BMPR-II mRNA Research Articles

Primary Pulmonary Hypertension Is Associated With Reduced Pulmonary Vascular Expression of Type II Bone Morphogenetic Protein Receptor

Mutations in the type II receptor for bone morphogenetic protein (BMPR-II), a receptor member of the transforming growth factor-beta (TGF-beta) superfamily, underlie many familial and sporadic cases of primary pulmonary hypertension (PPH). Because the sites of expression of BMPR-II in the normal and hypertensive lung are unknown, we studied the cellular localization of BMPR-II and the related type I and II receptors for TGF-beta by immunohistochemistry in lung sections from patients undergoing heart-lung transplantation for PPH (n=11, including 3 familial cases) or secondary pulmonary hypertension (n=6) and from unused donor lungs (n=4). In situ hybridization was performed for BMPR-II mRNA. Patients were screened for the presence of mutations in BMPR2. In normal lungs, BMPR-II expression was prominent on vascular endothelium, with minimal expression in airway and arterial smooth muscle. In pulmonary hypertension cases, the intensity of BMPR-II immunostaining varied between lesions but involved endothelial and myofibroblast components. Image analysis confirmed that expression of BMPR-II was markedly reduced in the peripheral lung of PPH patients, especially in those harboring heterozygous BMPR2 mutations. A less marked reduction was also observed in patients with secondary pulmonary hypertension. In contrast, there was no difference in level of staining for TGF-betaRII or the endothelial marker CD31. The cellular localization of BMPR-II is consistent with a role in the formation of pulmonary vascular lesions in PPH, and reduced BMPR-II expression may contribute to the process of vascular obliteration in severe pulmonary hypertension.

Seven BMPs and all their receptors are simultaneously expressed in osteosarcoma cells

Members of the bone morphogenetic protein (BMP) family and their receptors (BMPRs and activin receptors-ActRs) promote the development of bones with a fine regulation of their expression. Mutations in BMPs or BMPRs cause several diseases, as shown in knockout mice, such as skeletal defects, familial primary pulmonary hypertension and neoplasias. Osteosarcoma is the most frequent primary malignant tumor of bone. Due to their importance in bone development, BMPs, BMPRs and ActRs could also play a role in osteosarcoma growth and development. Previous data have shown that the overexpression of the BMPR-II was related to poor prognosis in malignant and metastatic bone tumors. We evaluate by reverse transcription-linked polymerase chain reaction analysis (RT-PCR) the expression pattern of BMPs, BMPRs and ActRs in five different human osteosarcoma cell lines (MG63, G292, HOS, SaOS and U2). Moreover, we performed the mutational screening of the complete BMPR-II mRNA by automated sequencing of the correspondent cDNA to evaluate the presence of point mutations in osteosarcoma cell lines. All the osteosarcoma cell lines studied simultaneously expressed the BMPs, BMPRs and ActRs investigated. No mutations were detected in the BMPR-II cDNA. Our results suggest the presence of a mechanism involving the simultaneous activation of the BMPs and their receptors in osteosarcoma cell lines.

Temporal expression patterns of BMP receptors and collagen II (B) during periosteal chondrogenesis

Articular cartilage has a limited ability to repair itself. Periosteal grafts have chondrogenic potential and are used clinically to repair defects in articular cartilage. An organ culture model system for in vitro rabbit periosteal chondrogenesis has been established to study the molecular events of periosteal chondrogenesis in vitro. In this model, bone morphogenetic protein-2 (BMP2) mRNA expression was found to be upregulated in the first 12 h. BMPs usually transduce their signals through a receptor complex that includes type II and either type IA or type IB BMP receptors. Receptors IA and IB play distinct roles during limb development. We have examined the temporal expression patterns for the mRNAs of these receptors using our experimental model. The mRNA expression patterns of these three BMP receptors differed from one another in periosteal explants during chondrogenesis. When these explants were cultured under chondrogenic conditions (agarose suspension with TGF-β1 added to the media for the first 2 days), the expression of BMPRII mRNA and that of BMPRIA mRNA varied only slightly and persisted over a long time. In contrast, the expression of BMPRIB mRNAwas upregulated within 12 h, peaked at day 5, and fell to a level that was barely detected beyond day 21. Moreover, the expression of BMPRIB mRNA preceded that of collagen type IIB mRNAs, a marker for matrix-depositing chondrocytes. These data support a role for coordinate expression of BMP2 and its receptors early during periosteal chondrogenesis.

Distribution of genes for bone morphogenetic protein-4, -6, growth differentiation factor-5, and bone morphogenetic protein receptors in the process of experimental spondylosis in mice.

Because little is known about the molecular mechanisms underlying the process of spondylosis, the authors examined the extent of genetic localization of several members of bone morphogenetic protein (BMP) and BMP receptors in chondrogenesis during the process of inducing spondylosis in their previously established experimental mice model. Experimental spondylosis was induced in 5-week-old ICR mice. The cervical spine was harvested chronologically, and histological sections were prepared. Messenger RNA for BMP-4, growth and differentiation (GDF)-5, BMP-6, and BMP receptors (ALK-3, -6, and BMP-RII) was localized in the tissue sections by in situ hybridization. In the early stage, BMP-4-derived mRNA was localized mainly in cells in the anterior margin of the cervical discs, together with ALK-6 and BMP-RII mRNA. No GDF-5 and BMP-6 mRNA was detected at this stage. In the late stage, cells positive for BMP-4 decreased, whereas GDF-5 and BMP-6 mRNA were localized in cells undergoing chondrogenesis. The ALK-3 mRNA began to appear in this stage, as did ALK-6 and BMP-RII. The localization of transcripts for BMP-4, -6, and GDF-5 as well as BMP receptors shown during the present experimental model indicate the possible involvement of molecular signaling by these BMPs in the chondrogenic progress in spondylosis.

Expression and Regulation of Type I BMP Receptors during Early Avian Sympathetic Ganglion Development

We have investigated the expression and regulation of the mRNAs for the type I BMP receptors, BMPR-IA and BMPR-IB, in quail embryos in vivo and in neural crest cultures in vitro. BMPR-IB mRNA was expressed in the primordial sympathetic ganglia at stage 17, soon after the first expression of Cash-1 mRNA, the avian homolog of the Drosophila transcription factor achaete-scute. BMP-4 mRNA was detected in the dorsal aorta at stage 17, coincident with BMPR-IB mRNA expression in the sympathetic ganglia. BMPR-IA mRNA was first expressed in the sympathetic ganglia at stage 18. Moreover, BMP-4 ligand mRNA was detected in the sympathetic ganglia starting at stage 18. BMPR-IA and BMPR-IB were differentially regulated in cultured neural crest cells. BMPR-IB was expressed in primary outgrowths of neural crest cells but was downregulated after primary outgrowths were harvested and replated in secondary cultures. In secondary cultures of neural crest cells, exogenous BMP-2 and BMP-4 increased the expression of BMPR-IA but decreased the expression of BMPR-IB. The expression of both type I BMP receptors was inhibited by exogenous TGF-β1. Our results suggest distinct roles for BMPR-IA and BMPR-IB in the development of the sympathoadrenal phenotype from cells of the neural crest.

Osteogenic protein-1 differentially regulates the mRNA expression of bone morphogenetic proteins and their receptors in primary cultures of osteoblasts.

The mRNA expression patterns of several bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in long-term primary cultures of fetal rat calvaria (FRC) cells were examined by Northern analysis. Their temporal orders of expression were correlated with those of several biochemical markers characteristic of osteoblastic cell differentiation. Distinct temporal patterns of expression of BMPs and BMPRs during osteoblastic cell differentiation were observed. BMP-2 and BMP-7 mRNA levels did not change significantly. BMP-4 mRNA expression increased and reached a peak prior to matrix formation. BMP-5 mRNA expression increased during the mineralization phase and BMP-6 mRNA expression increased throughout all phases of cell differentiation. Effects of BMP-7 (Osteogenic Protein-1; OP-1) on the expression patterns of several other members of the BMP family and the receptors were also studied. OP-1 downregulated the BMP-4, -5, and -6 mRNA levels by a maximal of 2-fold, 1.5-fold, and 6-fold, respectively. OP-1 did not change significantly the OP-1 and BMP-2 mRNA expression. Of the three type I BMPR examined, OP-1 upregulated ActR-I and BMPR-IA mRNA expression slightly but with statistical significance. OP-1 downregulated BMPR-IB mRNA expression slightly. OP-1 upregulated BMPR-II mRNA expression by a maximum of 2-fold. Our findings demonstrate that OP-1 differentially regulates the mRNA expression of several related members of the BMP family and their receptors in osteoblasts. The observations suggest that OP-1 action on osteoblastic cells involves a complex regulation of gene expression of related members of the BMP family and their receptors in a cell differentiation stage dependent manner.

Differential expression of BMP receptors in early mouse development.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of polypeptide signaling molecules. They function via binding to two types of transmembrane serine/threonine kinase receptors, type I and type II receptors, that are both necessary for signaling. The expression patterns of the type II BMP receptor (BMPR-II) and three type I BMP receptors (ActR-I, BMPR-IA and BMPR-IB) were examined in preimplantation embryos by means of heminested reverse transcription-polymerase chain reaction (RT-PCR). BMPR-II mRNA was detected in one-cell, two-cell and blastocyst stage embryos. ActR-I exhibited a similar expression pattern. BMPR-IA mRNA however was only detected in blastocysts, whereas BMPR-IB transcripts were detected at all stages from the one-cell zygote to the uncompacted morula, but not in the compacted morula and blastocyst. If translated into proteins, this suggests that different receptor complexes can be formed at different developmental stages. Transcripts for BMPs were not detected in preimplantation embryos, but were detected in the maternal tissues surrounding the embryos. BMPR-II, BMPR-IA and BMPR-IB mRNAs were also detected in undifferentiated and differentiated embryonal carcinoma and embryonic stem cells. In postimplantation embryos BMPR-II transcripts were first detected from 6.0 days post coitum. In situ hybridization analysis revealed that BMPR-II mRNA is ubiquitously expressed in the entire embryo at least until midgestation.

A Kinase Domain-truncated Type I Receptor Blocks Bone Morphogenetic Protein-2-induced Signal Transduction in C2C12 Myoblasts

Members of the transforming growth factor (TGF)-beta superfamily bind the transmembrane serine/threonine kinase complex consisting of type I and type II receptors. Their intracellular signals are propagated via respective type I receptors. Bone morphogenetic protein (BMP)-2, a member of the TGF-beta superfamily, induces ectopic bone formation when implanted into muscular tissues. Two type I receptors (BMPR-IA and BMPR-IB) have been identified for BMP-2. We have reported that BMP-2 inhibits the terminal differentiation of C2C12 myoblasts and converts their differentiation pathway into that of 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 involvement of functional BMP-2 type I receptors in signal transduction in C2C12 cells, which expressed mRNA for BMPR-IA, but not for BMPR-IB in Northern blotting. TGF-beta type I receptor (TbetaR-I) mRNA was also expressed in C2C12 cells. Subclonal cell lines of C2C12 that stably expressed a kinase domain-truncated BMPR-IA (DeltaBMPR-IA) differentiated into myosin heavy chain-expressing myotubes but not into alkaline phosphatase (ALP)-positive cells, even in the presence of BMP-2. In contrast, the differentiation of the DeltaBMPR-IA-transfected C2C12 cells into myotubes was suppressed by TGF-beta1, as in the parental C2C12 cells. BMP-2 did not efficiently suppress the mRNA expression of muscle-specific genes such as muscle creatine kinase, MyoD, and myogenin, nor did it induce the expression of ALP mRNA in the DeltaBMPR-IA-transfected C2C12 cells. In contrast, TGF-beta1 inhibited mRNA expression of the muscle-specific genes in those cells. When wild-type BMPR-IA was transiently transfected into the DeltaBMPR-IA-transfected C2C12 cells, a number of ALP-positive cells appeared in the presence of BMP-2. Transfection of wild-type BMPR-IB or TbetaR-I failed to increase the number of ALP-positive cells. These results suggest that the BMP-2-induced signals, which inhibit myogenic differentiation and induce osteoblast differentiation, are transduced via BMPR-IA in C2C12 myoblasts.

218 BMPR-II mRNA in the rat brain

218 BMPR-II mRNA in the rat brain

Molecular Cloning of Rat Bone Morphogenetic Protein (BMP) Type IA Receptor and Its Expression During Ectopic Bone Formation Induced by BMP

A cDNA for the rat bone morphogenetic protein (BMP) type IA receptor (BMPR-IA) was isolated from a dental pulp cell cDNA library. The rat BMPR-IA cDNA encodes a protein of 532 amino acids with a single transmembrane domain and a putative serine/threonine kinase domain. The overall amino acid sequence identity between the rat and human BMPR-IA was 97 %. Reverse transcriptase-polymerase chain reaction analysis revealed that BMPR-IA mRNA was highly expressed in the BMP-induced bone forming tissues throughout the stages tested.