Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.     

Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta

The effects of transforming growth factor-beta (TGF-beta) on the synthesis of cartilage-matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50 micrograms/ml ascorbic acid, and 2 x 10(-7) M hydrocortisone (Medium A). Various combinations of TGF-beta, insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF-beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF-beta became very elongated and formed distinct foci, and those grown with FGF and IGF-I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H with glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF-beta was similar to that in cells grown with FGF and IGF-I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF-beta or with FGF and IGF-I. However, FGF in combination with either TGF-beta or IGF-I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF-beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.     

Fibroblast growth factor 21 (FGF21) inhibits chondrocyte function and growth hormone action directly at the growth plate

Fibroblast growth factor 21 (FGF21) modulates glucose and lipid metabolism during fasting. In addition, previous evidence indicates that increased expression of FGF21 during chronic food restriction is associated with reduced bone growth and growth hormone (GH) insensitivity. In light of the inhibitory effects on growth plate chondrogenesis mediated by other FGFs, we hypothesized that FGF21 causes growth inhibition by acting directly at the long bones' growth plate. We first demonstrated the expression of FGF21, FGFR1 and FGFR3 (two receptors known to be activated by FGF21) and β-klotho (a co-receptor required for the FGF21-mediated receptor binding and activation) in fetal and 3-week-old mouse growth plate chondrocytes. We then cultured mouse growth plate chondrocytes in the presence of graded concentrations of rhFGF21 (0.01-10 μg/ml). Higher concentrations of FGF21 (5 and 10 μg/ml) inhibited chondrocyte thymidine incorporation and collagen X mRNA expression. 10 ng/ml GH stimulated chondrocyte thymidine incorporation and collagen X mRNA expression, with both effects prevented by the addition in the culture medium of FGF21 in a concentration-dependent manner. In addition, FGF21 reduced GH binding in cultured chondrocytes. In cells transfected with FGFR1 siRNA or ERK 1 siRNA, the antagonistic effects of FGF21 on GH action were all prevented, supporting a specific effect of this growth factor in chondrocytes. Our findings suggest that increased expression of FGF21 during food restriction causes growth attenuation by antagonizing the GH stimulatory effects on chondrogenesis directly at the growth plate. In addition, high concentrations of FGF21 may directly suppress growth plate chondrocyte proliferation and differentiation.     

Transforming growth factor (TGF)-beta-activated kinase 1 mimics and mediates TGF-beta-induced stimulation of type II collagen synthesis in chondrocytes independent of Col2a1 transcription and Smad3 signaling

Transforming growth factor (TGF)-beta, bone morphogenetic protein (BMP), and interleukin-1beta activate TGF-beta-activated kinase 1 (TAK1), which lies upstream of the p38 MAPK, JNK, and NF-kappaB pathways. Our knowledge remains incomplete of TAK1 target genes, requirement for cooperative signaling, and capacity for shared or segregated ligand-dependent responses. We show that adenoviral overexpression of TAK1a in articular chondrocytes stimulated type II collagen protein synthesis 3-6-fold and mimicked the response to TGF-beta1 and BMP2. Both factors activated endogenous TAK1 and its activating protein, TAB1, and the collagen response was inhibited by dominant-negative TAK1a. Isoform-specific antibodies to TGF-beta blocked the response to endogenous and exogenous TGF-beta but not the response to TAK1a. Expression of Smad3 did not stimulate type II collagen synthesis or enhance that caused by TGF-beta1 or TAK1a, in contrast to its effects on its endogenous targets, CTGF and plasminogen-activated inhibitor-1. TAK1a, overexpressed alone and immunoprecipitated, phosphorylated MKK6 and stimulated the plasminogen-activated inhibitor-1 promoter following transient transfection; both effects were enhanced by TAB1 coexpression, but type II collagen synthesis was not. Stimulation by TAK1a or TGF-beta did not require increased Col2a1 mRNA, and TAK1 actually reduced Col2a1 mRNA in parallel with the cartilage markers, SRY-type HMG box 9 (Sox9) and aggrecan. Thus, TAK1 increased target gene expression (Col2a1) by translational or posttranslational mechanisms as a Smad3-independent response shared by TGF-beta1 and BMP2.     

The effects of platelet-derived transforming growth factor beta on normal human diploid gingival fibroblasts

Studies of the effects of transforming growth factor (TGF) beta on normal human diploid gingival fibroblasts (HGF) have been carried out to determine possible physiological effects of this growth factor. Responses distinctly different from those characterized using established cell lines were observed. Whether alone, or in combination with EGF (2.5 ng/ml), human platelet-derived TGF-beta (0.1 ng/ml or 1.0 ng/ml) did not induce anchorage-independent growth of HGFs in soft agar assays. However, TGF-beta with EGF acted synergistically in promoting a 1.8-fold increase in anchorage-dependent proliferation of quiescent HGFs. At the same concentrations TGF-beta alone stimulated the incorporation of [35S]methionine into both cellular (cell-layer) and matrix (medium) proteins by as much as 3-fold and 1.7-fold respectively. Densitometric analysis of fluorographs of radiolabeled media proteins separated by SDS-PAGE revealed that the TGF-beta-stimulated protein synthesis was selective. However, synthesis of collagen, the major protein synthesized and secreted by HGFs, was stimulated by TGF-beta to the same extent as the average secreted protein. Protein synthesis and cell proliferation were significantly greater in subconfluent cells compared to confluent and multilayered cells. These effects are likely to reflect physiological activity of platelet-derived TGF-beta which may act to promote the wound healing response.     

Regulation of rat proximal tubule epithelial cell growth by fibroblast growth factors, insulin-like growth factor-1 and transforming growth factor-beta, and analysis of fibroblast growth factors in rat kidney.

Growth factors may play an important role in regulating the growth of the proximal tubule epithelium. To determine which growth factors could be involved, we have investigated the mitogenicity of various purified factors in rat kidney proximal tubule epithelial (RPTE) cells cultured in defined medium. Fibroblast growth factors, aFGF (acidic FGF) and bFGF (basic FGF), stimulate DNA synthesis in a dose-dependent manner, with ED50 values of 4.5 and 3.2 ng/ml, respectively; their effects are not additive. With cholera toxin in the medium, both aFGF and bFGF can replace insulin or epidermal growth factor (EGF) to attain the maximum level of cell growth, but they cannot replace cholera toxin. Cholera toxin specifically potentiates the effects of FGFs on DNA synthesis. At high cell density, both insulin and insulin-like growth factor 1 (IGF-1) induce DNA synthesis more effectively than EGF, FGFs and cholera toxin. The high concentration (0.2-1.0 microgram/ml) of insulin required for cell growth can be replaced by a low concentration of IGF-1 (10-20 ng/ml), indicating that insulin probably acts through a low affinity interaction with the IGF-1 receptor. Transforming growth factor-beta 1 (TGF-beta 1) inhibits DNA synthesis induced by individual factors and combinations of factors in a concentration-dependent manner. Northern blot analysis shows that mRNA for TGF-beta 1, IGF-1, and aFGF, but not bFGF are present in rat kidney. Western blot analysis and bioassay data confirmed that the majority of FGF-like protein in rat kidney is aFGF. The data suggest that in addition to EGF, IGFs, and TGF-beta, FGFs may also be important kidney-derived regulators of proximal tubule epithelial cell growth in vivo and in vitro.     

Modulation of cultured chicken growth plate chondrocytes by transforming growth factor-beta 1 and basic fibroblast growth factor.

Expression of several cellular and matrix proteins which increase significantly during the maturation of growth plate cartilage has been shown to be affected by various endocrine and autocrine factors. In the studies reported here, transforming growth factor-beta (TGF-beta 1) and basic fibroblast growth factor (bFGF) were administered to primary cultures of avian growth plate chondrocytes at pre- or post-confluent stages to study the interplay that occurs between these factors in modulating chondrocytic phenotype. Added continuously to pre-confluent chondrocytes, TGF-beta 1 stimulated the cells to produce abundant extracellular matrix and multilayered cell growth; cell morphology was altered to a more spherical configuration. These effects were generally mimicked by bFGF, but cell shape was not affected. Administered together with TGF-beta 1, bFGF caused additive stimulation of protein synthesis, and alkaline phosphatase (AP) activity was markedly, but transiently enhanced. During this pre-confluent stage, TGF-beta 1 also increased fibronectin secretion into the culture medium. Added to post-confluent cells, TGF-beta 1 alone caused a dosage-dependent suppression of AP activity, but bFGF alone did not. Under these conditions, TGF-beta 1 and bFGF had little effect on general protein synthesis, but TGF-beta 1 alone caused large, dosage-dependent increases in synthesis of fibronectin, and to some extent type II and X collagens. Given together with bFGF, TGF-beta 1 synergistically increased secretion of fibronectin. These findings reveal that regulation of phenotypic expression in maturing growth plate chondrocytes involves complex interactions between growth factors that are determined by timing, level, continuity, and length of exposure.     

Transforming growth factor-beta1 regulates basement membrane formation by alveolar epithelial cells in vitro

Immortalized alveolar type II epithelial (SV40-T2) cells formed a continuous, thin lamina densa when they were cultured on collagen fibrils with the supplement of 1.0 ng/ml TGF-beta1. Corresponding to lamina densa formation, immunohistochemical analysis of laminin, type IV collagen, perlecan, and entactin (nidogen) indicated integration of these components in a linear array beneath the SV40-T2 cells. Synthesis of these basement membrane constituents was significantly enhanced by TGF-beta1 in a dose-dependent manner. On the other hand, TGF-beta1 did not affect the synthesis of extracellular matrix-regulatory enzymes and their inhibitors, such as type II transglutaminase, matrix metalloproteinase-2, plasminogen activator inhibitor-1, or tissue inhibitor of matrix metalloproteinase-1. These results indicate that basement membrane formation in the presence of 1.0 ng/ml TGF-beta1 is attributable to enhanced synthesis of basement membrane constituents. However, formation of a continuous basement membrane was inhibited at a TGF-beta1 concentration of 5.0 ng/ml. Synthesis of the basement membrane constituents was further enhanced at this concentration and the extracellular matrix-regulatory enzymes remained unchanged. The deposits of cellular fibronectin and type I collagen beneath SV40-T2 cells were significantly augmented. Thus excessive production of interstitial extracellular matrix components appears to obstruct the integration of basement membrane constituents into a continuous architecture. These results indicate that the basement membrane formation by SV40-T2 cells is achieved at the optimal TGF-beta1 concentration.     

Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes

Summary The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggeted that CDF and MSA act on chondrocytes in an early G₁ phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G₁ phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.Abbreviations CDF cartilage-derived factor - MSA multiplication-stimulating activity - EGF epidermal growth factor - FGF fibroblast growth factor     

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-α in human articular chondrocytes in vitro

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-α in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-α. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-α or a combination of both (at 10 ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10 ng/ml TNF-α for 6 or 24 h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-α suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-α mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1β expression by TNF-α was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-α in chondrocytes.     

Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis in the rat femur

We have investigated the ability of exogenous transforming growth factor-beta (TGF-beta) to induce osteogenesis and chondrogenesis, critical events in both bone formation and fracture healing. Daily injections of TGF-beta 1 or 2 into the subperiosteal region of newborn rat femurs resulted in localized intramembranous bone formation and chondrogenesis. After cessation of the injections, endochondral ossification occurred, resulting in replacement of cartilage with bone. Gene expression of type II collagen and immunolocalization of types I and II collagen were detected within the TGF-beta-induced cartilage and bone. Moreover, injection of TGF-beta 2 stimulated synthesis of TGF-beta 1 in chondrocytes and osteoblasts within the newly induced bone and cartilage, suggesting positive autoregulation of TGF-beta. TGF-beta 2 was more active in vivo than TGF-beta 1, stimulating formation of a mass that was on the average 375% larger at a comparable dose (p less than 0.001). With either TGF-beta isoform, the dose of the growth factor determined which type of tissue formed, so that the ratio of cartilage formation to intramembranous bone formation decreased as the dose was lowered. For TGF-beta 1, reducing the daily dose from 200 to 20 ng decreased the cartilage/intramembranous bone formation ratio from 3.57 to zero (p less than 0.001). With TGF-beta 2, the same dose change decreased the ratio from 3.71 to 0.28 (p less than 0.001). These data demonstrate that mesenchymal precursor cells in the periosteum are stimulated by TGF-beta to proliferate and differentiate, as occurs in embryologic bone formation and early fracture healing.