Molecular cloning of rat efp: expression and regulation in primary osteoblasts.

We have previously identified an estrogen-responsive gene, efp (estrogen-responsive finger protein), by genomic binding-site cloning method. Here, we isolated a rat homologue of efp cDNA that encodes an open reading frame of 644 amino acids sharing high homology with human efp (69% identity at the protein level) and mouse efp (80% identity at the protein level). The efp protein has a RING finger, a variant type of zinc finger motif, B1 box and B2 box, each having a pair of zinc fingers, and coiled-coil domain, belonging to the RING finger-B box-Coiled Coil (RBCC) family. Several members of RBCC family including efp have characteristic C-terminal domain, forming a subfamily. Next, we detected efp mRNA in primary osteoblasts, one of estrogen target cells, derived from the calvariae of rat fetus. An anti-efp antibody revealed the efp protein is expressed and regulated by estrogen in the primary osteoblasts. Interestingly, the efp protein in primary osteoblasts is down-regulated by 1alpha,25-dihydroxyvitamin D(3) treatment that promotes the differentiation of the cells, whereas it is up-regulated by TGF-beta1 treatment that inhibits the differentiation of the cells. These findings suggest the possible involvement of the efp in the differentiation of osteoblastic cells.     

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12

When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-beta1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis, as a major TGF-beta1-responsive element binding protein induced by TGF-beta1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-beta1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-beta1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-beta1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-beta and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.     

A surface component on GH3 pituitary cells that recognizes transforming growth factor-beta, activin, and inhibin

We have examined the ability of various forms of activin and inhibin, which are structurally related to transforming growth factor-beta (TGF-beta), to interact with various types of cell surface TGF-beta binding sites. Activin AB, inhibin A, and inhibin B were unable to compete with 125I-TGF-beta 1 for binding to the TGF-beta receptor types I, II, or III that coexist in human skin fibroblasts, rat liver epithelial cells, and mink lung epithelial cells. In contrast, activins and inhibins effectively competed for TGF-beta 1 binding to GH3 rat pituitary tumor cells. Binding of TGF-beta 1 to GH3 cells was mediated by about 2700 sites/cell with a Kd = 90 pM. Affinity labeling of these GH3 binding sites by cross-linking to 125I-TGF-beta 1 yielded 70-74-kDa labeled complexes distinct from previously identified TGF-beta binding components. Labeling of these 70-74-kDa components with 125I-TGF-beta 1 was inhibited by TGF-beta 1, TGF-beta 2, activin AB, and inhibin B at concentrations in the high picomolar to low nanomolar range, but it was not significantly affected by other polypeptide hormones and growth factors tested. The 70-74-kDa labeled GH3 components represent a novel type of cell surface TGF-beta binding protein that is unique in its ability to recognize various other members of the TGF-beta family of bioactive polypeptides.     

Aldosterone promotes fibronectin production through a Smad2-dependent TGF-beta1 pathway in mesangial cells

Accumulating evidence demonstrates that aldosterone can cause extra-cellular matrix (ECM) accumulation, in addition to regulating sodium and potassium homeostasis. Increased extra-cellular matrix production by renal glomerular mesangial cells has been suggested to be involved in pathogenesis of glomerular sclerosis. The present studies examine whether aldosterone is also produced in renal mesangial cells, and the effect of aldosterone on ECM accumulation in these cells. In cultured renal mesangial cells, aldosterone synthase (CYP11B2), mineralocorticoid receptor (MR), and 11beta-HSD2 mRNA expressions were detected by RT-PCR. The ability of renal mesangial cells to produce aldosterone was confirmed by directly detecting aldosterone in culture medium via radioimmunoassay. Real-time RT-PCR showed that the expression of CYP11B2 mRNA in mesangial cells was significantly enhanced by AngII (P<0.001) and by potassium (P<0.05). Exposure of the cultured mesangial cells to aldosterone significantly increased fibronectin production from 12.4+/-1.9 to 74.6+/-16.8ng/ml (P<0.05). The aldosterone induced fibronectin production was abolished by aldosterone receptor antagonist spironolactone. Aldosterone also increased the TGF-beta1 reporter luciferase activity from 0.8+/-0.1 to 1.7+/-0.1 (P<0.05). Immunoblot showed TGF-beta1 protein expression was increased following aldosterone treatment. Blocking TGF-beta1 signaling pathway by knocking down Smad2 significantly blunted the aldosterone induced fibronectin production. The present studies indicate that renal mesangial cell is a target of local aldosterone action, which promotes ECM protein fibronectin production via TGF-beta1/Smad2 signaling pathway.     

SPARC inhibits epithelial cell proliferation in part through stimulation of the transforming growth factor-beta-signaling system

Secreted protein, acidic and rich in cysteine (SPARC) is a multifunctional secreted protein that regulates cell-cell and cell-matrix interactions, leading to alterations in cell adhesion, motility, and proliferation. Although SPARC is expressed in epithelial cells, its ability to regulate epithelial cell growth remains largely unknown. We show herein that SPARC strongly inhibited DNA synthesis in transforming growth factor (TGF)-beta-sensitive Mv1Lu cells, whereas moderately inhibiting that in TGF-beta-insensitive Mv1Lu cells (i.e., R1B cells). Overexpression of dominant-negative Smad3 in Mv1Lu cells, which abrogated growth arrest by TGF-beta, also attenuated growth arrest stimulated by SPARC. Moreover, the extracellular calcium-binding domain of SPARC (i.e., SPARC-EC) was sufficient to inhibit Mv1Lu cell proliferation but not that of R1B cells. Similar to TGF-beta and thrombospondin-1, treatment of Mv1Lu cells with SPARC or SPARC-EC stimulated Smad2 phosphorylation and Smad2/3 nuclear translocation: the latter response to all agonists was abrogated in R1B cells or by pretreatment of Mv1Lu cells with neutralizing TGF-beta antibodies. SPARC also stimulated Smad2 phosphorylation in MB114 endothelial cells but had no effect on bone morphogenetic protein-regulated Smad1 phosphorylation in either Mv1Lu or MB114 cells. Finally, SPARC and SPARC-EC stimulated TGF-beta-responsive reporter gene expression through a TGF-beta receptor- and Smad2/3-dependent pathway in Mv1Lu cells. Collectively, our findings identify a novel mechanism whereby SPARC inhibits epithelial cell proliferation by selectively commandeering the TGF-beta signaling system, doing so through coupling of SPARC-EC to a TGF-beta receptor- and Smad2/3-dependent pathway.