Stimulatory Effects Of TGF-β Research Articles

MicroRNA-155 attenuates profibrotic effects of transforming growth factor-beta on human lung fibroblasts.

Transforming growth factor-beta (TGF-β) functions in fibrogenesis as a profibrotic mediator, regulating cell proliferation, migration, apoptosis and collagen production of fibroblasts. microRNA-155 (miR-155), the expression of which has been related to bleomycin-induced idiopathic pulmonary fibrosis, has been involved in TGF-β induced epithelial-mesenchymal transition. Here, we found that miR-155 expression was decreased in human pulmonary fibroblasts by TGF-β treatment. We overexpressed miR-155 in fibroblasts to investigate the functional impact of miR-155 on TGF-β-induced fibrotic phenotype of fibroblasts. It is suggested that miR-155 overexpression attenuated the stimulatory effect of TGF-β on fibroblast proliferation, migration and collagen synthesis, by evidence from assessment of cell cycle, viability, apoptosis, migration and collagen content. Furthermore, quantitative measurement showed that SMAD1 gene expression was decreased following miR-155 inhibition, thereby demonstrating an indirect miRNA-SMAD interaction that links miR-155 to TGF-β signaling. Our work helped uncover an miRNA-mediated mechanism of fibroblast response to TGF-β. Moreover, it will help to achieve a better understanding of the regulatory roles of miR-155 in fibrogenesis.

Transforming growth factor-β stimulates human ovarian cancer cell migration by up-regulating connexin43 expression via Smad2/3 signaling

Reduced connexin43 (Cx43) expression is frequently detected in different types of human cancer. Cx43 has been shown to regulate cancer cell migration in a cell-type dependent manner. In both primary and recurrent human ovarian cancer, overexpression of TGF-β ligand and its receptors have been detected. TGF-β can regulate Cx43 expression in other cell types and stimulate human ovarian cancer cell migration. However, whether Cx43 can be regulated by TGF-β and is involved in TGF-β-stimulated cell migration in human ovarian cancer cells remain unknown. In this study, we demonstrate that TGF-β up-regulates Cx43 in two human ovarian cancer cell lines, SKOV3 and OVCAR4. The stimulatory effect of TGF-β on Cx43 expression is blocked by inhibition of TGF-β receptor. Treatment with TGF-β activates Smad2 and Smad3 signaling pathways in both ovarian cancer cell lines. In addition, siRNA-mediated knockdown of Smad2 or Smd3 abolishes TGF-β-induced up-regulation of Cx43 expression. Moreover, knockdown of Cx43 attenuates TGF-β-stimulated cell migration. This study demonstrates an important role for Cx43 in mediating the effects of TGF-β on human ovarian cancer cell migration.

Biphasic effects of transforming growth factor β on bone morphogenetic protein–induced osteoblast differentiation

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP-induced bone formation may lead to the development of more effective BMP-based therapies. To identify kinases mediating BMP-induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP-6-induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor β (TGF-β) generally is considered to antagonize BMP-induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF-β receptors displayed reduced BMP-6-induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF-β type I receptor impaired BMP-induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF-β further increased expression of osteoblast-specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF-β were found to depend on timing and duration of the costimulation. TGF-β inhibited BMP-induced activation of a BMP-Smad-dependent luciferase reporter, suggesting that the stimulatory effect of TGF-β is not due to increased BMP-Smad activity. TGF-β also inhibited the BMP-induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF-β, besides acting as an inhibitor, also can, by dampening the noggin-mediated negative-feedback loop, enhance BMP-induced osteoblast differentiation, which might be beneficial in fracture healing.

Molecular Cloning, Cellular Distribution and Regulation of Rat Steroidogenic Acute Regulatory Protein (StAR) in the Ovary.

Rat ovarian genes induced by the treatment of immature rats with pregnant mare serum gonadotropin (PMSG) were isolated by a subtraction cloning method. Amongst them was obtained a probable rat homologue of steroidogenic acute regulatory protein (StAR), which has been recently identified as a protein that is an acute regulator of the rate limiting transfer of cholesterol from the outer to the inner mitochondrial membrane. Structure of rat StAR was determined by nucleotide sequence analysis. Northern blot analysis revealed that StAR mRNA levels were rapidly and strongly increased by PMSG/hCG. In situ hybridization revealed that the expression of StAR mRNA was strongly induced by PMSG in theca interna cells as well as in corpora lutea. These findings indicate that expression of StAR mRNA is restricted to and induced in the ovarian steroidogenic cell types where cholesterol is used as a substrate for synthesis of steroid hormones. To study the mechanisms of regulation of StAR in rat granulosa cells, we used granulosa cells obtained from diethylstilbestrol-treated immature rats. Northern blot analysis revealed two major transcripts of about 3.6 kb and 1.6 kb of rat StAR mRNA. Rat StAR mRNA had strongly increased within 2 h due to the treatment of FSH or 8-Br-cAMP in this culture, a parallel increase of transcripts of both sizes was observed. Compared to the control, StAR mRNA levels increased in a dose-dependent manner in the presence of increasing concentrations of FSH (1-100 ng/ml) and 8-Br-cAMP (0.25-5 mM). Although co-treatment of rat granulosa cells with FSH and TGF-β did not change FSH-induced StAR mRNA levels, these levels in granulosa cells were markedly increased by pretreatment with TGF-β before being acutely (2 h) stimulated with an effective dose of FSH. The stimulatory effect of TGF-β was concentration-dependent (1-30 ng/ml).

Expression of steroidogenic acute regulatory protein (StAR) in rat granulosa cells

Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroids. To study the mechanisms of regulation of StAR in rat granulosa cells, we used granulosa cells obtained from diethylstilbestrol-treated immature rats. Northern blot analysis revealed two major transcripts of about 3.6 kb and 1.6 kb of rat StAR mRNA. Rat StAR mRNA had strongly increased within 2 h due to the treatment of FSH or 8-Br-cAMP in this culture, a parallel increase of transcripts of both sizes was observed. Compared to the control, StAR mRNA levels increased in a dose-dependent manner in the presence of increasing concentrations of FSH (1–100ng/ml) and 8-Br-cAMP (0.25–5 mM). Although co-treatment of rat granulosa cells with FSH and TGF-β did not change FSH-induced StAR mRNA levels, these levels in granulosa cells were markedly increased by pretreatment with TGF-β before being acutely (2 h) stimulated with an effective dose of FSH. The stimulatory effect of TGF-β was time- and concentration-dependent (1–30 ng/ml).

TGF-β Enhances Osteoclast Differentiation in Hematopoietic Cell Cultures Stimulated with RANKL and M-CSF

TGF-β has been shown to inhibit and stimulate osteoclastogenesis. The purpose of this study was to evaluate the effects of TGF-β in hematopoietic cell cultures stimulated with RANKL and M-CSF. In cocultures of hematopoietic cells and BALC cells (a calvarial-derived cell line), TGF-β inhibited tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell formation. In contrast, TGF-β enhanced TRAP-positive multinucleated cell formation up to 10-fold in hematopoietic cell cultures containing few osteoblastic/stromal cells. Likewise, TGF-β increased the number of calcitonin receptor (CTR)-positive multinucleated and mononucleated cells in a concentration-dependent manner. An increase in cell size and multinuclearity was also observed in the presence of TGF-β. The stimulatory effects of TGF-β were dependent on the presence of M-CSF and RANKL. When differentiated on bovine cortical bone slices, these cells formed resorption lacunae. These results suggest that TGF-β has a direct stimulatory effect on osteoclastogenesis in hematopoietic cells treated with RANKL and M-CSF.

Transforming growth factor β1-regulated gene expression of Ito cells

During liver fibrogenesis, Ito cells are regarded as the principal matrix synthesizing cells and transforming growth factor β 1 (TGF- β 1) appears to be the main fibrogenic mediator. This study analyzed the effects of TGF-β 1 on Ito cell activation, proliferation, and on the expression of a set of matrix proteins, antiproteases, and TGF-β receptors both in "early cultured" and "culture-activated" Ito cells. Rat liver Ito cells at day 2 of primary culture ("early cultured" cells) were mainly smooth muscle alpha actin (SMA)-negative, whereas cells at day 6 were judged as "activated" cells (SMA-positive). Following 24-hour exposure to 1 ng/mL TGF-β1, total protein synthesis, cell proliferation, and expression of the "activation" marker SMA were not significantly changed. In addition to previously described stimulatory effects on collagen types I and III, fibronectin, undulin, and proteoglycan-gene expression, TGF-β also dose-dependently increased synthesis and secretion of tenascin, laminin, entactin, collagen type IV, and α2-macroglobulin, but decreased C1-esterase inhibitor production by Ito cells, as revealed by immunoprecipitation of endogenously labeled proteins and by Northern blot analysis. The stimulatory effect of TGF-β was evident both in "early cultured" as well as "culture-activated" Ito cells. By reverse- transcription polymerase chain reaction (RT-PCR) analysis, TGF-β type II, III, and TGF-β/activin type I receptors were present in Ito cells, and their expression pattern was not changed upon TGF-β exposure. Northern blot analysis demonstrated that type I TGF-β/activin receptor was induced during in vitro activation and that TGF-β exposure resulted in a slight increase of type I and III receptor messenger RNAs. In summary, the data illustrate that TGF-β is an important fibrogenic mediator acting both on "early cultured" as well as "culture-activated" Ito cells, rather than a mitogenic or morphogenic mediator. The differential regulation of TGF-β/activin receptors during in vitro activation and their up-regulation by TGF-β1 might represent a mechanism by which the receptor complex regulates TGF-β signalling in Ito cells. (Hepatology 1996 Aug;24(2):352-60)

Alteration of the Chemotactic Response of Human Skin Fibroblasts to PDGF by Growth Factors

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for fibroblastic cells. In the early stage of wound healing, PDGF is released from aggregated platelets and it is believed that its chemotactic activity may play a key role in the influx of connective tissue cells into wound sites. Using the Boyden chamber assay, we investigated factors that alter the migratory response of human skin fibroblasts to PDGF. The response was related to the growth state of cells; that is, growing cells at low cell density exhibited a greater migratory response to PDGF than density-arrested quiescent cells. The level of random migration was also elevated in growing cells, compared to quiescent cells. The chemotactic response after 3-h exposure to serum was markedly decreased (>50%). Three-hour preincubation of the cells with transforming growth factor-β (TGF-β) or with epidermal growth factor reduced the migratory response to approximately half that in the nonstimulated control. In contrast, cells treated with TGF-β or basic fibroblast growth factor for 24 h exhibited a two- to threefold greater chemotactic response to PDGF than control cells. This stimulatory effect of TGF-β on the fibroblast migration induced by PDGF suggests that TGF-β acts synergistically with PDGF on the influx of connective tissue cells into human wound sites and may explain the potent wound healing activity of TGF-β in vivo.

Alteration of the chemotactic response of human skin fibroblasts to PDGF by growth factors.

Abstract Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for fibroblastic cells. In the early stage of wound healing, PDGF is released from aggregated platelets and it is believed that its chemotactic activity may play a key role in the influx of connective tissue cells into wound sites. Using the Boyden chamber assay, we investigated factors that alter the migratory response of human skin fibroblasts to PDGF. The response was related to the growth state of cells; that is, growing cells at low cell density exhibited a greater migratory response to PDGF than density-arrested quiescent cells. The level of random migration was also elevated in growing cells, compared to quiescent cells. The chemotactic response after 3-h exposure to serum was markedly decreased (>50%). Three-hour preincubation of the cells with transforming growth factor-β (TGF-β) or with epidermal growth factor reduced the migratory response to approximately half that in the nonstimulated control. In contrast, cells treated with TGF-β or basic fibroblast growth factor for 24 h exhibited a two- to threefold greater chemotactic response to PDGF than control cells. This stimulatory effect of TGF-β on the fibroblast migration induced by PDGF suggests that TGF-β acts synergistically with PDGF on the influx of connective tissue cells into human wound sites and may explain the potent wound healing activity of TGF-β in vivo.

Comparative effects of a novel Vitamin D Analogue MC-903 and 1,25-dihydroxyvitamin D 3 on alkaline phosphatase activity, osteocalcin and DNA synthesis by human osteoblastic cells in culture

MC-903 is a novel vitamin D analogue which has been shown to promote epidermal cell differentiation but is 100 times less active than 1,25 dihydroxyvitamin D 3 (1,25(OH) 2D) in causing hypercalcemia. In order to determine the activity of this compound on bone cells, we have compared the effects of MC-903 and 1,25 dihydroxyvitamin D 3 (1,25(OH) 2D) on parameters of cell proliferation and differentiation in cultured normal human osteoblastic cells derived by migration from trabecular bone fragments. Dose response curves showed that MC-903 was 10 to 100 times less effective than 1,25(OH) 2D in stimulating the synthesis of the osteoblast specific protein osteocalcin by human bone cells depending on the basal osteocalcin production. In cells showing high basal osteocalcin synthesis, 1,25(OH) 2D (10 −8 M) was 2- to 3-fold more potent than MC-903 (10 −8 M) in inducing osteocalcin from 48 to 96 h of treatment. The greater activity of 1,25(OH) 2D over MC-903 was observed in human bone cell cultures with elevated basal osteocalcin levels, indicating that the response to 1,25(OH) 2D but not to MC-903 was amplified in cells with the higher osteoblastic characteristics. The effects of MC-903 and 1,25(OH) 2D on alkaline phosphatase activity were not markedly different. Transforming Growth Factor-β (TGFβ) (0.5 ng/mL, 48 h) was found to completely suppress the osteocalcin synthesis induced by 1,25(OH) 2D (10 −8 and 10 −9 M), whereas the MC-903-induced osteocalcin synthesis was not affected, suggesting a negative interaction between TGFβ and 1,25(OH) 2D but not MC-903 on osteocalcin synthesis. In contrast, the stimulatory effect of TGFβ (0.5 ng/mL, 48 h) on alkaline phosphatase activity in quiescent human osteoblastic cells was suppressed by both MC-903 and 1,25(OH) 2D. The two vitamin D derivatives induced similar effects on DNA synthesis in human bone cells and did not affect the stimulation of [ 3H] thymidine incorporation into DNA induced by TGFβ (0.5 ng/mL). The results show that 1,25(OH) 2D and MC-903 exert equipotent effects on DNA synthesis and alkaline phosphatase activity but distinct effects on the synthesis of the bone specific protein osteocalcin. The finding that MC-903 has less potent effects than 1,25(OH) 2D on a specific osteoblastic function further indicates that this compound may be useful in the treatment of psoriatic lesions.

Effects of transforming growth factor type β on expression of cytoskeletal proteins in endosteal mouse osteoblastic cells

Transforming growth factor β (TGFβ) has been shown to influence the growth and differentiation of many cell types in vitro. We have examined the effects of TGFβ on cell morphology and cytoskeletal organization in relation to parameters of cell proliferation and differentiation in endosteal osteoblastic cells isolated from mouse caudal vertebrae. Treatment of mouse osteoblastic cells cultured in serum free medium for 24 hours with TGFβ (1.5–30 ng/mL) slightly (− 23%) inhibited alkaline phosphatase activity. In parallel, TGFβ (0.5–30 ng/mL, 24 hours) greatly increased cell replication as evaluated by [ 3H]-thymidine incorporation into DNA (157% to 325% of controls). At a median dose (1.5 ng/mL) that affected both alkaline phosphatase and DNA synthesis (235% of controls) TGFβ induced rapid (six hours) cell respreading of quiescent mouse osteoblastic cells. This effect was associated with increased polymerization of actin, α actinin, and tubulins, as evaluated by both biochemical and immunofluorescence methods. In addition, TGFβ (1.5 ng/mL) increased the de novo biosynthesis of actin, α actinin, vimentin, and tubulins, as determined by [ 35S] methionine labeling and fractionation of cytoskeletal proteins using two-dimensional gel electrophoresis. These effects were rapid and transient, as they occurred at six hours and were reversed after 24 hours of TGFβ exposure. The results indicate that the stimulatory effect of TGFβ on DNA synthesis in endosteal mouse osteoblastic cells is associated with a transient increase in cell spreading associated with enhanced polymerization and synthesis of cytoskeletal proteins.

Effects of transforming growth factor-beta on the production of immunoreactive insulin-like growth factor I and progesterone and on [3H]thymidine incorporation in porcine granulosa cell cultures.

Transforming growth factor-beta (TGF beta) has been reported to enhance many FSH-stimulated functions in rat granulosa cell cultures. We therefore, investigated the actions of TGF beta on cultured porcine granulosa cells. We evaluated the production of immunoreactive insulin-like growth factor I (iIGF-I) and progesterone in short term (3-day) and in longer term (7-day) cultures using porcine TGF beta 1 (pTGF beta 1). TGF beta had a biphasic effect on epidermal growth factor (EGF)-stimulated iIGF-I production in short term cultures. A modest stimulatory effect was apparent at 10 pg/ml; however, this end point was completely inhibited by 1-10 ng/ml. TGF beta also had a slight stimulatory effect on basal iIGF-I production at 1 pg/ml, but not at higher levels. In longer term cultures TGF beta did not have a significant effect on either basal or FSH-stimulated iIGF-I production. In both short and longer term cultures TGF beta markedly inhibited basal and FSH-stimulated progesterone production. We also evaluated the effects of TGF beta on the incorporation of [3H]thymidine into DNA and found that basal and growth factor-stimulated [3H]thymidine incorporation were inhibited. No stimulatory effects of TGF beta on progesterone production or [3H]thymidine incorporation could be detected over the dose range tested (1 pg/ml to 10 ng/ml). The effects of human TGF beta 1 and pTGF beta 2 were compared with those of pTGF beta 1 on basal and EGF-stimulated [3H]thymidine incorporation. Effects of the peptides were qualitatively similar, but pTGF beta 2 was somewhat less inhibitory to EGF-stimulated [3H]thymidine incorporation than pTGF beta 1. The present studies show that in contrast to the well documented stimulatory actions of TGF beta in cultured rat granulosa cells, this growth factor is a predominantly negative regulator of porcine granulosa cells. With the exception of a modest stimulation of iIGF-I production at very low doses, the effects of TGF beta were to potently inhibit both growth and differentiated function. The inhibitory nature of TGF beta should not be overlooked when considering the possible role of this peptide in ovarian development and differentiation.

Transforming growth factor β enhances basal and FSH-stimulated inhibin production by rat granulosa cells in vitro

Transforming growth factor β (TGFβ) caused a dose-dependent increase in both basal and follicletimulating hormone (FSH)-stimulated inhibin production by rat granulosa cells in culture. The TGFβ dose-response curve in the absence of FSH was approximately parallel to that in the presence of either a minimally effective dose (1 ng/ml) or a maximally effective dose (30 ng/ml) of FSH, suggesting an additive effect of these two agents on inhibin production. There was also a suggestion of an increased sensitivity of granulosa cell inhibin production to FSH when the cells were coincubated with TGFβ. The time course study showed that similar to FSH, the stimulatory effect of TGFβ on basal and FSH-stimulated inhibin production was evident on day 1 and was maximal by day 4. In addition, epidermal growth factor (EGF) reduced FSH-stimulated inhibin production with an ID 50 value of 1.3 ng/ml. Coincubation of cells with EGF and 1 ng TGFβ/ml enhanced greatly the inhibitory action of EGF on FSH-induced inhibin production (ID 50 < 0.1 ng/ml). It is concluded that: (1) TGFβ directly stimulates inhibin production by rat granulosa cells and the combined effect with FSH was largely additive, (2) the inhibitory effect of EGF on FSH-induced inhibin production was enhanced by TGFβ, (3) individual members of the TGFβ/inhibin gene family regulate ovarian function, not only by direct action on follicle cells but also indirectly by influencing the production rate of other members of that family.

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

The effects of the transforming growth factor β (TGF-β) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-β caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [ 3H]thymidine incorporation ( ED 50 = 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-β potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-β (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-β 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-β ( ED 50 = 0.1 ng/ml , maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-β (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-β-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-β caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes ( ED 50 = 0.3 ng/ml , maximum at 1 ng/ml). This stimulatory effect of TGF-β was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-β stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-β, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-β may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.

Reciprocal effects of epidermal growth factor and transforming growth factor β on the anchorage-dependent and -independent growth of A431 epidermoid carcinoma cells

The effects of epidermal growth factor (EGF) and transforming growth factor β (TGFβ) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGFβ. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGFβ on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGFβ. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.

Transforming growth factor type β can act as a potent competence factor for AKR-2B cells

Transforming growth factor type β (TGFβ) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGFβ. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGFβ during early to mid-G 1. In addition, TGFβ need be present only briefly (as little as l min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGFβ in the absence of EGF requires only an equally brief exposure to TGFβ. Use of homogeneous 125I-labeled TGFβ in a cell-binding assay demonstrates that TGFβ bound to cell-surface receptors can readily exchange into the culture medium T 1 2 = 120 min ), helping to rule out the possibility that persistent receptor-bound TGFβ is the source of a continuous stimulus. The data indicate that TGFβ exposure induces a stable state in the cell ( T 1 2 = 20 h ) similar to but distinct from the state of “competence” induced by platelet-derived growth factor (PDGF).