Kuber T. Sampath

Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development

Osteogenic protein-1 (OP-1; BMP-7) is a member of the bone morphogenetic protein subfamily. Because members of the TGF-beta superfamily have a role in tissue development, the distribution of OP-1 expression in developing human embryos (5-8 gestational weeks) and fetuses (8-14 gestational weeks) and mouse (9.5-17.5 gestational days) fetuses was examined. Northern hybridization with specific OP-1 probes revealed two mRNA species of 4 and 2.2 KB. Highest levels of OP-1 mRNA were found in human fetal kidney and heart between 12-14 weeks of gestation. By in situ hybridization, the OP-1 transcripts were found in various tissues, i.e., the ectodermal epithelium of the mouse fore- and hindlimbs, heart, teeth, intestinal epithelium, perichondrium, hypertrophic chondrocytes, and periosteum/osteoblast layer of developing human bones. In kidneys, transcripts were first detected in the epithelium of the branching uretheric buds, whereas at later stages glomeruli were the major site of OP-1 mRNA accumulation. These data suggest that, although OP-1 has been isolated from bone matrix, it may have additional regulatory roles in the morphogenesis and/or function of the kidney, limb bud, tooth, heart, and intestine.

Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro → Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.

Intracellular signaling of osteogenic protein-1 through Smad5 activation

Smad proteins play pivotal roles in the intracellular signaling of the multifunctional transforming growth factor‐β (TGF‐β) family members downstream of serine/threonine kinase type I and type II receptors. Smad2 and Smad3 are specific mediators of TGF‐β and activin, while Smad1 and Smad5 are involved in bone morphogenetic protein‐2 (BMP‐2) and BMP‐4 signaling. Here we report that osteogenic protein‐1 (OP‐1), also termed BMP‐7, binds predominantly to BMPR‐IB in the rat osteoprogenitor‐like cell line, ROB‐C26. Smad1, Smad5, and Smad8, but not Smad2 and Smad3, were found to stably interact with the kinase‐deficient BMPR‐IB after it was phosphorylated by the BMPR‐II kinase. In ROB‐C26 cells, which express Smad2, Smad3, Smad4, and Smad5, OP‐1 was found to stimulate the phosphorylation of Smad5. Whereas transfection of wild‐type Smad5 enhanced the OP‐1‐induced response, transfection of wild‐type Smad2 had no effect on OP‐1 signaling. A Smad5‐2SA mutant, in which the two most carboxy‐terminal serine residues were mutated to alanine residues, was found to act as a dominant negative inhibitor of OP‐1‐induced responses upon its transfection into various cell types, including ROB‐C26 cells, in contrast to ectopic expression of a Smad2‐2SA mutant which was without effect. Smad5, therefore, is a key component in the intracellular signaling of OP‐1. J. Cell. Physiol. 177:355–363, 1998.

Bone morphogenetic protein-7 reduces the severity of colon tissue damage and accelerates the healing of inflammatory bowel disease in rats

Bone morphogenetic protein‐7 (BMP‐7) is a growth and differentiation factor and belongs to the TGF‐β superfamily of proteins. Previous studies have shown an abundant expression of BMP‐7 in the developing intestine and an association with a perturbed BMP/SMAD downstream signaling leading to a malignant phenotype and inflammation in the gut. In the present study, we have evaluated the effect of systemically administered recombinant human BMP‐7 against trinitrobenzenesulfonic (TNBS) acid induced inflammatory bowel disease (IBD) in rats. The TNBS administered rats treated with BMP‐7 have developed much less severe form of colitis based on macroscopic and histological scoring when administered 1.5 h before or 24 h after colitis induction. Bioavailability studies in healthy rats have revealed that significant portion (3.6%) of i.v. administered BMP‐7 is targeted for BMP‐7 receptors in the stomach and ileum, respectively, suggesting its availability to target tissue upon administration. Immunohistochemical and RT‐PCR analyses have shown elevated expression of pro‐inflammatory (IL‐6, TNF‐β, ICAM‐1) and pro‐fibrogenic (TGF‐β) cytokines, and BMP‐7 treatment significantly reduced their expression in the intestine; among which the suppression of IL‐6 appeared to be the most important. Taken together, the results of this study suggest that BMP‐7 plays an important role in the regulation of anti‐inflammatory response in the adult gut tissue. J. Cell. Physiol. 196: 258–264, 2003.

Effects of osteogenic protein‐1 (OP‐1, BMP‐7) on bone matrix protein expression by fetal rat calvarial cells are differentiation stage specific

Bone morphogenetic proteins (BMPs) are a group of cytokines that are characterized by their ability to stimulate osteoblast differentiation and bone formation. However, the influence of BMPs on osteoblastic cells at different stages of differentiation is not known. Since bone matrix proteins are differentially regulated during bone formation we have studied the effects of recombinant human osteogenic protein‐1 (rhOP‐1; BMP‐7) on the expression of these proteins by fetal rat calvarial cells (FRCCs) at discrete stages of osteoblast differentiation. Continuous administration of rhOP‐1 to FRCCs, beginning at confluence (day 7), produced a dose‐dependent increase in the number, size and mineralization of bone‐like nodules formed in the presence of vitamin C and β‐glycerophosphate. Within 9 h of administration, rhOP‐1 stimulated a 3‐fold increase in OPN mRNA which was reflected in a comparable increase in the low phosphorylated, 55 kDa form of osteopontin. In contrast, changes in type I collagen, alkaline phosphatase and bone sialoprotein mRNAs followed the differentiation of preosteoblastic cells, and were increased 2‐, 4‐ and 5‐fold, respectively, after 8 days (day 15). When administered at intermediate stages of osteoblast differentiation (days 12, 15 and 18) BSP remained refractory to rhOP‐1 whereas the ALP was increased almost 2‐fold, independent of the constitutive levels of mRNA expression. To determine the effects on osteoblasts, FRCCs were first grown to the bone nodule‐forming stage (day 21) before rhOP‐1 was administered. Only modest, transient increases in the expression of ALP and OPN mRNAs were evident whereas OC expression was increased more than 3‐fold. In contrast, collagen type I and BSP mRNA levels were not changed significantly. These results suggest that rhOP‐1 increases bone formation by promoting osteoblastic differentiation, as indicated by the increased number of bone forming colonies and by increasing the number of osteoblastic cells in the colonies, but not by increasing matrix production by individual osteoblasts. It is also evident that the regulation of bone matrix proteins by rhOP‐1 is dependent upon the differentiated state of the cell.

Characterization of proliferating human skeletal muscle‐derived cells in vitro: Differential modulation of myoblast markers by TGF‐β2

Adult human skeletal muscle‐derived cells (HuSkMC) propagated in vitro are under investigation as a cell‐based therapy for the treatment of myocardial infarction. We have characterized HuSkMC with respect to cell identity and state of differentiation as a prerequisite to their clinical use. Flow cytometric analysis of propagated HuSkMC revealed a population of cells that expressed the myoblast markers CD56 and desmin. The presence of myoblasts in these cultures was further confirmed by their capacity to form myotubes and increase creatine kinase activity when cultured in low serum conditions. The non‐myoblast fraction of these propagated cells expressed TE7, a marker associated with the fibroblast phenotype. Spontaneous differentiation of myoblasts occurred during serial propagation of HuSkMC, as judged by myotube formation, thereby reducing the myoblast representative fraction with continued cell expansion. We examined transforming growth factor β2 (TGF‐β2) for its utility in controlling this spontaneous differentiation of adult human myoblasts in vitro. Propagation of HuSkMC in the presence of 1 ng/ml TGF‐β2 for 5 days decreased desmin expression within the myoblast population and caused a parallel reduction of creatine kinase activity. CD56 expression was unaffected, indicating a differential regulation of these myoblast markers. The reduction in desmin expression and creatine kinase activity was, however, reversible upon the removal of TGF‐β. These data collectively indicate that TGF‐β2 restrained differentiation of adult human skeletal myoblasts during propagation without causing irreversible loss of the myoblast phenotype, demonstrating the potential utility of using TGF‐β2 during cultivation and expansion of HuSkMC intended for therapeutic implantation.

Functional antagonism between activin and osteogenic protein‐1 in human embryonal carcinoma cells

Activin A and osteogenic protein‐1 (OP‐1) exerted antagonistic effects on each others responses on the human Tera‐2 embryonal carcinoma cell line. OP‐1 dose dependently inhibited activin A‐induced activation of p3TP‐Lux transcriptional reporter, containing part of the human plasminogen activator inhibitor‐1 (PAI‐1) promoter, while activin A inhibited OP‐1‐mediated alkaline phosphatase induction. Approximately equimolar concentrations of both growth factors resulted in 50% inhibition of the respective biological responses. Affinity cross‐linking studies using 125I‐activin A or 125I‐OP‐1 followed by receptor‐immunoprecipitations revealed that both ligands bound to the activin type II receptor (ActR‐II), but recruited different type I receptors. In addition, OP‐1 competed with binding of 125I‐activin A, and activin A competed with binding of 125I‐OP‐1 to ActR‐II. Transient transfection studies showed that competition between activin A and OP‐1 also occurred at the type I receptor (ActR‐1) level; constitutively active (CA)‐ActR‐I inhibited CA‐ActR‐IB‐mediated p3TP‐Lux reporter induction. There was no competition between activin A and OP‐1 for availability of Smad4, indicating that the concentration of this common signal transducer is not limiting for generating the observed biological responses. Overexpression of ActR‐II abolished the inhibitory effect of OP‐1 on activin A‐induced p3TP‐Lux activation and, surprisingly, led to OP‐1‐induced transcriptional reporter activity. Whereas the exact mechanism of competition is unclear, the role of ActR‐II in the competition between activin A and OP‐1 is discussed in light of the observed interference in downstream signaling by CA‐ActR‐I and CA‐ActR‐IB. J. Cell. Physiol. 180:141–149, 1999.

Bone Morphogenetic Proteins: From Local to Systemic Therapeutics

Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been commercially available in the United States since July 2002. It was initially approved for use in anterior lumbar interbody fusions with an interbody cage. It has been further approved for two additional clinical indications: fresh tibial fractures and certain oral maxillofacial procedures. Because of the compression resistance limitations of the ACS carrier, especially in challenging clinical environments where soft tissue compression is unavoidable, a secondgeneration rhBMP-2 carrier was developed. This carrier was termed the Compression Resistant Matrix (CRM). The CRM carrier is a composite sponge consisting of cross-linked Type I bovine collagen impregnated with biphasic calcium phosphate ceramic granules (15% hydroxyapatite [HA] and 85% β-tricalcium phosphate [β-TCP]). This chapter focuses on the identification, characterization, and pre-clinical testing of the rhBMP-2/CRM bone graft substitute.

Osteogenic Protein-1 Is Produced by Human Fetal Trophoblasts In Vivo and Regulates the Synthesis of Chorionic Gonadotropin and Progesterone by Trophoblasts In Vitro

Osteogenic protein-1 (OP-1/BMP-7), a member of the transforming growth factor (TGF-beta) superfamily of proteins, is shown to be expressed at sites of epithelial-mesenchymal tissue interaction during human fetal development. In the present study, we examined the expression of OP-1 in human placentas (5-11 weeks of gestation and full term) using in situ hybridization and immunohistochemistry methods. The results show that OP-1 was expressed in cytotrophoblasts (Langhans layer) of the chorionic villi in early and full term placentas. Employing highly purified cytotrophoblast cultures which fused to form functional syncytiotrophoblasts, we showed that exogenously added recombinant OP-1, in the presence of low serum concentration, reduced the secretion of chorionic gonadotropin and progesterone by 60% and 36%, respectively, compared with control cultures. The results suggest that osteogenic protein-1 is synthesized locally by cytotrophoblasts and may be involved in the regulation of human pregnancy by controlling reproductive hormonal secretion.

Bone morphogenetic protein-7 from serum of pregnant mice is available to the fetus through placental transfer during early stages of development.

Background: BMP-7 is an important mediator of metanephric mesenchyme differentiation during kidney development. Gene knockout studies have shown that BMP-7 null mutation mice die shortly after birth due to renal failure, although the induction of metanephric structures has initially occurred (E11–E13). Materials and Methods: Iodinated BMP-7 was injected into the tail vein of pregnant mice and its availability to tissues and fetuses was further analyzed by tissue uptake, LM autoradiography and SDS-PAGE electrophoresis. Results: Studies on the distribution of 125I-BMP-7 injected through the tail vein of pregnant mice indicated that 125I-BMP-7 passed across the placenta and localized in developing fetal organs, in particular kidneys, up to day 14 of gestation. At later stages of pregnancy 125I-BMP-7 did not pass the trophoblasts of the placental barrier and did not enter the fetal blood vessels. Conclusion: The analysis of the distribution of 125I-BMP-7 from pregnant mice to fetal organs, in particular the kidney, suggests a cross-over of maternal circulating BMP-7 to the fetus through the placental barrier.