Effect of IL‐1β, PGE2, and TGF‐β1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts

The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)‐1β, proftaglandin E₂ (PGE₂), and transforming growth factor‐β1 (TGF‐β1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real‐time PCR. In all cultures, OPG mRNA increased significantly in response to IL‐1β treatment and decreased in response to TGF‐β1 whereas PGE₂ treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL‐1β treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF‐β1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption. J. Cell. Biochem. 110: 304–310, 2010. © 2010 Wiley‐Liss, Inc.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

TGF-β1 is an organizer of responses to neurodgeneration

TGF-β1 mRNA and protein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF-β1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF-β1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF-β1 protein observed in βA/amyloid-containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims. Involvement of TGF-β1 in these human brain disorders is discussed in relation to the potent effects of TGF-β1 on wound healing and inflammatory responses in peripheral tissues. We hypothesize that TGF-β1 and possibly other TGF-β peptides have organizing roles in responses to neurodegeneration and brain injury that are similar to those observed in non-neural tissues. Work from many laboratories has shown that activities of TGF-β peptides on brain cells include chemotaxis, modification of extracellular matrix, and regulation of cytoskeletal gene expression and of neurotrophins. Similar activities of the TGF-β's are well established in other tissues.     

Modulation of gene expression in the preimplantation mouse embryo by TGF-α and TGF-β

The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously-added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF-α or TGF-β results in changes in the pattern of total protein synthesis as assessed by high-resolution two-dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up- or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up- or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF-α or TGF-β results in the increased expression of the b subunit of the F₀ ATPase. TGF-β also stimulates the expression of the DNA polymerase α. TGF-α treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin. © 1995 Wiley-Liss, Inc.     

Modulation of the catabolic effects of interleukin-1β on human articular chondrocytes by transforming growth factor-β

The effects of IL-1β and TGF-β on the biosynthesis of extracellular matrix structural components relative to the metalloproteinases and their inhibitor TIMP1 in human articular chondrocytes were investigated. It has been proposed that TGF-β, acting as a positive regulator of matrix accretion, can counteract the increased loss of cartilage matrix induced by IL-1β. To allow a comparison of their effects on mRNA levels for these different components, quantitation by competitive RT/PCR was employed. This method was found to give reproducible estimates of mRNA levels and the observed effects of IL-1β and TGF-β on individual components of this system agree with qualitative data obtained by northern blotting. IL-1β had a more pronounced effect on aggrecan mRNA levels than on those for type II collagen. Similar quantitative differences were observed between collagenase and stromelysin mRNA levels. TGF-β generally counteracted the effects of IL-1β, and new steady state levels were attained within 24 h. However, the reversal of IL-1β induced suppression of matrix protein mRNA levels appeared more effective than its suppression of the increase in stromelysin and collagenase mRNA levels. Similarly TGF-β did not reduce the extent of IL-1β induced secretion of stromelysin at the protein level. TIMP1 mRNA levels were only slightly reduced by IL-1β; however this cytokine effectively surpressed its induction by TGF-β. The higher concentrations of TGF-β and longer exposure times required to overcome the surpressive effects of IL-1β suggest that the interaction between IL-1β and TGF-β in the regulation of TIMP1 expression follows a different mechanism to that operating for the metalloproteinases and matrix proteins. Thus the overall potential of TGF-β to inhibit proteolysis is attenuated by its much slower effect on TIMP1 mRNA levels. © 1996 Wiley-Liss, Inc.     

Role for autocrine TGF-β1 in regulating differentiation of a human leukemic cell line toward osteoclast-like cells

Increasing evidence suggests that transforming growth factor-β (TGF-β) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF-β1 and that exogenous or autocrine TGF-β1 can induce the same features of osteoclastic-like cells, exerting its effects through the binding to TGF-β specific receptors. Scatchard analysis of ¹²⁵I-labeled TGF-β1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of ～25 pM and a binding capacity of ～900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF-β receptors. Stimulation of FLG 29.1 cells with low TGF-β1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF-β1 caused a significant and dose-dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF-β1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF-β1 mRNA expression and growth factor release. The majority of TGF-β1 secreted by TPA-treated cells was in its latent form. However, anti-TGF-β antibodies inhibited TGF-β1 and TPA-induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF-β1 and indicating that the cells can activate and respond to the TGF-β that they secrete. These findings support a potential autocrine role for TGF-β1 in osteoclast differentiation. © 1994 Wiley-Liss, Inc.     

Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-β to TGF-β

The role of receptor-bound urokinase-type plasminogen activator (uPA) in cellular activation of latent transforming growth factor-beta (LTGF-β) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR⁺). a human prouPA cDNA (LhuPA), or a control neomycinresistance cDNA (Lneo). When LhuPAR⁺ cells were co-cultured with LhuPA cells, the plasmin-dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co-cultures containing LhuPA and Lneo cells instead of the LhuPAR⁺ cells. The preferential activation of TGF-β by co-cultures with the greatest plasmin-generation potential, LhuPAR⁺ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF-β production, was observed with BAE cells treated with conditioned medium (CM) from co-cultures of LhuPA and LhuPAR⁺ cells. Inclusion of neutralizing antibodies to TGF-β abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF-β. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co-cultures blocked both the fibrinolytic activity and the generation of TGF-β activity in the CM. In the second assay, CM from co-cultures of LhuPA and LhuPAR⁺ cells inhibited the migration of BAE cells in a wound assay. Controls with anti-TGF-β IgG indicated that the inhibition was due to TGF-β. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co-cultures of LhuPA and LhuPAR⁺ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co-culture of LhuPA with LhuPAR^? cells. Excess mannose-6-phosphate (M6P) blocked the generation of TGF-β as assayed by both the BAE migration and PA assays, presumably because it interfered with cellsurface localization of LTGF-β. Additionally, small numbers of LhuPA and LhuPAR⁺ cells co-cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF-β. These results support the conclusion that plasmindependent activation LTGF-β by LB6 cells is promoted by the surface localization of uPA by its receptor. © 1994 Wiley-Liss, Inc.     

Induction of c-fos and c-jun mRNA at the M/G1 border is required for cell cycle progression

The proto-oncogenes c-fos and c-jun have been shown in numerous model systems to be induced within minutes of growth factor stimulation, during the G₀/G₁ transition. In this report we use the mitotic shake-off procedure to generate a population of highly synchronized Swiss 3T3 cells. We show that both of these immediate-early, competence genes are also induced during the M/G₁ transition, immediately after completion of mitosis. While c-fos mRNA levels drop to undetectable levels within 2 hr after division, c-jun mRNA levels are maintained at a basal level which is ～ 30% maximum throughout the remainder of G₁. In order to access the functional significance of these patterns of c-fos and c-jun expression, antisense oligodeoxynucleotides specific to c-fos or c-jun were added to either actively growing Swiss 3T3 cells or mitotically synchronized cells, and their ability to inhibit DNA synthesis and cell division determined. Our results show that treatment of Swiss 3T3 cells with either c-fos or c-jun antisense oligodeoxynucleotides, while actively growing, during mitosis, or in early G₁, results in a reduction in ability to enter S and subsequently divide. This was also true if Swiss 3T3 cells were treated during mid-G₁ with c-jun antisense oligodeoxynucleotides. These results demonstrate that the regulation of G₁ progression following mitosis is dependent upon the expression and function of the immediate-early, competence proto-oncogenes c-fos and c-jun. © 1994 Wiley-Liss, Inc.     

Transforming growth factor β1 induces mitogenesis in fetal rat brown adipocytes

The presence of transforming growth factor β₁ (TGF-β₁) for 24 or 48 h stimulated DNA synthesis, the percentage of cells in the S + G₂/M phases of the cell cycle, and cell number, as compared to quiescent cells. The mitogenic capacity of TGF-β₁ (1 pM) was similar to that shown by 10% fetal calf serum (FCS). TGF-β₁ for 48 h increased by 5-fold the percentage of cells containing (³H)thymidine-labeled nuclei as compared to quiescent cells. In addition, single fetal brown adipocytes, showing their typical multilocular fat droplets phenotype, become positive for (³H)thymidine-labeled nuclei in response to TGF-β₁. Moreover, TGF-β₁ induced the mRNA expression of a complete set of proliferation-related genes, such as c-fos (30 min), c-myc and β-actin (2 h), and H-ras, cdc2 kinase, and glucose 6-phosphate dehydrogenase (G6PD) at 4 and 8 h, as compared to quiescent cells. Concurrently, TGF-β₁ for 12 h increased the protein content of proliferating cellular nuclear antigen (PCNA) by 6-fold and p21-ras by 2-fold. Although our results demonstrate that TGF-β₁ induces the expression of very early genes related to cell proliferation, TGF-β₁ could be acting either as a mitogen or as a survival factor to induce proliferation in fetal brown adipocytes. © 1996 Wiley-Liss, Inc.     

Differential expression and biological activity of retinoic acid–induced TGFβ isoforms in embryonic palate mesenchymal cells

The effect of retinoic acid (RA) on TGF-β mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-β bioassay in association with TGF-β isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-β protein released into the culture medium. RA had little or no effect on TGF-β1 mRNA expression and protein production. In contrast, RA increased TGF-β2 and β3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-β released was increased relative to the total increase in TGF-β released, suggesting that RA treatment stimulated activation of latent TGF-β. RA also increased TGF-β2 mRNA expression; we have previously shown that RA upregulates TGF-β3 mRNA in these cells. RA and TGF-β individually inhibited ³H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit ³H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-β2 or β3-specific neutralizing antibodies partially relieved the inhibitory effect on ³H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-β. Simultaneous treatment with RA and TGF-β also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-β treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-β in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-β in retinoid-induced cleft palate. J. Cell. Physiol. 177:36–46, 1998. © 1998 Wiley-Liss, Inc.     

Retinoids potentiate transforming growth factor-β activity in bovine endothelial cells through up-regulating the expression of transforming growth factor-β receptors

Retinoic acid (RA) induces the activation of latent transforming growth factor-β (TGF-β) in bovine aortic endothelial cells (BAECs) via enhancement of cellular plasminogen activator (PA)/plasmin levels. The resultant TGF-β suppresses the excessive fibrinolytic activity by decreasing PA expression and stimulating expression of the PA inhibitor, PA inhibitor-1 (PAI-1), and inhibits cell proliferation. Here, we report that, in this regulatory system, RA simultaneously up-regulates the expression of TGF-β receptor types I and II, resulting in enhancement of TGF-β activity in the cells. RA increased the numbers of high- and low-affinity binding sites for ¹²⁵I-TGF-β1 2.1-fold and 1.5-fold, respectively, without alteration of their Kd values. Affinity labeling and Western and Northern blotting studies showed that, following RA treatment, surface levels of both type I and type II receptors increased due to augmentation in their mRNA levels. The effect was dose- and time-dependent. Treatment with 1 μM RA for 15 hr increased mRNA levels of type I and II receptor threefold and eightfold, respectively. Pretreatment of BAECs with either RA or retinol lowered the concentration of TGF-β1 required to suppress PA levels, to enhance PAI-1 levels, and to inhibit cell proliferation. Thus, retinoids may regulate cellular functions of BAECs not only by inducing the formation of active TGF-β but also by stimulating TGF-β receptor expression. This regulatory mechanism may sustain TGF-β-mediated regulation of EC function at a focal site where RA is acting. J. Cell. Physiol. 176:565–573, 1998. © 1998 Wiley-Liss, Inc.     

Differential regulation of p34cdc2 and p33cdk2 by transforming growth factor-β1 in murine mammary epithelial cells

Cyclin-dependent kinases (cdks) are a family of proteins whose function plays a critical role in cell cycle traverse. Transforming growth factor-β₁ (TGF-β₁) is a potent growth inhibitor of epithelial cells. Since cdks have been suggested as possible biochemical markers for TGF-β growth inhibition, we investigated the effect of TGF-β₁ on cdc2 and cdk2 in a normal mouse mammary epithelial cell line (MME) and a TGF-β-resistant MME cell line (BG18.2). TGF-β₁ decreases newly synthesized cdc2 protein levels within 6 h after addition. Coincident with this decrease in newly synthesized cdc2 protein was a marked reduction in its ability to phosphorylate histone H1. This decrease in kinase activity is not due to a change in steady-state levels of cdc2 protein, since mRNA and total protein levels of cdc2 are not reduced until 12 h after TGF-β₁ addition. This suggests that the kinase activity of cdc2 is dependent on newly synthesized cdc2 protien. Moreover, the protein synthesis of another cyclin-dependent kinase, cdk2, is not effected by TGF-β₁ addition, but its kinase activity is substantially reduced. Thus, it appears that TGF-β decreases the kinase activity of both cdc2 and cdk2 by distinct mechanisms.