Understanding the molecular mechanisms by which cartilage formation is regulated is essential toward understanding the physiology of both embryonic bone development and postnatal bone growth. Although much is known about growth factor signaling in cartilage formation, the regulatory role of noncollagenous matrix proteins in this process are still largely unknown. In the present studies, we present evidence for a critical role of DMP1 (dentin matrix protein 1) in postnatal chondrogenesis. The Dmp1 gene was originally identified from a rat incisor cDNA library and has been shown to play an important role in late stage dentinogenesis. Whereas no apparent abnormalities were observed in prenatal bone development, Dmp1-deficient (Dmp1-/-) mice unexpectedly develop a severe defect in cartilage formation during postnatal chondrogenesis. Vertebrae and long bones in Dmp1-deficient (Dmp1-/-) mice are shorter and wider with delayed and malformed secondary ossification centers and an irregular and highly expanded growth plate, results of both a highly expanded proliferation and a highly expanded hypertrophic zone creating a phenotype resembling dwarfism with chondrodysplasia. This phenotype appears to be due to increased cell proliferation in the proliferating zone and reduced apoptosis in the hypertrophic zone. In addition, blood vessel invasion is impaired in the epiphyses of Dmp1-/- mice. These findings show that DMP1 is essential for normal postnatal chondrogenesis and subsequent osteogenesis.

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.

Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the α1β1 and α2β1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)‐2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP‐2 promoter driving SV40 T‐antigen. These cells require exogenous BMP‐2, as well as ascorbic acid and β‐glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen‐I affect BMP‐2 signaling, function‐perturbing anti‐rat α1 and/or α2 integrin subunit, or anti‐type I collagen (Col‐I), antibodies were added to human recombinant (hr)BMP‐2–treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti‐Collagen‐I or both anti–integrin‐α1 and ‐α2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5–1.8% control) of these cells. In all cases, adding anti‐α1 or anti‐α2 antibodies separately produced partial effects, while their combined effect approached that of anti‐Collagen‐I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR‐IB) showed elevated ALP activity without hrBMP‐2; this constitutive activity was also suppressed by α1 and α2 integrin antibodies and by anti‐Col‐I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR‐IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.