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Biomedical subjects

V Rosen

Publications and source records attributed to V Rosen.

At least 37 records · Page 2Linked to original sources
PubMed1997

Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family.

Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF-beta gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis.

Amino Acid Sequence↗
PubMed1997

Bone morphogenetic protein-2 inhibits terminal differentiation of myogenic cells by suppressing the transcriptional activity of MyoD and myogenin.

Bone morphogenetic protein (BMP) is a family of cytokines that induce ectopic bone formation when implanted into muscular tissues. We reported that BMP-2 inhibits the terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells (Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., and Suda, T. (1994) J. Cell Biol. 127, 1755-1766). In the present study, we examined the molecular mechanism of the inhibitory effect of BMP-2 on terminal differentiation of myogenic cells. When either MyoD or myogenin cDNA was introduced into C3H10T1/2 (10T1/2) cells with a muscle-specific CAT reporter containing four copies of the right E-box of muscle creatine kinase (MCK) enhancer, the CAT activity was dose-dependently suppressed by BMP-2. Furthermore, BMP-2 inhibited the terminal differentiation of these subclonal 10T1/2 cells that stably expressed MyoD or myogenin into mature myotubes that expressed myosin heavy chain and troponin T. The differentiation of a subclone of the MyoD-transfected NIH3T3 cells into mature muscle cells was also inhibited by BMP-2. BMP-2 induced alkaline phosphatase activity in 10T1/2-derived, but not in NIH3T3-derived MyoD-transfected cells. These cells constitutively expressed exogenous MyoD and myogenin, which were localized exclusively in the nuclei irrespective of the presence and the absence of BMP-2. However, these cells failed to express the mRNAs of endogenous myogenic factors and MCK when cultured with BMP-2. In the electrophoresis mobility shift assay using nuclear extracts of the myogenic cells, MyoD and myogenin bound to the right E-box in the enhancer region of the MCK gene even in the presence of BMP-2. These results suggest that BMP-2 inhibits the terminal differentiation of myogenic cells by suppressing the transcriptional activity of the myogenic factors.

3T3 Cells↗
PubMed1996

Bone morphogenetic protein-12 and -13 inhibit terminal differentiation of myoblasts, but do not induce their differentiation into osteoblasts.

Effects of bone morphogenetic protein (BMP)-12 and BMP-13, new members of the BMP family which belong to the transforming growth factor (TGF)-beta superfamily, on terminal differentiation of myoblasts were examined in C2C12 and L-6 myoblasts. When the myoblasts were cultured with BMP-12 or BMP-13, the expression of the myosin heavy chain and the formation of multinucleated myotubes mRNA in L-6 cells. The inhibitory effects of BMP-12 and BMP-13 on myogenic differentiation were similar to the effects of BMP-2, though their potencies were lower than BMP-2. Unlike BMP-2, neither BMP-12 nor BMP-13 induced alkaline phosphatase activity in C2C12 myoblasts. The differences in the biological activities of these new BMPs suggest that the intracellular signalling pathway used by BMP-12 and BMP-13 differs from that of BMP-2.

Alkaline Phosphatase↗
PubMed1996

Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6.

The effects of bone morphogenetic protein-2 (BMP-2) on osteoblastic differentiation of bone marrow stromal cells were investigated using two bone marrow stromal cell lines, ST2 and MC3T3-G2/PA6 (PA6). BMP-2 stimulated ALP activity and induced parathyroid hormone (PTH)-dependent production of cAMP in both ST2 and PA6 cells, but these effects were more apparent in ST2 cells than in PA6 cells. BMP-2 induced the production of osteocalcin in ST2 cells, but not in PA6 cells. BMP-4 and BMP-6 stimulated ALP activity in ST2 cells, but the effect of BMP-6 was less marked than that of BMP-2 and BMP-4. BMP-4 induced PTH-dependent cAMP production of cAMP in ST2 cells, but BMP-6 did not. When ST2 cells were transplanted into the peritoneal cavities of athymic mice with BMP-2 in diffusion chambers, these cells generated mineralized bone in the chambers. These results indicate that BMPs induce the differentiation of bone marrow stromal cells into osteoblasts. However, the effects differ among the BMPs and among the types of cell exposed to these proteins.

Alkaline Phosphatase↗
PubMed1996

The effects of aging on the bone inductive activity of recombinant human bone morphogenetic protein-2.

We examined the effects of gain on the ectopic bone-forming ability of recombinant human BMP-2 (rhBMP-2) in rats and investigated the mechanism by which aging might affect this type of bone. Bone formation induced after 12 days of sc implantation of 5 micrograms rhBMP-2 was reduced as animals aged from 1-16 months. The osteocalcin messenger RNA levels of implants also declined in aging animals. When the implant period was doubled, 16-month-old rats formed amounts of bone equivalent to those in 3-month-old rats. Increasing the dose of rhBMP-2 increased bone formation in older rats. To get a response comparable to that seen in 1-month-old rats given 5 micrograms rhBMP-2 for 12 days, 3-month-old rats required 30 micrograms rhBMP-2, whereas 16-month-old rats required 60 micrograms. Treatment with either GH or 1,25-dihydroxyvitamin D3 during the 12-day implantation period returned the bone formation in 16-month-olds rats to that in 3-month-old rats. These studies show that aging blunts rhBMP-2 inducted bone formation in rats. We speculate that the decreased response may be due in part to a decrease in the number of mesenchymal stem cells present in order rats or to a change in the responsiveness of these target cells to rhBMP-2.

Aging↗
PubMed1996

Identification of ovarian granulosa cells as a novel site of expression for bone morphogenetic protein-3 (BMP-3/osteogenin) and regulation of BMP-3 messenger ribonucleic acids by chorionic gonadotropin in cultured human granulosa-luteal cells.

Bone morphogenetic proteins (BMP) belong structurally to the transforming growth factor-beta superfamily comprising several growth and differentiation factors such as inhibin, activin, and Müllerian inhibitory factor that regulate ovarian function. We studied here the potential expression of BMP-2, -3, and -4 messenger RNAs (mRNAs) in isolated human granulosa cells obtained at oocyte retrieval for in vitro fertilization. Freshly isolated granulosa cells were found to express BMP-3 (also known as osteogenin) mRNAs but not those of BMP-2 or -4. The BMP-3 transcripts were detected with RT-PCR amplification followed by Southern blot hybridization, as well as by Northern and dot blot hybridization analyses. To investigate whether BMP-3 mRNAs are hormonally regulated, cultures of human granulosa-luteal (GL) cells were treated with different concentrations of purified human chorionic gonadotropin (hCG) at varying stages of culture. hCG decreased BMP-3 mRNA levels from the first day of the culture up to day 5. Time-dependence studies showed that a clear decrease in BMP-3 mRNA levels was evident at 24 h after hCG treatment, and that the effect of hCG was concentration dependent with 3 ng/mL hCG decreasing significantly (P < 0.05) BMP-3 mRNA levels. Furthermore, the cAMP analog, 8-bromo-cAMP (8-Br-cAMP), which activates protein kinase-A, and 12-0-tetradecanoylphorbol 13-acetate, an activator of protein kinase-C, both markedly decreased BMP-3 mRNA levels in an 8-h treatment. We conclude that: 1) BMP-3 mRNAs are expressed in human granulosa cells; 2) their steady state levels are hormonally regulated in cultured human GL cells as evidenced by the ability of hCG to markedly decrease BMP-3 transcript levels; and (3) that activation of both protein kinase-A-and protein kinase-C-mediated signaling pathways also results in a decrease in BMP-3 mRNA levels in GL cells. We suggest that BMP-3, like several other members of the transforming growth factor-beta superfamily, is a potential local regulator of female gonadal function.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo.

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.

Alkaline Phosphatase↗
PubMed1996

Recombinant human bone morphogenetic protein-2 induces a hematopoietic microenvironment in the rat that supports the growth of stem cells.

In the mammalian bone marrow, stromal components support the growth and differentiation of blood cells. To study this complex system, we used a rat model in which ectopic hematopoietic tissue was induced to form after subcutaneous implantation of recombinant human bone morphogenetic protein (rhBMP-2). We showed that this organoid contained clonogenic precursors of both erythroid and myeloid lineages and progenitors competent to regenerate splenic lymphopoiesis. Furthermore, stem cells derived from ectopic foci conferred both short-term (30 day) and long-term (>6-month) protection in vivo against radiation-induced marrow aplasia. Lead shielding of the ectopic marrow in situ also permitted endogenous recovery of hematopoiesis after sublethal irradiation. Extending previous observations that most fibroblastoid cells of the marrow stain with the anti-ST3 antibody (but minimally with anti-ST4), whereas those growing from nonhematopoietic tissues react with anti-ST4, we found that analogous cells of the ectopic foci stained predominantly with anti-ST3. The ability to induce formation of a hematopoietic microenvironment from mesenchymal precursors may make possible the development of new strategies for the treatment of primary disorders of stem cells and irreversible stromal injury.

Animals↗
PubMed1995

Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells.

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-beta gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.

Adipose Tissue↗
PubMed1995

Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype.

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.

Animals↗
PubMed1995

Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation.

A new member of the transforming growth factor (TGF)-beta superfamily, BMP-9, has recently been identified and shown to be expressed in the developing mouse liver. This report demonstrates that human HepG2 liver tumor cells bind recombinant human BMP-9 (rhBMP-9) with high affinity. Cross-linking analysis indicates that HepG2 cells express two BMP-9 receptors of approximately 54 and 80 kilodaltons, similar in size to the Type I and Type II receptors reported by others for TGF-beta and BMP-4. However, cross-competition experiments demonstrate that the BMP-9 receptors on HepG2 cells do not bind other BMPs or TGF-beta s, indicating that these are novel receptors with binding specificity for BMP-9. In functional studies, rhBMP-9 stimulates HepG2 cell proliferation as indicated by [3H]thymidine incorporation and cell counting assays. A proliferative effect of rh-BMP-9 was also observed on primary rat hepatocytes. In contrast, TGF-beta had no effect on HepG2 cell proliferation and inhibited proliferation in primary hepatocytes. These results suggest that BMP-9, acting through a novel set of receptors, may play a regulatory role in hepatic growth and function.

Animals↗
PubMed1994

A mammalian serine/threonine kinase receptor specifically binds BMP-2 and BMP-4.

Bone morphogenetic proteins (BMPs) are a class of related growth and differentiation factors within the TGF-beta superfamily of proteins which are known to induce cartilage and bone formation in adult animals and to be involved in many inductive events throughout embryonic development. Here we describe the molecular cloning and characterization of a mammalian receptor, CFK-43a, which specifically binds BMP-2 and BMP-4. This molecule is a member of the serine/threonine kinase receptor family which includes receptors for other TGF-beta superfamily members. CFK-43a binds other BMP family members with lower affinity, but does not bind TGF-beta. During embryogenesis, in situ hybridization analysis indicates that CFK-43a mRNA is localized in developing skeletal tissues in a complementary fashion to the transcripts for its ligands.

Amino Acid Sequence↗
PubMed1994

Immunohistochemical detection of bone morphogenetic proteins in bone and soft-tissue sarcomas.

BACKGROUND: Bone morphogenetic proteins (BMPs) are potent inducers of bone formation. Functional and immunohistochemical studies have identified BMPs in a subset of osteosarcomas. In the present study, the authors extend the analysis of BMP expression to other bone and soft tissue sarcomas. METHODS: Monoclonal antibody AbH3b2/17 against human BMP-2 and BMP-4 was used in avidin-biotin-immunoperoxidase assays with frozen sections of bone tumors (71 specimens), soft tissue sarcomas (69 specimens), and normal tissues. RESULTS: Among bone tumors, BMP was detected in osteosarcomas (17 of 29 samples), malignant fibrous histiocytomas (MFHs) (6 of 6), and the spindle cell sarcomatous components of spindle cell (dedifferentiated) chondrosarcomas (4 of 4), but not in conventional chondrosarcomas (0 of 10) or Ewing's sarcomas (0 of 14). Histologic subtypes of osteosarcoma differed for BMP expression, with 8 of 9 fibrohistiocytic, 9 of 13 osteoblastic, and 0 of 5 chondroblastic lesions showing immunostaining. In all BMP-positive bone tumors, immunostaining was localized in the cytoplasm of primitive mesenchymal cells, with little or no staining in tumor matrix and more mature osteoblastic/chondrocytic cells. Among soft tissue sarcomas, MFHs (11 of 12), liposarcomas (5 of 11), leiomyosarcomas (3 of 9), and malignant schwannomas (3 of 8) showed cytoplasmic BMP immunostaining. Synovial sarcomas (0 of 9), rhabdomyosarcomas (0 of 8), and fibrosarcomas (0 of 7) were BMP-negative. All normal human tissues tested, including the tissues of a 16-week-old fetus, lacked BMP immunoreactivity. CONCLUSIONS: Bone morphogenetic protein is expressed in a subset of osteosarcomas, a high proportion of MFHs of bone and soft tissue, and in spindle cell chondrosarcomas. In these tumors, BMP is localized predominantly to the cytoplasm of malignant cells with primitive mesenchymal features; no or little BMP is detected in the more differentiated elements of bone and soft tissue sarcomas. Different histologic types of bone and soft tissue sarcomas may mimic discrete stages of mesenchymal differentiation as defined by BMP expression and histologic criteria.

Antibodies, Monoclonal↗
PubMed1994

Expression of bone morphogenetic proteins in human osteosarcoma. Immunohistochemical detection with monoclonal antibody.

BACKGROUND: Bone morphogenetic proteins (BMP) induce ectopic bone formation in vivo and may play a role in normal bone development. In addition, bone morphogenetic activity, as measured in a bone-forming assay in immunodeficient, athymic nu/nu mice, is present in a proportion of osteosarcomas; this activity, which may be mediated by BMP, is correlated with a poor prognosis. METHODS: The development of a monoclonal antibody against recombinant human BMP-2, AbH3b2/17, has allowed immunohistochemical localization of BMP in tumor tissues. Cryostat sections of osteosarcomas (21 tumor samples), chondrosarcomas (5 samples), and Ewing's sarcomas of bone (5 samples) were examined with AbH3b2/17 using the avidin-biotin-immunoperoxidase method. RESULTS: The authors found AbH3b2/17 immunoreactivity in 12 of the 21 osteosarcoma samples (57% sensitivity) obtained from 20 patients. For one patient, samples of the primary lesion and a subsequent metastasis were tested, and only the latter showed AbH3b2/17 immunoreactivity. The chondrosarcomas and Ewing's sarcomas examined showed no immunoreactivity. In antigen-positive osteosarcomas, AbH3b2/17 immunostaining was localized predominantly in the cytoplasm of tumor cells. Moreover, the proportion of AbH3b2/17-reactive cells varied among osteosarcomas with disparate histologic features. CONCLUSIONS: The authors identified a rapid and widely applicable method for detecting BMP expression in intact tissues, which may complement and enhance the bone-forming assay in nu/nu mice as a prognostic procedure in osteosarcomas.

Adolescent↗
PubMed1994

Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes.

There is growing evidence to suggest that BMPs are among the signals necessary to create the embryonic skeleton, but how these regulatory molecules enter the pathways of embryonic bone formation remains to be defined. The earliest steps of endochondral bone formation, consisting of mesenchymal condensation and chondrogenesis, have been shown to result directly from BMP-2 action. To determine whether the transition from chondrogenesis to osteogenesis occurring later in endochondral bone formation is also the result of BMP activity, we tested the effects of BMP-2 on immortalized endochondral skeletal progenitor cells derived from mouse limb bud. The cell lines established by this process were found to fall into three general categories: undifferentiated skeletal progenitor cells, which in the presence of BMP-2 first express cartilage matrix proteins and then switch to production of bone matrix proteins; prechondroblast-like cells that constitutively express a subset of markers associated with chondrogenesis and, in the presence of BMP-2, shut off synthesis of these molecules and are induced to produce bone matrix molecules; and osteoblast-like cells that are not significantly affected by BMP-2 treatment. These data suggest that BMP-2 initiates the differentiation of limb bud cells into cells of both the cartilage and bone lineages in a sequential manner, making BMP-2 a potent regulator of skeletal cell differentiation.

Aggrecans↗
PubMed1994

Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage.

The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP-2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)-positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.

Alkaline Phosphatase↗