Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells

Transforming growth factor beta (TGF-beta) activates Ras/MAPK signaling in many cell types. Because TGF-beta and BMP-2 exert similar effects, we examined if this signaling is stimulated by both factors and analyzed the relationship between this signaling and the Smads in osteoblasts. BMP-2 and TGF-beta stimulated Ras, MAPK, and AP-1 activities. The DNA binding activities of c-Fos, FosB/Delta FosB, Fra-1, Fra-2, and JunB were up-regulated whereas JunD activity was decreased. c-Fos, FosB/Delta FosB, and JunB were associated with Smad4. The stimulation of AP-1 by BMP-2 and TGF-beta was dependent on Smad signaling, and anti-Smad4 antibody interfered with AP-1 activity. Thus, BMP-2 and TGF-beta activate both Ras/MAPK/AP-1 and Smad signaling in osteoblasts with Smads modulating AP-1 activity. To determine the roles of MAPK in BMP-2 and TGF-beta function, we analyzed the effect of ERK and p38 inhibitors on the regulation of bone matrix protein expression and JunB and JunD levels by these two factors. ERK and p38 mediated TGF-beta suppression of osteocalcin and JunD as well as stimulation of JunB. p38 was essential in BMP-2 up-regulation of type I collagen, fibronectin, osteopontin, osteocalcin, and alkaline phosphatase activity whereas ERK mediated BMP-2 stimulation of fibronectin and osteopontin. Thus, ERK and p38 differentially mediate TGF-beta and BMP-2 function in osteoblasts.     

Increased expression of heme oxygenase-1 in human retinal pigment epithelial cells by transforming growth factor-β

Antibodies specific for heme oxygenase-1 (HO-1) were produced in rabbits, using the multiple antigen peptide (MAP) technique, and were employed to investigate the ability of transforming growth factor-β1 (TGF-β1) to induce the HO-1 protein in cultured human retinal pigment epithelial (RPE) cells. Western blot analyses showed that the cytokine induced HO-1 in these cells in a time- and dose-dependent manner. TGF-β1 also increased the mRNA for HO-1 in treated cells prior to the increase in HO-1 protein. The induction was effectively blocked by a neutralizing antibody preparation against TGF-β1. When tested under similar conditions, other growth factors such as basic fibroblast growth factor-I, plateletderived growth factor, insulin-like growth factor, transforming growth factor-α, and epidermal growth factor did not show appreciable induction of HO-1. Lipopolysaccharide, tumor necrosis factor-α, and interferon-γ were also not inducers, although TGF-β2 effectively induced HO-1. Heavy metal ions and thiol reagents were also highly potent inducers of HO-1 in human RPE cells. The induction of HO-1 by TGF-β1 was also observed in bovine choroid fibroblasts, but not in HELA, HEL or bovine corneal fibroblasts. Our results demonstrate for the first time that HO-1 can be induced by an important cytokine, TGF-β1, causing an increase in the expression of both HO-1 message and protein in specific neuroepithelial and fibroblast cells. © 1994 wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Transforming growth factor-β, osteogenin,and bone morphogenetic protein-2 inhibit intercellular communication and alter cell proliferation in MC3T3-E1 cells

Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues—processes which are regulated, in part, by peptide growth factors, including transforming growth factor-beta (TGF-β) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-β and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3-E1 cells were cultured in the presence of bone morphogenetic protein-2 (BMP-2) or TGF-β for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by ³H-thymidine (³H-TdR) incorporation. After times ranging from 6 to 48 hr, BMP-2 significantly inhibited uptake of ³H-TdR at doses of 50–800 ng/ml. Similarly, TGF-β inhibited uptake of ³H-TdR at doses of 2–32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell-cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 μg/ml) or higher dose of TGF-β (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP-2 and TGF-β on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-β (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP-2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose-dependent effects of BMP-2 and TGF-β on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-β at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC₅₀) calculated for BMP-2 and TGF-β at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF-β. The observation may suggest that TGF-β may have effects upon cytoskeletal elements in osseous tissues. © 1996 Wiley-Liss, Inc.     

Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells

To improve recombinant human bone morphogenetic protein-2 (rhBMP-2) yield, cell lines stably expressing hBMP2 were cultured in the presence of polyarginine peptide IND-1 and showed up to 6-fold increase in the yield of mature BMP-2. Repeated addition of IND-1 to cell cultures consistently improved BMP-2 yields over 53 days without affecting cell growth and viability. Investigation of its mechanism of action showed that IND-1 inhibited pro-protein convertase (PC) activity when incubated with cell lysates. However, when intact cells were cultured with IND-1, no change in cellular PC activity was observed. Furthermore, knockdown of furin (a prototypical member of the PCs) in cells did not affect their BMP-2 yields, suggesting furin/PC inhibition is unlikely the mechanism by which IND-1 enhances BMP-2 yields. IND-1 as a medium additive thus enhances BMP-2 production in mammalian cell expression systems.     

Lipopolysaccharide inhibits activation of latent transforming growth factor-β in bovine endothelial cells

The activation of latent transforming growth factor-β (TGF-β) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-β levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-β activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 μM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-β secreted into the culture modium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-β expression as well as the suppression of surface activation of latent TGF-β. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-β did not occur in cells maintained in LPS-contaminated culture medium. © 1995 Wiley-Liss, Inc.     

Transforming growth factor-β1 in reproduction

Expression patterns of TGF-βs during embryogenesis and in adult reproductive organs, as well as the activities of these molecules in in vitro assays of biological processes relating to reproduction and development, have suggested that TGF-βs may play a role in both reproductive function and embryonic development. To investigate the function of TGF-β1 in vivo, the murine TGF-β1 gene was disrupted by gene targeting, and animals that lacked TGF-β1 activity were generated. Homozygous mutant animals were obtained which exhibited a multifocal inflammatory disease. However, the observed numbers of homozygous mutant offspring were less than expected, suggesting the occurrence of some type of prenatal lethality. This paper reviews the proposed role of the TGF-βs in reproductive and developmental processes and discusses observations obtained from the TGF-β1 gene-targeting experiments as they relate to these processes. © 1994 Wiley-Liss, Inc.     

Effects of dexamethasone on the expression of transforming growth factor-β in mouse embryonic palatal mesenchymal cells

The central role of TGF-β in the development of the embryonic palate has been well characterized. TGF-β inhibits mesenchymal cell proliferation, induces medial edge epithelial cell differentiation, and modulates the expression of extracellular matrix proteins as well as the proteases that act upon them. Mechanisms by which TGF-β expression itself is regulated are less well understood. Glucocorticoids are recognized in several cellular systems as able to regulate the expression of TGF-β. This study was therefore designed to examine whether glucocorticoids affect the expression of TGF-β isoforms in embryonic palatal cells. Based on flow cytometric analysis and viability determination, confluent primary cultures of mouse embryonic palate mesenchymal (MEPM) cells exposed to up to 10⁻⁶ M dexamethasone (dex) exhibited no signs of cytotoxicity after 24 hours of exposure. Northern blot analyses revealed that dexamethasone reduced steady-state mRNA levels of TGF-β3 in a dose-dependent manner as early as 4 hours after treatment but had little effect on TGF-β1 and TGF-β2 expression up to 24 hours of dex exposure. Dex also reduced the synthesis of both latent and mature forms of TGF-β protein by approximately four-fold as determined by the mink lung epithelial cell growth inhibition bioassay. Assessment of the ratio of mature to latent protein found in conditioned medium of control compared to dex-treated cultures indicated that dexamethasone may reduce the activation of latent TGF-β to mature biologically active TGF-β. Dexamethasone inhibited the proliferation of MEPM cells despite the down-regulation of TGF-β suggesting that dex-induced growth inhibition of MEPM cells is not mediated by TGF-β. These data suggest that dex modulates TGF-β signaling pathways directly by down-regulating TGF-β expression and possibly indirectly by altering the availability of mature TGF-β necessary to exert its biological effects in the developing palate. © 1996 Wiley-Liss, Inc.     

The role of transforming growth factor-β1, -β2, and -β3 in androgen-responsive growth of NRP-152 rat prostatic epithelial cells

We have investigated the role of autocrine/paracrine TGF-β secretion in the regulation of cell growth by androgens as demonstrated by its inhibition by two androgen response modifiers; the nonsteroidal antiandrogen hydroxyflutamide (OHF), believed to act by inhibiting androgen binding to androgen receptors, or finasteride, an inhibitor of 5α-reductase, the enzyme necessary for the conversion of testosterone to 5α-dihydrotestosterone (DHT), using the nontumorigenic rat prostatic epithelial cell line NRP-152. Growth of these cells was stimulated three- to sixfold over control by either testosterone or DHT under serum-free culture conditions. This was accompanied by a two- to threefold decrease in the secretion rate of TGF-β1, -β2, and -β3. Finasteride reversed the ability of testosterone but not DHT to stimulate growth and downregulate expression of TGF-β1, -β2, and -β3 in a dose-dependent fashion, suggesting that this activity of testosterone required its conversion to DHT. OHF antagonized the stimulatory effects of DHT on NRP-152 cell growth but could reverse the inhibitory effects of DHT only on TGF-β2 and TGF-β3 and not TGF-β1 secretion. This suggests that either TGF-β1 regulation by DHT or the androgen antagonism of OHF occurs independent of androgen receptor binding. Neutralizing antibodies to TGF-β (pantropic and isoform-specific) were able to block the ability of finasteride to antagonize the effects of testosterone nearly completely while only partially inhibiting the antiandrogenic effects of OHF. Thus, the ability of androgens to stimulate growth of NRP-152 cells involves the downregulation of the production of TGF-β1, -β2, and -β3 in addition to other growth-stimulatory mechanisms. J. Cell. Physiol. 175:184–192, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Mechanism of retinoid-induced activation of latent transforming growth factor-β in bovine endothelial cells

Cell-associated plasmin is a putative physiological activator of latent transforming growth factor-β (LTGF-β). Since retinoids enhance the production of plasminogen activator (PA) and thereby increase cell-associated plasmin activity, we tested the possibility that retinoids might induce the activation of LTGF-β using bovine endothelial cells (ECs) as a model system. ECs treated with physiological concentrations of retinol or retinoic acid formed active TGF-β in the culture media in a dose- and time-dependent fashion. Cells were treated with 2 μM retinol for 24 h, and the amount of TGF-β produced during a subsequent 12-h incubation period was measured. Out of a total of 14 pM LTGF-β secreted, 0.7 pM was converted to active TGF-β. Northern blot analyses showed that mRNA levels for TGF-β2 but not for TGF-β1 increased in cells treated with retinol. Inclusion of either inhibitors of PA or of plasmin or antibody against PA in the culture medium as well as depletion of plasminogen from the serum blocked the formation of TGF-β, suggesting that PA, plasminogen, and the resulting plasmin are essential for activation of LTGF-β in retinoid-stimulated cells. Antibody against the LTGF-β binding protein blocked activation implying that localization of LTGF-β through its binding protein may be important. However, inhibition of binding of LTGF-β to the cell surface mannose 6-phosphate receptor did not prevent activation. These data indicate that retinoids up-regulate the production of LTGF-β in ECs and induce activation of LTGF-β, perhaps, by increasing PA and plasmin levels. Thus, TGF-β might be a local mediator of some of the biological activities of retinoids both in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

Cell-associated activation of latent transforming growth factor-β by calpain

Transforming growth factor-β (TGF-β) is normally secreted in a latent form, and plasmin-mediated proteolytic cleavage of latency-associated peptide (LAP), a component of latent TGF-β complex that makes the complex inactive, activates latent TGF-β. In the present study, we investigated the possible involvement of calpain, one of the cysteine proteases, in the activation of latent TGF-β. When recombinant latent TGF-β was incubated with calpain (1–10 u/ml) in a test tube, calpain cleaved LAP and released mature TGF-β from the latent complex. When calpain was applied to cultured bovine capillary endothelial (BCE) cells, a low concentration of calpain (0.05–0.1 u/ml) inhibited the migration and proliferation of the cells, and these inhibitory effects were abrogated by anti-TGF-β antibody as well as by calpain inhibitor peptide, but not by α₂-antiplasmin, a specific inhibitor of plasmin. Active TGF-β was detected in the conditioned medium of BCE cells collected in the presence of calpain. Chemical cross-linking of ¹²⁵I-calpain to BCE cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that calpain bound to the cell surface through chondroitinase ABC-sensitive proteoglycan. In addition, treatment of the BCE cells with chondroitinase ABC abrogated the inhibitory effect of calpain on the migration of these cells. Our data thus suggest that calpain is able to activate latent TGF-β through a mechanism independent of plasmin. This activation is efficient in the presence of cells, and calpain binds to the cell surface via proteoglycan and activates latent TGF-β, which is targeted to the same surface. J. Cell. Physiol. 174:186–193, 1998. © 1998 Wiley-Liss, Inc.     

Density-dependent inhibitory effect of transforming growth factor-β on human fibroblasts involves the down-regulation of platelet-derived growth factor α-receptors

We have previously found that transforming growth factor-β1 (TGF-β1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-β1 on the PDGF α-receptor, which binds all PDGF isoforms, as well as on the β-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-β1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-β1, there was an complete inhibition of ³H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-β1 was seen in PDGF-BB treated cells, although less pronounced. The binding of ¹²⁵I-labeled PDGF-AA and PDGF-BB to the α-receptor was significantly reduced after treatment with TGF-β1 in dense cultures, whereas the sparse cultures were less affected. A decrease of α-receptor mRNA was also seen. The levels of β-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-β1 is cell density-dependent and involves down-regulation of PDGF α-receptors. © 1993 Wiley-Liss, Inc.     

Transforming growth factor-β inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-β (TGF-β). In this paper, we show that TGF-β inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-β-treated chondrocytes show reduced ¹²⁵I-IL-1 binding that returns to a normal level when TGF-β is removed from the culture medium. The inhibitory effect of TGF-β is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor—ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of M_r =116 and 80 kDa and a minor band of M_r =100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-β treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-β to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

Pro-inflammatory cytokines downregulate platelet derived growth factor-α receptor gene expression in human osteoblastic cells

Platelet derived growth factor (PDGF) is thought to play a significant role in bone repair and regeneration. We previously demonstrated that PDGF-AA binding can be modulated by interleukin-1 (IL-1). We now report that TNF-α significantly reduces PDGF-AA binding by decreasing the number of PDGF-α receptor subunits on the surface of normal human osteoblastic cells. This inhibition is likely due to a decrease in synthesis of PDGF-α receptors since TNF-α causes a relatively rapid decrease in PDGF-α receptor mRNA levels as determined by Northern blot analysis. The physiologic importance of this inhibition is demonstrated by a TNF-α induced decrease in PDGF-AA stimulated tyrosine kinase activity. When saturating concentrations of TNF-α were used, the addition of IL-1 further inhibited PDGF-AA binding and further decreased surface expression of PDGF-α receptors. In contrast, other mediators such as IL-6, PTH, 1,25(OH)₂ vit D₃, hydrocortisone, PGE₂, bFGF, and IGF-1 had no effect. These results suggest that binding to the PDGF-α receptor is decreased by the strong pro-inflammatory cytokines such as IL-1β and TNF-α rather than as a general response to mediators important in bone resorption or bone formation. TNF-α and IL-1 are often co-expressed during destructive inflammatory processes. Thus, TNF-α and IL-1 may work in concert to limit the response of osteoblastic cells to PDGF-AA during periods of osseous inflammation. © 1996 Wiley-Liss, Inc.     

Transforming growth factor-β receptor expression on human skin fibroblasts: Dimeric complex formation of type I and type II receptors and identification of glycosyl phosphatidylinositol-anchored transforming growth factor-β binding proteins

Fibroblasts play a critical role in wound repair and in the development of fibrotic diseases, and transforming growth factor-β (TGF-β) has been shown to profoundly modulate fibroblast function. However, there is limited information on the TGF-β receptor types, isoform specificity, and complex formation in skin fibroblasts. Here, we report that normal adult human skin fibroblasts display two isoform-specific, cell surface glycosyl phosphatidylinositol (GPI)-anchored, TGF-β binding proteins in addition to the type I, II, and III TGF-β receptors. The identities of these proteins are confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific antireceptor antibodies, and other biochemical analyses. Immunoprecipitation results also indicated oligomeric complex formation between type I and II and between type II and III TGF-β receptors. Furthermore, by using affinity labeling and two-dimensional electrophoresis, we demonstrate the occurrence of type I and II heterodimers and type I homodimers of TGF-β receptors on these cells. Because the type I receptor does not bind TGF-β in the absence of type II receptor, these results indicate that one molecule of TGF-β induces the formation of a heterooligomeric complex containing more than one molecule each of type I and II TGF-β receptors on these cells. These cells respond to TGF-β by markedly down-regulating all five binding proteins and by potently augmenting DNA synthesis. These results allow the expansion of the proposed heteromeric TGF-β receptor signaling paradigm using mutantcells that are unresponsive to TGF-β and cell lines that have been transfected to overexpress these receptors, to include normal TGF-β-responsive cells. In addition, the definition of TGF-β receptor profiles in human skin fibroblasts provides important information for studying their alterations in these cells in various skin diseases. J. Cell. Physiol. 176:553–564, 1998. © 1998 Wiley-Liss, Inc.