Roles of stromal cell RANKL, OPG, and M-CSF expression in biphasic TGF-beta regulation of osteoclast differentiation

To better understand the complex roles of transforming growth factor-beta (TGF-beta) in bone metabolism, we examined the impact of a range of TGF-beta concentrations on osteoclast differentiation. In co-cultures of support cells and spleen or marrow osteoclast precursors, low TGF-beta concentrations stimulated while high concentrations inhibited differentiation. We investigated the influences of TGF-beta on macrophage colony stimulating factor (M-CSF), receptor activator of NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) expression and found a dose dependent inhibition of M-CSF expression. RANKL expression was elevated at low TGF-beta concentrations with a less dramatic increase in OPG. Addition of OPG blocked differentiation at the stimulatory TGF-beta dose. Thus, low TGF-beta concentrations elevated the RANKL/OPG ratio while high concentrations did not, supporting that, at low TGF-beta concentrations, there is sufficient M-CSF and a high RANKL/OPG ratio to stimulate differentiation. At high TGF-beta concentrations, the RANKL/OPG ratio and M-CSF expression were both repressed and there was no differentiation. We examined whether TGF-beta-mediated repression of osteoclasts differentiation is due to these changes by adding M-CSF and/or RANKL and did not observe any impact on differentiation repression. We studied direct TGF-beta impacts on osteoclast precursors by culturing spleen or marrow cells with M-CSF and RANKL. TGF-beta treatment dose-dependently stimulated osteoclast differentiation. These data indicate that low TGF-beta levels stimulate osteoclast differentiation by impacting the RANKL/OPG ratio while high TGF-beta levels repress osteoclast differentiation by multiple avenues including mechanisms independent of the RANKL/OPG ratio or M-CSF expression regulation.     

Upstream stimulatory factor (USF) proteins induce human TGF-beta1 gene activation via the glucose-response element-1013/-1002 in mesangial cells: up-regulation of USF activity by the hexosamine biosynthetic pathway

The hyperglycemia-enhanced flux through the hexosamine biosynthetic pathway (HBP) has been implicated in the up-regulated gene expression of transforming growth factor-beta1 (TGF-beta1) in mesangial cells, thus leading to mesangial matrix expansion and diabetic glomerulosclerosis. Since the -1013 to -1002 region of the TGF-beta1 promoter shows high homology to glucose-response elements (GlRE) formerly described in genes involved in glucose metabolism, we studied the function of the GlRE in the high glucose-induced TGF-beta1 gene activation in mesangial cells. We found that high glucose concentrations enhanced the nuclear amount of upstream stimulatory factors (USF) and their binding to this sequence. Fusion of the GlRE to the thymidine kinase promoter resulted in glucose responsiveness of this promoter construct. Overexpression of either USF-1 or USF-2 increased TGF-beta1 promoter activity 2-fold, which was prevented by mutation or deletion of the GlRE. The high glucose-induced activation of the GlRE is mediated by the HBP; increased flux through the HBP induced by high glucose concentrations, by glutamine, or by overexpression of the rate-limiting enzyme glutamine:fructose-6-phosphate aminotransferase (GFAT) particularly activated USF-2 expression. GFAT-overexpressing cells showed higher USF binding activity to the GlRE and enhanced promoter activation via the GlRE. Increasing O-GlcNAc modification of proteins by streptozotocin, thereby mimicking HBP activation, also resulted in increased mRNA and nuclear protein levels of USF-2, leading to enhanced DNA binding activity to the GlRE. USF proteins themselves were not found to be O-GlcNAc-modified. Thus, we have provided evidence for a new molecular mechanism linking high glucose-enhanced HBP activity with increased nuclear USF protein levels and DNA binding activity and with up-regulated TGF-beta1 promoter activity.     

Sequence specific protein binding to and activation of the TGF-beta 3 promoter through a repeated TCCC motif.

We have previously characterized the TGF-beta 3 promoter and shown that the activity of this promoter is highly variable in different cell types. Although the promoter contains a proximal cAMP responsive element, which is critical to basal and forskolin-induced promoter activity, this element is not responsible for the variable, cell-specific regulation of the promoter. In this paper, we identify a 25 base pair sequence in the proximal region of the TGF-beta 3 promoter that binds a novel DNA-binding protein. This region includes the sequence T-CCCTCCCTCCC, (3 x TCCC), and mutation of these T-CCC repeats inhibits protein binding. Further, we show that in the cell line A375, which we have previously shown expresses high levels of TGF-beta 3 mRNA, this region is responsible for mediating high level TGF-beta 3 promoter activity. Immediately 3' to the 3 x TCCC sequence is a consensus AP-2 binding site, however, we show that this region does not bind AP-2, and AP-2 does not transactivate the TGF-beta 3 promoter. Therefore, we provide strong evidence that high level expression of TGF-beta 3 in A375 cells results from transactivation of the TGF-beta 3 promoter by a protein that binds to a repeated TCCC motif in the promoter and suggest that this DNA-binding protein likely also regulates aspects of developmental and tissue-specific expression of this cytokine.     

Regulation of osteoclastogenesis and RANK expression by TGF-beta1

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta1 (0.01-5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-beta1 resulted in a significant increase in the percentage of RANK+ RAW cells (P < 0.05), as well as an increase in the fluorescence intensity per cell (P < 0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-beta directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression.     

Cytokine-specific induction of the TGF-beta inducible early gene (TIEG): regulation by specific members of the TGF-beta family

Select members of the TGF-beta family of cytokines play key regulatory roles in skeletal development, structure, and turnover. This laboratory has previously reported that TGF-beta treatment of immortalized normal human fetal osteoblast (hFOB) cells results in the rapid induction of the mRNA levels of a TGF-beta inducible early gene (TIEG) followed by changes in cell proliferation and bone matrix protein production. Previous studies have also shown that nonmembers of the TGF-beta superfamily showed little or no induction of TIEG mRNA. This article further addresses the cytokine specificity of this TIEG induction by examining whether activin and select bone morphogenetic proteins, (BMP-2, BMP-4, and BMP-6), which are representative of different subfamilies of this superfamily, also induce the expression of TIEG in hFOB cells. However, TGF-beta remained the most potent of these cytokines, inducing TIEG mRNA steady-state levels at 0.1 ng/ml, with a maximum induction of 24-fold at 2.0 ng/ml. The BMP-2 (16-fold), BMP-4 (4-fold), and activin (1-3-fold) also induced TIEG mRNA levels, but at reduced degrees compared to TGF-beta (24-fold), and only at much higher cytokine concentrations, e.g., 50-100 ng/ml, compared to 2 ng/ml for TGF-beta. BMP-6 showed no effect on TIEG mRNA levels. The TIEG protein levels generally correlated with the mRNA steady-state levels. As with TGF-beta, BMP-2 treatment of hFOB cells was shown by confocal microscopy to induce a rapid translocation of the TIEG protein to the nucleus. In summary, the relative potencies of these TGF-beta family members to induce TIEG expression generally follows the general osteoinductive capacity of these cytokines, with TGF-beta > BMP-2 > BMP-4 > activin > BMP-6.     

Phosphodiesterase 4 inhibitor regulates the TRANCE/OPG ratio via COX-2 expression in a manner similar to PTH in osteoblasts

Phosphodiesterase 4 (PDE4) inhibitors stimulate osteoclast formation by increasing the TRANCE/OPG mRNA ratio via cAMP-mediated pathways in a manner similar to parathyroid hormone (PTH) in osteoblasts. We investigated the role of cyclooxygenase-2 (COX-2) in osteoclast formation induced by the PDE4 inhibitor rolipram. Rolipram induced COX-2 expression in mRNA and protein levels, followed by increased prostaglandin E(2) production in osteoblasts. PKA, ERK, and p38 MAPK pathways regulate COX-2 mRNA expression induced by rolipram, in which PKA is a central regulator of the ERK and p38 MAPK pathways. A COX-2 inhibitor reversed the up-regulation of the TRANCE/OPG mRNA ratio induced by rolipram in osteoblasts, resulting in decreased osteoclast formation. These data suggest that COX-2 mediates rolipram induced osteoclast formation by regulating the TRANCE/OPG mRNA ratio in osteoblasts. Furthermore, the effects of the PDE4 inhibitor on osteoblasts were very similar to those of PTH, indicating that the PDE4 inhibitor largely shares the biological actions of PTH in osteoblasts.     

Convergence of cAMP, TGF-beta and retinoic acid signaling pathways in cells of the embryonic palate.

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-beta) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-beta1, cyclic adenosine 3', 5'-monophosphate (cAMP) and RA signaling systems. TGF-beta1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-beta mRNA in MEPM cells, though only TGF-beta1 inhibited RA-induced RAR-beta protein expression. Forskolin, but not TGF-beta1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-beta gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-beta-responsive, element. Thus, a putative TGF-beta Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-beta promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.     

Abscisic acid-responsive sequences from the em gene of wheat.

We demonstrate that a chimeric gene containing the beta-glucuronidase (GUS) reporter gene linked to a 646-base pair 5' fragment (-554 to +92) from the abscisic acid (ABA)-regulated Em gene from wheat is correctly expressed in transgenic tobacco. We observe high activity only in embryos of mature seeds, and immature seeds cultured on ABA show enhanced expression. Using a rice transient assay, we identify a 260-base pair fragment (-168 to +92) that accounts for the ABA-specific 15-fold to 20-fold increase in GUS expression. A 50-base pair sequence (-152 to -103) fused 5' in either orientation to a truncated cauliflower mosaic virus promoter (35S) increases GUS activity threefold in the presence of ABA. Insertion of the Em 5'-untranslated region (+6 to +86) between the 35S promoter and the ATG of GUS results in a 10-fold increase in GUS activity in the absence of ABA. These results suggest the following two functional fragments of the Em 5' region: an ABA response element from -152 to -103 and an element between +6 and +86 that quantitatively increases the ABA response.     

Ribozyme to human TGF-beta1 mRNA inhibits the proliferation of human vascular smooth muscle cells

Transforming growth factor-beta (TGF-beta) has been reported to be involved in the pathogenesis of cardiovascular proliferative diseases such as hypertensive vascular disease, atherosclerosis, and arterial restenosis after angioplasty. We designed a 38-base DNA-RNA chimeric hammerhead ribozyme to cleave human TGF-beta1 mRNA as a gene therapy for human arterial proliferative diseases. In the presence of MgCl(2), synthetic ribozyme to human TGF-beta1 mRNA cleaved the synthetic target RNA into two RNA fragments of predicted size. A control mismatch ribozyme, with one different base in the catalytic loop region, was inactive. DNA-RNA chimeric ribozyme (0. 01-1.0 microM) significantly inhibited angiotensin II (Ang II)-stimulated DNA synthesis in a dose-dependent manner in human vascular smooth muscle cells (VSMC). The mismatch ribozyme did not affect Ang II-stimulated DNA synthesis in the cells. DNA-RNA chimeric ribozyme (1.0 microM) inhibited the proliferation of human VSMC in the presence of Ang II. DNA-RNA chimeric ribozyme (1.0 microM) significantly inhibited Ang II-stimulated TGF-beta1 mRNA and protein expression in human VSMC. These results indicate that the designed DNA-RNA chimeric hammerhead ribozyme targeted to human TGF-beta1 mRNA can effectively and potentially inhibit growth of human VSMC by cleaving the TGF-beta1 mRNA. This finding suggests that this ribozyme will be useful in the gene therapy of arterial proliferative diseases.