected with Smad1 siRNA, treated with 50 ng/ml of BMP-2 and analyzed by Western blot. BMP-2 treatment increased OA mRNA and protein expression in a dose-dependent manner and this upregulation was blocked in Smad1 siRNA transfected cultures. We next examined whether the role of OA as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Osteoblast cultures were transfected with OA antisense oligonucleotides and treated with 50 ng/ml of BMP-2. Cultures transfected with OA antisense oligonucleotides and treated with BMP-2 showed a reduction of OA expression associated with a significant reduction in early and late differentiation markers induced by BMP-2. Therefore, OA acts, at least in part, as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Our findings suggest that BMP-2 regulates OA expression through the Smad1 signaling pathway. Our data also emphasize that OA protein acts as a downstream mediator of BMP-2 effects on osteoblast differentiation and function. J. Cell. Physiol. 210: 26–37, 2007. © 2006 Wiley-Liss, Inc.
Osteoactivin (OA) is a novel factor that was initially identified from studies using an animal model of Osteopetrosis (op), the mutation in rats. Using the technique of mRNA differential display, the expression of OA cDNA was highly upregulated in op compared to normal bone (Safadi et al., 2002). OA has high homology to human glycoprotein nmb (gpnmb) (Watermann et al., 1995), and mouse DC-HIL (dendritic cell-associated, heparan sulfate proteoglycan dependent-integrin ligand) (Shikano et al., 2001). It has two isoforms, one is transmembrane type I with a MW of 65 kDa and the other is a secreted glycoprotein with MW of 115 kDa (Safadi et al., 2002). OA was found to be highly expressed in various malignant tumors such as in glioma (Loging et al., 2000), and hepatocellular carcinoma (Onaga et al., 2003). It has been shown that overexpression of OA in glioma cell lines (Rich et al., 2003), as well as in hepatoma cell lines (Onaga et al., 2003), permits tumor invasiveness. The OA protein has been found to modulate osteoblast differentiation and function in vitro by stimulating osteoblast differentiation markers, including alkaline phosphatase (ALP) activity, nodule formation, osteocalcin production, and matrix mineralization, without affecting cell proliferation or viability (Selim et al., 2003).
Bone morphogenetic proteins (BMPs) are secreted growth factors, which form a subgroup of the transforming growth factor (TGF-β) superfamily based on amino acid homology of a highly conserved seven-cysteine domain in the carboxy-terminal region of the proteins (Kingsley, 1994; Eimon and Harland, 1999). BMPs were originally known by their ability to induce ectopic bone and cartilage formation in vivo (Urist, 1965), but recently it became evident that BMPs also act as multifunctional regulators in morphogenesis during development in vertebrates (Wozney, 1998). BMP dimers initiate signaling by binding to both type I and type II serine/threonine kinase receptors and the phosphorylation of type I receptors upon ligand binding (Miyazono et al., 2000). Receptor-regulated Smads (R-Smads) (Smad 1, 5, 8) are activated by type I receptors (BMPR-IA or BMPR-IB) (Kawai et al., 2000), associate with Smad4, and translocate to the nucleus where they interact with transcription factors to regulate the transcription of target genes.
It is known that BMP proteins initiate the cascade of endochondral bone formation where mesenchymal stem cells differentiate into chondrocytes which lay down cartilage that is replaced by bone tissue (Reddi, 1994). BMPs can also act as local factors in the regulation of osteoblast differentiation (Katagiri et al., 1994). Several BMP knockout experiments in mice have contributed to elucidate the role of BMPs in bone formation and development. For example, BMP-2 deficient mice had amnion/chorion malformation and defects in cardiac development, and died during embryonic development (Zhang and Bradley, 1996b). Number of studies have shown that BMP-2, -3, -4,and -7 can upregulate differentiation markers of the mature osteoblast, including short term such as ALP activity, and long-term surrogates such as osteocalcin expression (Zhou et al., 1993). In addition, studies have demonstrated increased expression of osteoblast markers in pluripotent mesenchymal stem cell cultures after stimulation with BMP-2, suggesting that BMPs may regulate specific differentiation pathways in uncommitted cells (Wang et al., 1993).
The similarity in the temporal expression patterns of BMP-2 and OA during osteoblast differentiation and the fact that both of these factors play a role in osteoblast function in vitro led us to examine the relationship between BMP2 and OA in osteoblasts. In this study, we were interested in determining whether BMP-2 regulates the expression of OA through the Smad-1 signaling pathway, and whether OA acts as a downstream mediator of BMP-2 effects on osteoblast development and function.
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- MATERIALS AND METHODS
- LITERATURE CITED
As previously described, OA is homologous to other family members of trans-membrane proteins such as GPNMB (Wetermann et al., 1995), DC-HIL (Shikano et al., 2001), PMEL17, and human growth factor inducible neurokinin (HGFIN) (Metz et al., 2005). These family members play a role in differentiation of multiple cell types, such as DC-HIL in dendritic cells (Shikano et al., 2001), PMEL17 in melanocytes (Berson et al., 2001), and HGFIN in differentiation of lymphohematopoietic stem cells (Bandari et al., 2003). In this report, we examined the regulation of OA expression by BMP-2 during osteoblast differentiation and whether OA modulates BMP-2 effects on early and terminal osteoblast differentiation. The stages of osteoblast development have been well characterized in numerous previous studies. Primary osteoblasts in vitro undergo three distinct stages beginning with cell proliferation (Days 0–7), followed by nodule formation, collagen deposition and matrix maturation (Days 7–14), and ending with osteoblast differentiation and matrix mineralization (Days 14–21) (Aronow et al., 1990; Safadi et al., 2003). The fact that OA and BMP-2 have similar pattern of expression during osteoblast development in culture (see Figs. 1 and 2) and both of these factors have been shown to exhibit an overlapping effect in regulating osteoblast differentiation and function. (Urist, 1965; Selim et al., 2003). Data presented in this report suggest a relationship between OA and BMP-2 in regulating osteoblast function.
The OA protein has two isoforms, one is secreted (glycosylated at 115 kDa) and one is trans-membrane (native at 65 kDa) (Safadi et al., 2002). As primary osteoblasts develop in culture, the secreted isoform of OA reaches its highest level during the terminal differentiation of osteoblasts, while the trans-membrane isoform reaches its lowest levels during terminal differentiation (3 weeks in culture). In support of our findings, another group reported similar findings for DC-HIL, the mouse ortholog of OA (Shikano et al., 2001). They showed that in SX52, a long-term mouse dentritic cell line, DC-HIL is detected in both the cytosolic fraction that represents the secreted isoform and the membranous fraction that represents the transmembrane isoform. They also showed that the transmembrane isoform of DC-HIL mediates the adhesion of SVEC, mouse vascular endothelial cell line. Our group has previously shown that neutralizing the secreted isoform of OA using an anti-osteoactivin antibody inhibited osteoblast differentiation as evidenced by decreasing ALP activity, nodule formation, osteocalcin production, and matrix mineralization (Selim et al., 2003). Thus, these findings indicate possible dual roles for both isoforms of OA during osteoblast differentiation, an adhesion role for the transmembrane isoform and a differentiation role for the secreted isoform. However, more experiments are warranted to explore these possibilities.
The results in this study also showed that expression of both isoforms of OA are upregulated by BMP-2 in a dose-dependent manner, reaching maximum levels at 50 ng/ml of BMP-2. The fact that BMP-2 upregulates the expression of OA isoforms during the different stages of osteoblast development in culture suggests that OA may play a role as a downstream mediator of BMP-2 during osteoblast development in vitro. Other factors have been reported to be regulated by BMP-2 and are key regulators of osteoblast differentiation including, BIG-3 (Gori and Demay, 2005), Runx-2 (Karsenty and Wagner, 2002), and Osterix (Nakashima et al., 2002).
It has been well documented that BMP stimulation of osteoblast cell differentiation is mediated by heterotetrameric serine/threonine kinase receptors and the downstream transcription factors Smad1, 5, 8 (Sykaras and Opperman, 2003). We showed here that OA is regulated by BMP-2 and this regulation is mediated through the Smad-1 signaling pathway. Smad1 is an essential intracellular component that is specifically phosphorylated by BMP receptors and translocated into the nucleus upon ligand stimulation (Yang et al., 2000). Phosphorylation of Smad1 involves serines in the carboxy-terminal motif. These residues are phosphorylated directly by a BMP type I receptor in vitro. Mutation of these carboxy-terminal serines prevents Smad1 association with the related protein, accumulation in the nucleus, and gain of transcriptional activity (Kretzschmar et al., 1997). Transgenic mice expressing the Smad1 domain, termed Smad1C, show increased skeletal bone mineral density compared to their littermates. Bone histomorphometric analysis of transgenic mouse tibiae showed that Smad1C significantly increases trabecular bone area and length of trabecular surface covered with osteoid, and upregulates several osteoblast-related genes in cultured osteoblasts derived from Smad1C transgenic mouse (Liu et al., 2004). Targeted deletion of the Smad1 gene results in early embryonic lethality due to failure of the allantois to fuse to the chorion (Lechleider et al., 2001; Tremblay et al., 2001). In conclusion, our results demonstrate that BMP-2 signaling plays an important role in the regulation of OA expression. By close analysis of the OA promoter, multiple Smad1 binding motifs (CAGAC) (Lopez-Rovira et al., 2002) have been identified. These motifs are located in tandem, 1,442 base pairs upstream from the ATG starting codon (data not shown). Further analysis of the OA promoter by generating deletion constructs of the Smad1 binding motifs will clearly demonstrate the regulation of OA expression by BMP-2. Our study indicates that Smad1 inhibition in osteoblast cultures by Smad1 siRNA at a dose of 50 nM inhibited OA expression, a result that suggests regulation of OA expression by BMP-2 is mediated by (Smad 1) signaling.
We were also interested to examine whether the effects of BMP-2 on early and late osteoblast differentiation are OA dependent. In order to test this possibility, we used an antisense approach that has been shown previously to be effective in blocking different factors in osteoblast and other cell types (Bonnelye et al., 2001; Galindo et al., 2005). Using OA antisense oligonucleotides, we were able to inhibit OA expression significantly in cultures terminated at Day 14 and Day 21. We have also previously shown that neutralizing the constitutively secreted OA protein in primary osteoblast cultures with anti-OA antibody inhibited osteoblast differentiation (Selim et al., 2003). In this study, we demonstrated that short-term treatment of osteoblast cultures with BMP-2 under serum-free condition increased osteoblast ALP production (Day 14), nodule formation and mineralization (Day 21). Similar results were reported by Hay et al. (1999), where short-term treatment with BMP-2 in human neonatal primary osteoblasts stimulated cell differentiation markers. However, when OA expression was blocked in our cultures, BMP-2-induced early and late markers of differentiation were decreased to levels comparable to control. These data suggest that BMP-2-induced osteoblast function is, at least in part, OA dependent.
The mechanism whereby OA acts downstream of BMP-2 effects on osteoblast function is not fully understood. Our findings suggest that OA production is required for BMP-2-mediated osteoblast maturation and mineralization in primary osteoblast cultures. One possible mechanism is that a downregulation of OA expression inhibits osteoblast differentiation markers indirectly. The inhibition of OA expression could activate some BMP-2 antagonistic/inhibitory regulatory pathways that influence osteoblast differentiation. For example, the soluble BMP-2 antagonists such as, noggin, chordin, chordin-like, cerebrus, and gremlin bind BMP-s in the extracellular space and mask receptor binding interfaces for BMP type I and type II receptors (Balemans and Van Hul, 2002; Groppe et al., 2002). Another alternative mechanism whereby OA acts downstream mediator of BMP-2 effects on osteoblast maturation and terminal differentiation could be explained by the fact that the inhibitory effects of OA antisense oligonucleotides could decrease the expression/phosphorylation of regulatory Smads (1, 5, and 8) or increase the expression/phosphorylation of inhibitory Smads (Smad 6 and 7). The latter Smads inhibit signaling by either interacting with phosphorylated BMP type I receptors to prevent activation of receptor-activated Smads (Imamura et al., 1997; Nakao et al., 1997; Souchelnytskyi et al., 1998), or through competition to prevent formation of the receptor-activated Smad/co-Smad complex (Hata et al., 1998). Data from our laboratory showed that transfection of primary osteoblasts with OA antisense oligonucleotides resulted in a dramatic reduction in the amount and the phosphorylation levels of Smad1, 5, 8 and an increase in the amount and phosphorylation levels of Smad-7 (un-published observations), suggesting that OA acts, at least in part, as downstream mediator of BMP-2 actions on osteoblasts through modulating regulatory (Smad1, 5, 8) and inhibitory (Smad7) signaling molecules (Fig. 7).
Figure 7. Schematic diagram of the relationship between BMP-2 and osteoactivin in osteoblasts. BMP-2 dimers bind to serine/threonine receptors and induce phosphorylation (P) of Smad1. P-Smad1 binds to Smad4 in multimeric complex then translocates into nucleus. P-Smad1 might activate the transcription of OA gene through binding to Smad1 response elements in the OA promoter. The secreted OA protein induces alkaline phosphatase activity, nodule formation and matrix mineralization. Smad1 siRNA inhibits the expression of OA through inhibition of Smad1 expression. OA antisense inhibits OA expression that resulting in blocking BMP-2-induced alkaline phosphatase activity, nodule formation, and matrix mineralization.
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Another possibility is that downregulation of OA expression might stimulate other intracellular molecules, such as Smurf1 and Smurf2 (Smad ubiquinitation regulatory factors), which selectively target activated type I receptors and Smad proteins for degradation (Zhu et al., 1999; Kavsak et al., 2000; Zhang et al., 2001). Several transcription factors, such as Runx-2 (Gersbach et al., 2004), and growth factors, such as Wnt3a (Rawadi et al., 2003) modulate at least partially, the effects of BMP-2 on osteoblast differentiation and function. Similar results were presented where connective tissue growth factor (CTGF), a factor that plays a role in osteoblast differentiation in vitro and in vivo (Safadi et al., 2003). CTGF expression is regulated by TGF-β and acts as a downstream mediator of TGF-β-induced effects such as matrix production and differentiation of osteoblasts and other cell types (Qi et al., 2005; Arnott et al., 2006).
In this study, we have examined OA expression and its regulation by BMP-2 and have explored regulatory interaction between these two proteins. We also showed that the effects of BMP-2 on ALP activity, nodule formation and matrix mineralization in osteoblasts are partially mediated through OA protein. Further dissection of the relationship between BMP-2 and OA in osteoblasts will lead to a better understanding of the role of OA in osteoblast differentiation in vitro and bone formation in vivo.