We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and <em>bone</em> formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic beta subunits of the 20S proteasome stimulated <em>bone</em> formation in <em>bone</em> organ cultures in concentrations as low as <em>10</em> nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor-1 increased <em>bone</em> volume and <em>bone</em> formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on <em>bone</em> formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed <em>bone</em> formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 <em>protein</em>. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and <em>bone</em> formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of <em>bone</em> loss.

BACKGROUND

Osteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.

METHODS

To investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13fx/fx (Mmp13Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were <em>10</em> weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on <em>10</em>-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.

RESULTS

The OA progression was decelerated in Mmp13Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (>85%) or bone morphogenetic protein 2 (BMP2)-treated (>90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.

CONCLUSIONS

Mmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.

Periostin was originally isolated as a osteoblast-specific factor that functions as a cell adhesion molecule for preosteoblasts and is thought to be involved in osteoblast recruitment, attachment and spreading. Additionally, periostin expression has previously been shown to be significantly increased by both transforming growth factor beta-1(TGFbeta1) and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2. Likewise the endocardial cushions that form within embryonic heart tube (embryonic day (E)<em>10</em>-13) are formed by the recruitment, attachment and spreading of endocardial cells into the overlying extracellular matrix, in response to secreted growth factors of the TGFbeta and BMP families. In order to determine whether periostin is similarly involved in heart morphogenesis, in situ hybridization and reverse transcription-polymerase chain reaction were used to detect periostin mRNA expression in the developing mouse heart. We show for the first time that periostin mRNA is expressed in the developing mouse embryonic and fetal heart, and that it is localized to the endocardial cushions that ultimately divide the primitive heart tube into a four-chambered heart.

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -<em>10</em>, and -18, hepatocyte growth factor, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-<em>10</em>); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding <em>proteins</em>. Octasaccharides with the high affinity to FGF-2 and FGF-<em>10</em> had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS.

BACKGROUND

Recombinant human bone morphogenetic proteins (rhBMPs) can induce bone formation, but the inability to identify an ideal delivery system limits their clinical application. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone-marrow cells, which allow delivery of the BMP-2 to a specific anatomical site. The autologous BMP-2-producing bone-marrow cells then were used to heal a critical-sized femoral segmental defect in syngeneic rats.

METHODS

Femoral defects in five groups of rats were filled with 5 x 10(6) BMP-2-producing bone-marrow cells, created through adenoviral gene transfer (twenty-four femora, Group I); twenty micrograms of rhBMP-2 (sixteen femora, Group II); 5 x 10(6) beta-galactosidase-producing rat-bone-marrow cells, created through adenoviral gene transfer of the lacZ gene (twelve femora, Group III); 5 x 10(6) uninfected rat-bone-marrow cells (ten femora, Group IV); or guanidine hydrochloride-extracted demineralized bone matrix only (ten femora, Group V). Guanidine hydrochloride-extracted demineralized bone matrix served as a substrate in all experimental groups. Specimens that were removed two months postoperatively underwent histological and histomorphometric analysis as well as biomechanical testing.

RESULTS

Twenty-two of the twenty-four defects in Group I (BMP-2-producing bone-marrow cells) and all sixteen defects in Group II (rhBMP-2) had healed radiographically at two months postoperatively compared with only one of the thirty-two defects in the three control groups (beta-galactosidase-producing rat-bone-marrow cells, uninfected rat-bone-marrow cells, and guanidine hydrochloride-extracted demineralized bone matrix alone). Histological analysis of the specimens revealed that defects that had received BMP-2-producing bone-marrow cells (Group I) were filled with coarse trabecular bone at two months postoperatively, whereas in those that had received rhBMP-2 (Group II) the bone was thin and lace-like. Defects that had been treated with bone-marrow cells producing beta-galactosidase (Group III), uninfected bone-marrow cells (Group IV), or guanidine hydrochloride-extracted demineralized bone matrix only (Group V) demonstrated little or no bone formation. Histomorphometric analysis revealed a significantly greater total area of bone formation in the defects treated with the BMP-2-producing bone-marrow cells than in those treated with the rhBMP-2 (p = 0.036). Biomechanical testing demonstrated no significant differences, with the numbers available, between the healed femora that had received BMP-2-producing bone-marrow cells and the untreated (control) femora with respect to ultimate torque to failure or energy to failure.

CONCLUSIONS

This study demonstrated that BMP-2-producing bone-marrow cells created by means of adenoviral gene transfer produce sufficient protein to heal a segmental femoral defect. We also established the feasibility of ex vivo gene transfer with the use of biologically acute autologous short-term cultures of bone-marrow cells.

BACKGROUND

Most patients with primary pulmonary hypertension are thought to have sporadic, not inherited, disease. Because clinical disease develops in only <em>10</em> to 20 percent of persons carrying the gene for familial primary pulmonary hypertension, we hypothesized that many patients with apparently sporadic primary pulmonary hypertension may actually have familial primary pulmonary hypertension.

METHODS

In a study conducted over 20 years, we developed a registry of 67 families affected by familial primary pulmonary hypertension. Through patient referrals, extensive family histories, and correlation of family pedigrees, we discovered shared ancestry among five subfamilies. We established the diagnosis of primary pulmonary hypertension by direct evaluation of patients and review of autopsy material and medical records. We assessed some family members for mutations in the gene encoding bone morphogenetic protein receptor II (BMPR2), which has recently been found to cause familial primary pulmonary hypertension.

RESULTS

We linked five separately identified subfamilies that included 394 known members spanning seven generations, which were traced back to a founding couple in the mid-1800s. Familial primary pulmonary hypertension has been diagnosed in 18 family members, 12 of whom were first thought to have sporadic disease. The conditions of 7 of the 18 were initially misdiagnosed as other cardiopulmonary diseases. Six members affected with familial primary pulmonary hypertension and 6 of <em>10</em> at risk for carriage have been undergone genotype analysis, and they have the same mutation in BMPR2, a transversion of thymine to guanine at position 354 in exon 3.

CONCLUSIONS

Many cases of apparently sporadic primary pulmonary hypertension may be familial. Failure to detect familial primary pulmonary hypertension results from incomplete expression within families, skipped generations, and incomplete family pedigrees. The recent discovery of mutations in BMPR2 should make it possible to identify those with susceptibility to disease.

METHODS

In vivo study of the effect of injection of osteogenic protein-1 (OP-1) on a rabbit anular needle puncture model of intervertebral disc (IVD) degeneration.

OBJECTIVE

To study radiographic, magnetic resonance imaging (MRI), biochemical, and histologic changes in the rabbit IVD after injection of OP-1 into the nucleus pulposus in a needle puncture disc degeneration model.

BACKGROUND

Growth factors, such as OP-1, have the ability to stimulate synthesis of proteoglycans and collagen in vitro. The in vivo injection of OP-1 into the normal rabbit IVD has increased disc height and proteoglycan content in the anulus fibrosus and nucleus pulposus. However, to our knowledge, no attempts have yet been made to determine the effects of these growth factors in an in vivo model of disc degeneration.

METHODS

New Zealand adolescent white rabbits (n = 90, 8 for baseline evaluation, 82 at 8 times) received an anular puncture in 2 noncontiguous discs with an 18-gauge needle to induce disc degeneration. Four weeks later, either 5% lactose (10 microL) or OP-1 (100 microg in 10 microL 5% lactose) was injected into the center of the nucleus pulposus. The disc height was followed radiographically for up to 24 weeks after the injections. At the 2, 4, 8, 12, and 24-week times after the injection, rabbits were euthanized, and MRI of the harvested spinal columns was obtained to grade the degeneration. The discs injected with OP-1 or lactose and noninjected discs were subjected to biochemical and histologic analysis. The specimens at the 24-week time were limited to histologic evaluation.

RESULTS

The anular puncture with a needle induced a consistent disc narrowing within 4 weeks. The injection of OP-1 induced a restoration of disc height at 6 weeks, which was sustained for the entire experimental period, up to 24 weeks after the injection. The injection of lactose alone did not change the course of disc narrowing over the same time. MRI grading score showed significant differences between the OP-1 and lactose groups at the 8, 12, and 24-week times, suggesting an increase in water content in the nucleus pulposus of the OP-1 group. The proteoglycan content of the nucleus pulposus and anulus fibrosus was significantly higher in the OP-1 group than in the control group. The degeneration grades of the punctured discs in the OP-1 group were significantly lower than those in the lactose group.

CONCLUSIONS

The results of this study show the feasibility of restoring degenerative rabbit discs by a single injection of OP-1 into the nucleus pulposus. Importantly, the effects of the OP-1 injection on disc height were sustained for up to 24 weeks. The metabolic changes in the cells, following a single injection, might be sustained and, thus, induce long-term changes in disc structure. An efficacy study in large animals is required to show further that the intradiscal injection of OP-1, or bone morphogenetic proteins or growth factors with similar properties would be useful for the structural restoration of the IVD in humans.

We have reported previously that Noggin is a heparin-binding <em>protein</em> and associates with the cell surface through heparan sulfate proteoglycans, where it remains functional for the binding of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs). Here we report that the binding of Noggin to the cell surface is highly selective for heparan sulfate and that specific structural features are required for the interaction. Noggin binds most efficiently to heparin sequences composed of <em>10</em> or more monosaccharides; N-, 6-O-, and 2-O-sulfates contribute to this interaction. In addition, we have shown that the developmentally regulated endosulfatase Qsulf1 selectively removes sulfate groups from the 6-O position of sugars within the most highly sulfated S domains of heparan sulfate, whereas 6-O-sulfates in the NA/NS domains are not substrates for the enzyme. The activity of Qsulf1 in cells in culture results in the release of Noggin from the cell surface and a restoration of BMP responsiveness to the cells. This shows that Noggin binds to the S domains of heparan sulfate and provides evidence that, in addition to modulating Wnt signaling in vivo by the release of heparan sulfate bound Wnt, Qsulf1 also modulates BMP signaling by the release of surface-bound Noggin.

Both latent transforming growth factor-beta (TGF-beta)-binding <em>proteins</em> fibrillins are components of microfibril networks, and both interact with members of the TGF-beta family of growth factors. Interactions between latent TGF-beta-binding <em>protein</em>-1 and TGF-beta and between fibrillin-1 and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) are mediated by the prodomain of growth factor complexes. To extend this information, investigations were performed to test whether stable complexes are formed by additional selected TGF-beta family members. Using velocity sedimentation in sucrose gradients as an assay, complex formation was demonstrated for BMP-7 and growth and differentiation factor-8 (GDF-8), which are known to exist in prodomain/growth factor complexes. Comparison of these results with complex formation by BMP-2, BMP-4 (full-length and shortened propeptides), BMP-<em>10</em>, and GDF-5 allowed us to conclude that all, except for BMP-2 and the short BMP-4 propeptides, formed complexes with their growth factors. Using surface plasmon resonance, binding affinities between fibrillin and all propeptides were determined. Binding studies revealed that the N-terminal end of fibrillin-1 serves as a universal high affinity docking site for the propeptides of BMP-2, -4, -7, and -<em>10</em> and GDF-5, but not GDF-8, and located the BMP/GDF binding site within the N-terminal domain in fibrillin-1. Rotary shadowing electron microscopy of molecules of BMP-7 complex bound to fibrillin-1 confirmed these findings and also showed that prodomain binding targets the growth factor to fibrillin. Immunolocalization of BMP-4 demonstrated fibrillar staining limited to certain tissues, indicating tissue-specific targeting of BMP-4. These data implicate the fibrillin microfibril network in the extracellular control of BMP signaling and demonstrate differences in how prodomains target their growth factors to the extracellular space.

BACKGROUND

Autogenous iliac crest bone is the gold-standard graft for spinal fusion surgery. Unfortunately, there is a frequent incidence of graft site pain that persists well into the postoperative period with complication rates reported in 2.8-39% of patients. Persistent pain lasting at least 2 years is reported in 15-39% of patients.

OBJECTIVE

The objective of this work was to determine the incidence of acute and persistent pain as well as patient assessment of graft site appearance following iliac crest bone graft harvest for anterior lumbar interbody fusion (ALIF). The control arms of four randomized prospective multicenter clinical trials evaluating recombinant human bone morphogenetic protein-2 (rhBMP-2) versus autogenous iliac crest bone graft were combined. Two hundred eight patients underwent iliac crest bone graft harvest for ALIF in threaded cylindrical cages or threaded bone dowels. Patients were assessed at each postoperative visit with questionnaires evaluating graft site pain intensity, duration, and appearance.

METHODS

Two hundred eight patients underwent iliac crest bone graft harvest for ALIF as the control group of four randomized prospective multicenter clinical trials evaluating rhBMP-2 versus autogenous iliac crest bone graft in threaded cylindrical cages or threaded bone dowels. Most patients in the control group had anterior iliac crest graft harvest, equally from the right and left side. Three grafts (1.4%) were taken from the posterior crest and six (2.9%) were tricortical. Follow-up was obtained at hospital discharge, 6 weeks, 3, 6, 12, and 24 months. A pain evaluation score was made up of two Visual Analog Scales: one scale measuring intensity, the other frequency of pain. For the intensity scale, a rating of "0" meant no pain and "10" was "as bad as it could be." For the frequency scale, a rating of "0" meant pain was present "none of the time" and "10" meant it was present "all the time." Combining these scales, a total score of 20 indicated the worst pain was present all the time. Patients were also questioned about the graft site appearance.

RESULTS

Two hundred eight patients underwent iliac crest harvest, and prospective data were available on 202 patients. At hospital discharge, 2 patients (1%) had no pain; this increased to 34 of 199 (17%) at 6 weeks postoperatively and to 85 of 199 patients (43%) at 3 months. However, 41% of patients reported pain at 6 months postoperatively (79/192), and 33% of 168 reported pain at 1 year. One hundred forty-one of 208 patients completed a survey at 24 months, with 31% reporting some level of pain. At hospital discharge, the graft site pain score ranged from 0 to 20 with a mean of 12.8, decreasing to 7.3 at 6 weeks, to 3.8 at 3 months, and to 2.9, 2.4, and 1.8 at 6, 12, and 24 months, respectively. At all time intervals, P values from t tests comparing the mean with 0 were <0.001. Graft site appearance at discharge was good in 49% of patients, fair in 40%, and poor in 11%. At 6 weeks, appearance was good in 69%, fair in 27%, and poor in 5%; at 3 months, it was good in 75%, fair in 24%, and poor in 2%; at 6 months, it was good in 82%, fair in 15%, and poor in 3%; at 12 months, it was good in 82% and fair or poor in 19%; and at 24 months, it was good in 84% and fair or poor in 16%. There was no significant difference between posterior and anterior harvest sites or bicortical grafts, and all were included in the analysis. Right or left side demonstrated no differences.

CONCLUSIONS

This is the first study known to the authors presenting results of prospective data collected in a multicenter study evaluating iliac crest harvest site pain, both intensity and frequency, as well as graft site appearance. The results demonstrate that 31% of patients had persistent pain at 24 months postoperatively and 16% reported fair or poor appearance of their graft site.

CONCLUSIONS

Persistent donor site pain remains a problem with harvest of autogenous iliac crest bone graft for spinal fusion. This prospective study, the first such study reported for ALIF, confirms that donor site pain remains a significant postoperative management problem.

Multipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the alpha3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to <em>10</em> of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, <em>protein</em>uria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice.

Adult vertebrates require a continuous supply of osteoblasts for both <em>bone</em> remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the stem cells within the fibroblastic component of <em>bone</em> marrow stroma. Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the <em>bone</em>-specific matrix <em>proteins</em> osteocalcin and <em>bone</em> sialo<em>protein</em> and the capacity for matrix mineralization. We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured <em>bone</em> marrow stromal cells. Osteoblastic differentiation was induced by continuous culture with <em>10</em>(-8) M dexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well as osteopontin, <em>bone</em> sialo<em>protein</em>, and osteocalcin mRNA by Day 8 of culture; coaddition of <em>10</em>(-8) M 1,25-dihydroxyvitamin D3 (vitamin D) with dex was essential for high osteocalcin mRNA expression. Recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs. Using in situ hybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4. Quantitation of cell surface AP <em>protein</em> by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone. When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix. Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers. These data demonstrate that stem cells within the stromal compartment of <em>bone</em> marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.

The TGF-beta family of growth factors has been extensively studied and found to play major roles in <em>bone</em> physiology and disease. A novel, TGF-beta-inducible early gene (TIEG) in normal human fetal osteoblasts (hFOB) has been identified using differential-display PCR. Using this differentially expressed cDNA fragment of TIEG to screen a hOB cDNA library, a near full-length cDNA for this gene was isolated. Northern analyses indicated that the steady-state levels of the 3.5 kb TIEG mRNA increased within 30 min of TGF-beta treatment of human osteoblasts and reached a maximum of <em>10</em>-fold above control levels at 120 min post-treatment. This regulation was independent of new <em>protein</em> synthesis. Computer sequence analyses indicates that TIEG mRNA encodes for a 480 amino-acid <em>protein</em>. The TIEG <em>protein</em> contains three zinc finger motifs, several proline-rich src homology-3 (SH3) binding domains at the C-terminal end, and is homologous in this region to the zinc finger-containing transcription factor family of genes. A growth factor/cytokine-specific induction of TIEG has been shown. TIEG expression in hFOB cells was highly induced by TGF-beta and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2), with a moderate induction by epidermal growth factor (EGF), but no induction by other growth factors/cytokines was observed. In addition to osteoblastic cells, high levels of TIEG expression were detected in skeletal muscle tissue, while low or no detectable levels were found in brain, lung, liver or kidney. Because TIEG is an early induced putative transcription factor gene, and shows a growth factor induction and tissue specificity, its <em>protein</em> product might play an important role as a signalling molecule in osteoblastic cells.

Recent studies have indicated a critical role for vascular endothelial growth factor (VEGF) during the process of endochondral ossification, in particular in coupling cartilage resorption with <em>bone</em> formation. Therefore, we studied the chemoattractive and proliferative properties of human VEGF-A on primary human osteoblasts (PHO) and compared these data with the effects of human basic fibroblast growth factor (bFGF) and human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2). Furthermore, initial experiments were carried out to characterize VEGF-binding <em>proteins</em> on osteoblastic cells possibly involved in the response. For the first time, to our knowledge, we could demonstrate a chemoattractive effect of VEGF-A, but not VEGF-E, on primary human osteoblasts. The effect of VEGF-A was dose-dependent and did not reach a maximum within the concentration range tested (up to <em>10</em> ng/mL). The maximal effect observed was a chemotactic index (CI) of 2 at a concentration of <em>10</em> ng/mL. bFGF and BMP-2 exhibited maxima at 1.0 ng/mL with CI values of 2.5 and 2, respectively. In addition to its effect on cell migration, VEGF-A stimulated cell proliferation by up to 70%. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of VEGF receptors VEGFR-1 (Flt-1), VEGFR-2 (Kdr), and VEGFR-3 (Flt-4), as well as neuropilin-1 and -2. An in vitro kinase assay failed to demonstrate activation of VEGFR-2 upon stimulation with either VEGF-E or VEGF-A, consistent with the idea that the effect of VEGF-A on primary human osteoblasts is mediated via VEGFR-1. Taken together, our data establish that human osteoblasts respond to VEGF-A, suggesting a functional role for this growth factor in <em>bone</em> formation and remodeling.

Osteoporotic fractures are a major cause of morbidity and mortality in ageing populations. Osteoporosis, defined as low <em>bone</em> mineral density (BMD) and associated fractures, have significant genetic components that are largely unknown. Linkage analysis in a large number of extended osteoporosis families in Iceland, using a phenotype that combines osteoporotic fractures and BMD measurements, showed linkage to Chromosome 20p12.3 (multipoint allele-sharing LOD, 5.<em>10</em>; p value, 6.3 x <em>10</em>(-7)), results that are statistically significant after adjusting for the number of phenotypes tested and the genome-wide search. A follow-up association analysis using closely spaced polymorphic markers was performed. Three variants in the <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) gene, a missense polymorphism and two anonymous single nucleotide polymorphism haplotypes, were determined to be associated with osteoporosis in the Icelandic patients. The association is seen with many definitions of an osteoporotic phenotype, including osteoporotic fractures as well as low BMD, both before and after menopause. A replication study with a Danish cohort of postmenopausal women was conducted to confirm the contribution of the three identified variants. In conclusion, we find that a region on the short arm of Chromosome 20 contains a gene or genes that appear to be a major risk factor for osteoporosis and osteoporotic fractures, and our evidence supports the view that BMP2 is at least one of these genes.

Coordinated growth and differentiation of external genitalia generates a proximodistally elongated structure suitable for copulation and efficient fertilization. The differentiation of external genitalia incorporates a unique process, i.e. the formation of the urethral plate and the urethral tube. Despite significant progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes for external genitalia. The sonic hedgehog (Shh) gene and its signaling genes have been found to be dynamically expressed during murine external genitalia development. Functional analysis by organ culture revealed that Shh could regulate mesenchymally expressed genes, patched 1 (Ptch1), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (Bmp4), Hoxd13 and fibroblast growth factor <em>10</em> (Fgf<em>10</em>), in the anlage: the genital tubercle (GT). Activities of Shh for both GT outgrowth and differentiation were also demonstrated. Shh(-/-) mice displayed complete GT agenesis, which is compatible with such observations. Furthermore, the regulation of apoptosis during GT formation was revealed for the first time. Increased cell death and reduced cell proliferation of the Shh(-/-) mice GT were shown. A search for alterations of Shh downstream gene expression identified a dramatic shift of Bmp4 gene expression from the mesenchyme to the epithelium of the Shh mutant before GT outgrowth. Regulation of mesenchymal Fgf<em>10</em> gene expression by the epithelial Shh was indicated during late GT development. These results suggest a dual mode of Shh function, first by the regulation of initiating GT outgrowth, and second, by subsequent GT differentiation.

Endoglin (CD<em>10</em>5), a transmembrane <em>protein</em> of the transforming growth factor β superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to <em>bone</em> <em>morphogenetic</em> <em>proteins</em> 9 and <em>10</em> (BMP9 and BMP<em>10</em>) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the <em>protein</em>. The BMP9/BMP<em>10</em> binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the <em>bone</em> effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat <em>bone</em> marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the statin was observed at relatively low doses (significant at <em>10</em>(-8) M and maximal at <em>10</em>(-7) M). Northern blot analysis showed that the statin (<em>10</em>(-7) M) increased in <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. Simvastatin (<em>10</em>(-7) M) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 14 and 22 of culture. These results indicate that simvastatin has anabolic effects on <em>bone</em> through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic <em>bone</em> diseases such as osteoporosis.

We have shown that osteogenic <em>protein</em>-1 (OP-1) (<em>bone</em> <em>morphogenetic</em> <em>protein</em>-7) is responsible for the induction of nephrogenic mesenchyme during embryonic kidney development. Gene knock-out studies showed that OP-1 null mutant mice die of renal failure within the first day of postnatal life. In the present study, we evaluated the effect of recombinant human OP-1 for the treatment of acute renal failure after 60 min bilateral renal artery occlusion in rats. Bioavailability studies in normal rats indicate that approximately 1.4 microg OP-1/ml is available in the circulation 1 min after intravenous administration of 250 microg/kg, which then declines steadily with a half life of 30 min. About 0.5% of the administered OP-1 dose/g tissue is targeted for OP-1 receptors in the kidney. We show that OP-1 preserves kidney function, as determined by reduced blood urea nitrogen and serum creatinine, and increased survival rate when administered <em>10</em> min before or 1 or 16 h after ischemia, and then at 24-h intervals up to 72 h after reperfusion. Histochemical and molecular analyses demonstrate that OP-1: (a) minimizes infarction and cell necrosis, and decreases the number of plugged tubules; (b) suppresses inflammation by downregulating the expression of intercellular adhesive molecule, and prevents the accumulation and activity of neutrophils; (c) maintains the expression of the vascular smooth muscle cell phenotype in pericellular capillaries; and (d) reduces programmed cell death during the recovery. Collectively, these data suggest that OP-1 prevents the loss of kidney function associated with ischemic injury and may provide a basis for the treatment of acute renal failure.

Retinoic acid has profound effects on vertebrate limb morphogenesis (refs 1-6, reviewed in refs 7-9), including in the mouse, where it can act as a teratogen generating phocomelia and <em>bone</em> defects. A retinoic acid gradient, possibly amplified by a graded distribution of cellular retinoic acid-binding <em>protein</em> (CRABP), could provide positional information across the antero-posterior axis of the chick limb bud. The discovery of nuclear retinoic acid receptors (RARs) acting as retinoic acid-inducible enhancer factors provided a basis for understanding how retinoic acid signals could be transduced at the level of gene expression. We have now used in situ hybridization to study the distribution of messenger RNA transcripts of the three murine receptors (mRARs) and CRABP during mouse limb development. Both mRAR alpha and mRAR gamma transcripts, but not those for mRAR beta, are present and uniformly distributed in the limb bud at day <em>10</em> post-coitum, whereas CRABP transcripts have a graded proximo-distal distribution, indicating that differential expression of CRABP, but not of mRAR alpha or mRAR gamma, could participate in the establishment of the <em>morphogenetic</em> field. At later stages, mRAR gamma transcripts become specific to the cartilage cell lineage and to the differentiating skin and mRAR beta transcripts are mostly restricted to the interdigital mesenchyme. CRABP transcripts, however, are excluded from regions expressing mRAR gamma and mRAR beta. These results indicate that all three RARs and CRABP have specific functions during morphogenesis and differentiation of the mouse limb.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt <em>bone</em> metastases in patients with>> or = <em>10</em> years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc(+) breast cancer cells under basal and transforming growth factor-beta (TGF-beta)-stimulated conditions. In addition, exogenous addition of BMP7 to TGF-beta-stimulated MDA-231 cells inhibited Smad-mediated TGF-beta signaling. Furthermore, in a well-established <em>bone</em> metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic <em>bone</em> metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (<em>10</em>0 mug/kg/d) significantly inhibited orthotopic and intra<em>bone</em> growth of MDA-231-B/Luc(+) cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a <em>bone</em> metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in <em>bone</em>, BMP7 may represent a novel therapeutic molecule for repression of local and <em>bone</em> metastatic growth of breast cancer.

<em>Proteins</em> in the TGF-beta superfamily transduce their effects through binding to type I and type II serine/threonine kinase receptors. Osteogenic <em>protein</em>-1 (OP-1, also known as <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 or BMP-7), a member of the TGF-beta superfamily which belongs to the BMP subfamily, was found to bind activin receptor type I (ActR-I), and BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB) in the presence of activin receptors type II (ActR-II) and type IIB (ActR-IIB). The binding affinity of OP-1 to ActR-II was two- to threefold lower than that of activin A. A transcriptional activation signal was transduced after binding of OP-1 to the complex of ActR-I and ActR-II, or that of BMPR-IB and ActR-II. These results indicate that ActR-II can act as a functional type II receptor for OP-1, as well as for activins. Some of the known biological effects of activin were observed for OP-1, including growth inhibition and erythroid differentiation induction. Compared to activin, OP-1 was shown to be a poor inducer of mesoderm in Xenopus embryos. Moreover, follistatin, an inhibitor of activins, was found to inhibit the effects of OP-1, if added at a <em>10</em>-fold excess. However, certain effects of activin, like induction of follicle stimulating hormone secretion in rat pituitary cells were not observed for OP-1. OP-1 has overlapping binding specificities with activins, and shares certain but not all of the functional effects of activins. Thus, OP-1 may have broader effects in vivo than hitherto recognized.

TGF-beta family <em>proteins</em> with a cystine knot motif serve as ligands for diverse families of plasma membrane receptors. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) antagonists represent a subgroup of these <em>proteins</em>, some of which bind BMPs and antagonize their actions during development and morphogenesis. Availability of completed genome sequences from diverse organisms allows bioinformatic analysis of the evolution of BMP antagonists and facilitates their classification. Using a regular expression algorithm (http://BioRegEx.stanford.edu), an exhaustive search of the human genome identified all cystine knot-containing BMP antagonists. Based on the size of the cystine ring, these <em>proteins</em> were divided into three subfamilies: CAN (eight-membered ring), twisted gastrulation (nine-membered ring), as well as chordin and noggin (<em>10</em>-membered ring). The CAN family can be divided further into four subgroups based on a conserved arrangement of additional cysteine residues-gremlin and PRDC, cerberus and coco, and DAN, together with USAG-1 and sclerostin. We searched for orthologs of human BMP antagonists in the genomes of model organisms and analyzed their phylogenetic relationship. New human paralogs were identified together with the verification of orthologous relationships of known genes. We also discuss the physiological roles of the CAN subfamily of BMP antagonists and the associated genetic defects. Based on the known three-dimensional structure of key cystine knot <em>proteins</em>, we postulated disulfide bondings for eight-membered ring BMP antagonists to predict their potential folding and dimerization.

OBJECTIVE

The finding of bone morphogenetic protein (BMP) receptor 1a mutations in juvenile polyposis suggests that BMPs are important in colorectal cancer (CRC). We investigated the BMP pathway in sporadic CRC.

METHODS

We investigated BMP receptor (BMPR) expression using immunoblotting and sequenced BMPR2 in CRC cell lines. We assessed the expression of BMPRs, SMAD4, and pSMAD1/5/8 in 72 sporadic CRCs using a tissue microarray and immunohistochemistry. We assessed the effect of reintroduction of wild-type BMPR2 on BMP pathway activity and the effect of wild-type or mutated BMPR2 3' untranslated region (UTR) sequences on protein expression by attachment to pCMV-Luc.

RESULTS

BMPR2 and SMAD4 protein expression is abrogated in microsatellite unstable (MSI) and microsatellite stable (MSS) cell lines, respectively. BMPR2 3'UTR is mutated in all MSI and in none of the MSS cell lines. Mutant BMPR2 3'UTR sequences reduced luciferase expression 10-fold compared with wild-type BMPR2 3'UTR. BMPR2 expression is impaired more frequently in MSI CRCs than MSS (85% vs 29%; P < .0001) and shows a mutually exclusive pattern of impaired expression compared with SMAD4. Nine of 11 MSI cancers with impaired expression of BMPR2 have microsatellite mutations. The BMP pathway is inactivated, as judged by nuclear pSMAD1/5/8 expression, in 70% of CRCs, and this correlates with BMPR and SMAD4 loss.

CONCLUSIONS

Our data suggest that the BMP pathway is inactivated in the majority of sporadic CRCs. In MSI CRC this is associated predominantly with impaired BMPR2 expression and in MSS CRC with impaired SMAD4 expression.