Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS؊/؊) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS؊/؊ femurs showed bone volume and bone formation rate was reduced by up to 45% (P < 0.01) and 52% (P < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P < 0.05) and spinal bone mineral densities (P < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS؊/؊ mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17␤-estradiol and had an attenuated chemotactic response to transforming growth factor-␤. Bone is a vital dynamic connective tissue that has evolved to maintain a balance between its two major functions: provision of mechanical integrity for locomotion and modulation and control of mineral homeostasis. 1 Mineralized bone is continuously resorbed by osteoclasts and new bone is formed by osteoblasts. This process, known as bone remodeling, is highly regulated with maintenance of normal integrity and structure. 2 Systemic hormones including calcitonin, parathyroid hormone, and sex steroids, particularly estrogen, are known to be important regulators of bone cell function. Their effects on bone turnover are in general exerted by activation of local mediators and second messengers present within bone cells. 3 Recent investigations have focused on the role of nitric oxide (NO) as one of these possible local regulators of bone metabolism and bone cell activity. NO is a shortlived radical gas generated from L-arginine by nitric oxide synthase (NOS) isoenzymes. 4 Three distinct isoforms of NOS have been identified: a neuronal form (type I; nNOS) originally isolated from brain, 5 an endothelial form (type III; eNOS) originally isolated from bovine aortic endothelial cells, 6 and an inducible form (type II; iNOS) originally isolated from murine macrophages. 7 Both eNOS and nNOS are expressed constitutively and are characterized by highly regulated rapid but low-output NO production. 4 In contrast the iNOS pathway is generally only activated after stimulation by certain pro-inflammatory cytokines such as interferon-␥, interleukin-1␤, and tumor necrosis factor-␣. The inducible NOS isoform is characterized by production of persistent and high concentrations of NO. 8 There is now am...

Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-␤ (TGF␤) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426 -428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGF␤ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity.Skeletal development and bone formation require coordinated activities of multiple signaling pathways that include bone morphogenetic protein 2 (BMP2) 5 and transforming growth factor-␤ (TGF␤). Transduction of these signals results in the activation of target genes that are essential for bone development. Specific receptor-regulated SMADs (R-SMADs) serve as substrates for the BMP and TGF␤/activin/Nodal receptors. SMAD-1, -2, -3, and -5 transduce, whereas Smad-4 serves as a common partner for all R-SMADs to provide the DNA binding property (1, 2). The structural and functional domains of SMAD proteins are well characterized with binding sites for SMAD ubiquitination-related factor (SMURF) ubiquitin ligases, and phosphorylation sites for several classes of protein kinases (3). The MH2 domain mediates interactions with transcriptional activators and repressors for signal transduction; including co-regulators of skeletal development (4 -7).Several studies suggest that the principle activity of BMP and TGF␤ SMADs for the control of skeletogenesis is mediated by their interaction with RUNX2 (CBFA1/AML3). This runt-related transcription factor is critical for osteogenic lineage commitment and formation of the skeleton (8 -12). Mutations in the human RUNX2 cause cleidocranial dysplasia (13,14). Targeted disruption of Runx2 in mice results in the maturational arrest of osteoblasts and a complete lack of mineralized bone (15-17). The gene regulatory properties of RUNX factors are mediated not only by DNA binding to cognate elements, but also through the formation of selective co-regulatory protein interactions with co-activator and co-repressor proteins (9, 10). The C terminus of RUNX proteins contains a 31-amino acid nuclear matrix targeting si...

The coordinated activity of Runx2 and BMP/TGFbeta-activated Smads is critical for formation of the skeleton, but the precise structural basis for the Runx2/Smad interaction has not been resolved. By deletion mutagenesis, we have defined the Runx2 motif required for physical and functional interaction with either BMP or TGFbeta responsive Smads. Smad responsive transcriptional activity was retained upon deletion of the C-terminus to amino acid (aa) 432 but lost with deletion to aa 391. Thus the Smad interacting domain (SMID) of Runx2 (432-391) is embedded in the well-defined nuclear matrix targeting signal (NMTS) that mediates intranuclear trafficking. The SMID suffices as an interacting module when fused to the heterologous Gal-4 protein. Formation of the Runx2 and Smad complex is dependent on Runx2 phosphorylation through the MAPK signaling pathway, as determined by co-immunoprecipitation studies. We established that all SMID/NMTS deficient Runx2 mutants do not show in situ association with Smad in the nucleus nor do they support BMP2-mediated osteogenic induction of the mesenchymal C2C12 cell line. Thus, we provide direct evidence that the SMID/NMTS domain (391-432) of Runx2 is essential for BMP2-mediated osteoblast differentiation. Our findings suggest that TGFbeta/ BMP2 signaling, MAPK dependent phosphorylation, and Runx2 subnuclear targeting converge to induce the osteogenic phenotype.

BMP2 signaling and RUNX2 regulatory pathways converge for transcriptional control of bone formation in vivo. SMAD proteins are recruited to RUNX2 regulatory complexes via an overlapping nuclear matrix targeting signal/Smad interacting domain sequence (391–432) in Runx2. To establish the contribution of RUNX2-SMAD interaction to osteoblastogenesis, we characterized a number of point mutants. Only a triple mutation of amino acids 426–428 (HTY-AAA) results in loss of RUNX2 interactions with either BMP2- or TGF-β- responsive SMADs and fails to integrate the BMP2/TGF-β signal on target gene promoters. In a Runx2 null cell reconstitution assay, the HTY mutant did not activate the program of osteoblast differentiation (alkaline phosphatase, collagen type 1, osteopontin, bone sialoprotein and osteocalcin) in response to BMP2 signaling. Thus, subnuclear targeting function and formation of a RUNX2-SMAD osteogenic complex are functionally inseparable. Taken together, these studies provide direct evidence that RUNX2 is essential for execution and completion of BMP2 signaling for osteoblast differentiation.

It has been shown previously that osteoblast differentiation and maintenance of bone mass are impaired in endothelial nitric oxide synthase gene knockout mice. The present study shows by analysis of messenger RNA expression that the transcription factor Cbfa-1/Runx-2 and the bone matrix protein osteocalcin, which are fundamental to osteoblast differentiation, are significantly reduced in neonatal calvarial osteoblasts from these gene knockout mice. Expression of these genes could be restored to wild-type levels by exogenous supply of the photoactivatable nitric oxide donor potassium nitrosylpentachlorouthenate, but this was dependent on the timing of its activation and recovery in gene expression was only evident during the latter stages of osteoblast differentiation associated with its mineralizing activity. Calvarial, femoral/pelvic, spinal, and total bone mineral density, together with bone microhardness and expression of osteocalcin in whole femurs, were all reduced significantly in gene knockout mice at 8 weeks of age, but not at 12 weeks, where all of these indices of bone integrity were comparable to wild type. In accordance with these temporal effects, reduced bone mineral density, bone microhardness, and osteocalcin expression could be restored to normal, wild-type values after 21 days in vivo administration of the nitric oxide donor glyceryl trinitrate to 4-week-old endothelial nitric oxide synthase knockout mice, but there was no significant effect in older animals. Taken together, these results further demonstrate the importance of endothelial nitric oxide synthase in the regulation of osteoblast metabolism. In particular, they show that nitric oxide is involved in co-ordinating specific phases of osteoblast differentiation and bone formation: this could be relevant to its therapeutic actions on bone turnover.