OP-1 cDNA encodes an osteogenic protein in the TGF-beta family.

Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF-beta (transforming growth factor beta) supergene family, DPP (decapentaplegic protein) of Drosophila and Vg-1 (vegetal protein) of Xenopus. Building upon this information we constructed a synthetic consensus gene to use as a probe to screen human genomic libraries. This resulted in the isolation of three interrelated genes. Among these were BMP-2b and BMP-3 which have recently been described by others. The third gene, termed OP-1 (osteogenic protein one), is new and was subsequently shown to encode the human homolog of a major component of bovine osteogenic protein. The genomic clones were used to isolate the corresponding complementary DNA (cDNA) clones. Sequence analysis indicates that OP-1 is a relative of the murine Vgr-1 (Vg-1 related gene). This report describes the cDNA structure and putative amino acid sequence of OP-1.     

Osteogenic protein-2. A new member of the transforming growth factor-beta superfamily expressed early in embryogenesis.

Osteogenic protein-2, OP-2, a new member of the transforming growth factor-beta (TGF-beta) superfamily, closely related to the osteogenic/bone morphogenetic proteins, was discovered in mouse embryo and human hippocampus cDNA libraries. The TGF-beta domain of OP-2 shows 74% identity to OP-1, 75% to Vgr-1, and 76% to BMP-5, hence OP-2 may also have bone inductive activity. The genomic locus of OP-2 has seven exons, like OP-1, and spans more than 27 kilobases (kb). In the C-terminal TGF-beta domain, OP-2 has a unique additional cysteine. Mouse embryos express relatively high levels of OP-2 mRNA at 8 days, two species of 3 and 5 kb. A careful study of mRNA expression of the osteogenic proteins in specific organs revealed discrete mRNA species for BMP-3, BMP-4, BMP-5, and BMP-6/Vgr-1 in lung or liver of young and adult mice. OP-1 is expressed in kidney; however, OP-2 and BMP-2 mRNAs were not detected in any organs studied, suggesting an early developmental role.     

Identification of a novel member (GDF-1) of the transforming growth factor-beta superfamily.

A cDNA clone encoding a new member (designated GDF-1) of the transforming growth factor-beta (TGF beta) superfamily was isolated from a library prepared from day 8.5 mouse embryos. The nucleotide sequence of GDF-1 predicts a protein of 357 amino acids with a mol wt of 38,600. The sequence contains a pair of arginine residues at positions 236-237, which is likely to represent a site for proteolytic processing. The C-terminus following the presumed dibasic cleavage site shows significant homology with the known members of the TGF beta superfamily, matching the other family members at all of the invariant positions, including the seven cysteine residues with their characteristic spacing. GDF-1 is most homologous to Xenopus Vg-1 (52%), but is not likely to be the murine homolog of Vg-1. In vitro translation experiments were consistent with GDF-1 being a secreted glycoprotein. Genomic Southern analysis indicated that GDF-1 may be highly conserved across species. These results suggest that GDF-1 is most likely an extracellular factor mediating cell differentiation events during embryonic development.     

Bone morphogenetic protein: chromosomal localization of human genes for BMP1, BMP2A, and BMP3

Bone morphogenetic protein (BMP) induces endochondral bone formation in vivo. The human genes have been cloned for a group of proteins containing BMP activity (BMP1, BMP2A, and BMP3). Two of the proteins are members of the transforming growth factor-beta supergene family (BMP2A and BMP3), while BMP1 is a novel regulatory protein. Using somatic cell hybrid lines, cDNA probes were used to map BMP1 to chromosome 8, BMP2A to chromosome 20, and BMP3 to the p14-q21 region of chromosome 4. This analysis reveals that the BMP2A and BMP3 genes map to conserved regions between mouse and human, while the BMP1 gene does not. The locations of the BMP genes were found to overlap with the loci for several disorders of cartilage and bone formation.     

Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures.

Osteogenin and related bone morphogenetic proteins are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and bone formation in vivo. The influence of osteogenin, purified from bovine bone, and of recombinant human bone morphogenetic protein-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor I, 10 ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.     

Complementary DNA cloning of the murine transforming growth factor-beta 3 (TGF beta 3) precursor and the comparative expression of TGF beta 3 and TGF beta 1 messenger RNA in murine embryos and adult tissues

Murine transforming growth factor-beta 3 (TGF beta 3) cDNAs were isolated from a TGF beta 2-induced AKR-2B cDNA library. The composite cDNA sequence is 2894 nucleotides long, including 610-nucleotide and 1054-nucleotide 5' and 3' untranslated sequences, respectively. The murine TGF beta 3-coding region is 1230 nucleotides in length and encodes a precursor protein of 410 amino acids, with a 96% peptide sequence identity with the human TGF beta 3 precursor. Examination of TGF beta 1 and TGF beta 3 mRNA levels in adult murine tissues showed that TGF beta 1 mRNA expression is predominant in spleen, lung, and placenta. In contrast, TGF beta 3 RNA was present in substantial amounts in brain, heart, adipose tissue, and testis. TGF beta 3 mRNA is also observed in adult mouse lung and placenta. Both TGF beta 1 and TGF beta 3 RNAs were present in all stages of mouse fetal development studied from 10.5-17.5 days postcoitum, with higher levels observed in the latter stages. The differential expression of these TGF beta genes suggests that the various TGF beta species may have distinct physiological roles in vivo.     

hch-1, a gene required for normal hatching and normal migration of a neuroblast in C. elegans, encodes a protein related to TOLLOID and BMP-1.

Proteins of the tolloid/bone morphogenetic protein (BMP)-1 family play important roles in the differentiation of cell fates. Among those proteins are BMP-1, which plays a role in cartilage and bone formation in mammals, the TOLLOID protein, which is required for the establishment of the dorsoventral axis of Drosophila embryos and BP10/SpAN, which are thought to act in the morphogenesis of sea urchins. These proteins have some properties in common. First, they contain the astacin metalloprotease domain, the CUB domain and the epidermal growth factor-like domain. Second, they are expressed in embryos at stages expected for their role in cell differentiation. Third, at least BMP-1 and TOLLOID are thought to interact with proteins of the transforming growth factor-beta family. We report that the hch-1 gene of the nematode Caenorhabditis elegans encodes a tolloid/BMP-1 family protein. The protein has the characteristic domains common to the tolloid/ BMP-1 family. Like other members of the family, it is expressed in embryos. However, the phenotype of hch-1 mutants shows that it is required for normal hatching and normal migration of a post-embryonic neuroblast. Furthermore, in spite of its expression in embryogenesis, it is not required for the viability of embryos. These results show new functions of the tolloid/BMP-1 family proteins and give insight into their evolution.     

Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissues and embryos

Murine transforming growth factor-beta 2 (TGF-beta 2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5'-untranslated sequence, and encodes a murine TGF-beta 2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3'-untranslated sequence. Five TGF-beta 2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-beta 2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-beta 2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.     

Developmental changes in extracellular matrix messenger RNAs in the mouse placenta during the second half of pregnancy: possible factors involved in the regulation of placental extracellular matrix expression

Expression of procollagens (Col1a1/2, Col3a1, Col4a1/2, Col5a1/2) and fibronectin 1 (Fn1) in the mouse fetal placental tissue was examined during the second half of pregnancy. Ribonuclease protection assays (RPAs) revealed that levels of these mRNAs noticeably increased between Days 10 and 14 of pregnancy, and they remained at relatively constant levels thereafter. In situ hyridization showed that Col1a1 and Col4a1 mainly localized in the labyrinth, whereas Fn1 was expressed mainly in the spongiotrophoblast. Since members of the transforming growth factor-beta (TGFB) superfamily are involved in the regulation of extracellular matrix (ECM) expression in various tissues, mRNA levels of TGFB family members and their binding proteins were also examined by RPAs. Transforming growth factor-beta1-3 (Tgfb1-3), activin subunits (Inhba, Inhbb), follistatin (Fst), and follistatin-like 3 (Fstl3) were expressed in the placenta, whereas significant expression of myostatin (Mstn) was not detected. Although the expression patterns of Tgfb1-3 and Inhba in the placenta suggest possible involvement of TGFBs and activin A in the regulation of placental ECM expression, neither TGFBs nor activin A affected ECM mRNA levels in vitro. On the other hand, hypoxia significantly decreased Col1a1/2 and Col4a1/2 mRNAs in cultured placental cells, and a high-glucose condition significantly increased Col1a1 and Col3a1 mRNAs. Fn1 expression was increased under the high-glucose condition, although hypoxia also increased Fn1 expression to a lesser degree. These data suggest that an increase in oxygen tension and nutrient supply during placentation rather than TGFB family members may be responsible for the increase in the placental ECM mRNA expression.     

Cloning and expression of guinea pig TIMP-2. Expression in normal and hyperoxic lung injury

Tissue inhibitors of metalloproteinases (TIMPs) play a key regulatory role in extracellular matrix remodeling. By screening a lung library with a human TIMP-2 cDNA probe, we have isolated the cDNA corresponding to guinea pig TIMP-2. The 3.5-kb cDNA presents an open reading frame that predicts a protein of 220 amino acids showing 97.2, 96.8, 97.2, and 77.3% overall identity with human, mouse, rat, and chicken TIMP-2, respectively. Guinea pig TIMP-2 cDNA was expressed in CHO-K1 cells, showing a protein with the expected molecular weight and activity. Northern blot analysis revealed TIMP-2 expression in brain, kidney, intestine, spleen, heart, and lung. Transforming growth factor-beta downregulated TIMP-2 mRNA in guinea pig lung fibroblasts, whereas a variety of other stimuli showed no effect. In normal and hyperoxia-exposed lungs, TIMP-2 mRNA was mainly localized in alveolar macrophages and epithelial cells. No quantitative differences were found by Northern blot. These results confirm that TIMP-2 is highly conserved in mammals and largely expressed in lungs.     

Accumulation of a form of p27(Kip1) not associated with Cdk-cyclin complexes in transforming growth factor-beta-arrested Mv1Lu cells

The p27(Kip1) cyclin-dependent kinase inhibitor translocates in response to transforming growth factor-beta to a Cdk2-cyclin E complex inhibiting its catalytic activity, but the p27(Kip1) protein levels are unaffected [1]. We show here that transforming growth factor-beta induces the accumulation of a form of p27(Kip1) representing a subpopulation of total p27(Kip1) in growth-arrested Mv1Lu epithelial cells. The inducible p27(Kip1) is detectable only by a specific p27(Kip1) monoclonal antibody recognizing a native form of p27(Kip1). The increase in this subset of p27(Kip1) correlates with G(1) arrest and withdrawal of the cells from the cycle induced by transforming growth factor-beta, serum starvation, or contact inhibition. In contrast to the majority of p27(Kip1) in the cells, the transforming growth factor-beta-inducible p27(Kip1) is devoid of cyclin-dependent kinase/cyclin interactions. The results indicate that growth arresting treatments induce the accumulation of non-cyclin-dependent kinase-bound p27(Kip1), which may function as a reservoir for inhibition of Cdk2-cyclin E activities.     

Interleukin-1 beta- and forskolin-induced synthesis and secretion of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by transforming growth factor-beta 2.

Interleukin-1 beta and forskolin induce prostaglandin E2 release as well as 14-kDa group II phospholipase A2 gene expression and secretion of the enzyme from rat glomerular mesangial cells. We now report that pretreatment of mesangial cells with transforming growth factor-beta 2 prior to stimulation with interleukin-1 beta or forskolin inhibits the induced release of prostaglandin E2. At the same time the secretion of group II phospholipase A2, measured both as enzyme activity with sn-2-labeled phosphatidylethanolamine as substrate and as enzyme protein in immunoblot experiments, is dose-dependently inhibited by pretreatment of the cells with transforming growth factor-beta 2. Analyses of enzyme activity and enzyme protein levels in the cells indicated that this is not due to inhibition of enzyme secretion with a concomitant increase in cellular levels of the enzyme. Rather, pretreatment of the cells with transforming growth factor-beta 2 largely prevented both the interleukin-1 beta- and the forskolin-induced synthesis of phospholipase A2. This is the first report indicating an inhibition of group II phospholipase A2 gene expression by transforming growth factor-beta 2. In line with those results, transforming growth factor-beta 2 did not induce the synthesis and secretion of group II phospholipase A2. However, under conditions where the interleukin-1 beta-induced expression of group II phospholipase A2 is fully suppressed by transforming growth factor-beta 2, the growth factor itself stimulated prostaglandin E2 synthesis by a mechanism apparently not involving group II phospholipase A2. The immunochemical identification of the inducible and secretable phospholipase A2 from rat mesangial cells as a group II enzyme was confirmed by purification and N-terminal amino acid sequence determination.