Proliferation of 3T3-L1 preadipocytes in a completely defined serum-free medium

We have developed a completely defined serum-free medium that supports the growth of Swiss 3T3-L1 fibroblasts to nearly the same extent as Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. With ASF301 medium [former name, RITC 80-7; Yamane et al. Exp. Cell Res. 134, 470 (1981)], most of the 3T3-L1 cells survived for at least 10 days, but did not grow. ASF301 medium contains insulin and mouse-epidermal growth factor as growth factors, which are termed "progression factors". So we examined the effects of "competent factors" on the proliferation of 3T3-L1 preadipocytes. Growth in the medium supplemented with competent factors reached a confluent monolayer in 6-7 days. Furthermore, it was confirmed that 3T3-L1 cells grown in the serum-free medium retained the properties of differentiation into adipocytes. Our serum-free medium should be a useful tool for research on the growth and differentiation of 3T3-L1 preadipocytes.     

Transforming growth factor-beta,basic fibroblast growth factor,and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells

Transforming growth factor beta-1 (1GF-β) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serumcontaining medium by 58%. The effect of TGF-β on cell proliferation in serumfree medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-β inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-β in serum-free medium was not different than TGF-β alone. TGF-β depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-β did not affect proliferation. TGF-β inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-β and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-β and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-β interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-β on proliferation of clonally derived porcine satelite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF. © 1993 Wiley-Liss, Inc.     

Characterization of a serum-free culture system comparing growth factor requirements of transformed and untransformed cells

We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [3H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF a or b, or TGF-beta 1) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-beta 1 (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-beta 1 and FGF a or b were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state.     

Regulation of the Balb/c-3T3 cell cycle-effects of growth factors

The platelet-derived growth factor (PDGF), which is found in serum but not in plasma, has been purified to homogeneity; it stimulates replication at a concentration of 10^?10M. Brief treatment with PDGF causes densityinhibited Balb/c-3T3 cells to become competent to synthesize DNA; pituitary fibroblast growth factor (FGF) or precipitates of calcium phosphate also induce competence. Continuous treatment with plasma allows competent, but not incompetent, cells to synthesize DNA. A critical component of plasma is somatomedin, a group of hormones with insulin-like activity; multiplication-stimulating activity (MSA) or insulin replace plasma somatomedin in promoting DNA synthesis. We have studied the molecular correlates of competence and the role of SV40 gene A products in regulating DNA synthesis. Treatment of quiescent cells with pure PDGF or FGF causes the preferential synthesis of five cytoplasmic proteins (approximate molecular weight 29,000, 35,000, 45,000, 60,000, and 72,000 detected by SDS-PAGE under reducing conditions). Two of these competence-associated proteins (29,000 and 35,000 daltons) are found within 40 min of PDGF addition; they are not induced by plasma, insulin, or epidermal growth factor (EGF), PDGF, FGF, or calcium phosphate induce an ultrastructure change within the centriole of 3T3 cells; this ultrastructural modification of the centriole is detectable by immunofluorescence within 2 h of PDGF treatment. Plasma, EGF, or MSA do not modify the centriole. SV40 induces replicative DNA synthesis in growth-arrested 3T3 cells but does not cause this alteration in centriole structure. Gene A variants of SV40, including a mutant with temperature-sensitive (ts) T-antigen (ts A209), a deletion in t-antigen (dl 884), and several ts A209 strains containing t-antigen deletions were used to induce DNA synthesis in Balb/c-3T3 cells. Like wild type SV40, all strains induced DNA synthesis equally well under permissive or nonpermissive conditions. Addition of PDGF or plasma had little effect on SV40-induced DNA synthesis. Thus, the viral function that induces replicative DNA synthesis in Balb/c-3T3 cells is not t and is not temperature sensitive. This SV40 gene function overrides the cellular requirement for hormonal growth factors. It does not induce transient centriole deciliation, a hormonally regulated event.     

Defined medium for normal adult human prostatic stromal cells

Summary Stromal-epithelial interactions are pivotal in many aspects of prostatic biology. A defined culture system is critical for the investigation of factors that regulate the growth and differentiation of human prostatic stromal cells. We have identified conditions which promote stromal cell attachment and proliferation in serum-free medium. MCDB 201, originally developed for the clonal growth of chick embryo fibroblasts, proved to be a superior basal medium of those that we tested. Supplementation of MCDB 201 with basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) permitted attachment and exponential growth of cells throughout a 7-d period with an initial inoculum as low as 10³ cells per well of a 96-well microtiter dish. Using these assay conditions, we subsequently verified that basic FGF and IGF, but not PDGF, were required for optimal growth. No activity was found for heparin, transferrin, or the androgen R1881. Epidermal growth factor (EGF) didn’t stimulate growth when added to medium containing basic FGF and IGF, but was moderately stimulatory when added to basal medium alone. Cholera toxin inhibited growth. This simple and efficient culture medium provides a suitable assay system for more extensive studies of growth regulation and differentiation of human prostatic stromal cells, and will provide the basis for future development of a defined medium that supports clonal growth. Characterization of stromal-epithelial interactions will be facilitated by the use of this defined culture system for stromal cells in conjunction with the serum-free culture systems previously developed for human prostatic epithelial cells.     

Growth-stimulatory effects of androgen, high concentration of glucocorticoid or fibroblast growth factors on a cloned cell line from Shionogi carcinoma 115 cells in a serum-free medium

The effects of various kinds of growth factors or steroids on the proliferation of Shionogi carcinoma 115 (SC115) cells were investigated in cell culture. In a serum-free medium [Ham's F-12:Eagle's minimum essential medium (1:1, vol/vol) containing 0.1% bovine serum albumin], the proliferation of SC-3 cells (a cloned cell line from SC115 cells) estimated by [3H]thymidine incorporation into DNA and cell number reached a plateau at 10(-8) M testosterone (up to 200-fold), 10(-7) M dexamethasone (up to 30-fold) or 1 ng/ml of fibroblast growth factors (FGF; up to 50-fold). However, the proliferation in the serum-free medium was not significantly stimulated by the addition of low to very high concentrations of progesterone, oestradiol-17 beta, epidermal growth factor, platelet derived growth factor or insulin; transforming growth factor beta slightly stimulated the growth (up to 5-fold) but markedly inhibited the growth stimulation induced by testosterone. Furthermore, an epithelial appearance of SC-3 cells grown in the absence of growth factors or steroids was changed to a fibroblast-like appearance only by the addition of testosterone, high concentrations of dexamethasone or FGF. By investigating various kinds of growth factors or steroids, the present study demonstrates that androgen, high concentration of glucocorticoid or FGF alone significantly stimulates the proliferation of SC-3 cells with a change of morphology in the serum-free medium.     

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.     

Induction of the nuclear protein ''cyclin'' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.     

Competence growth factors can cause modification in higher-order chromatin structure in mouse embryo 3T3 fibroblasts

The authors compared sedimentation rates of nucleoids from mouse embryo 3T3 fibroblasts cultured in the presence or absence of different cell growth factors. The results clearly showed that rapidly sedimenting nucleoids are obtained only when cells are supplied with any of the following competence growth factors: platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or the product of the oncogene v-sis (a peptide homologous to PDGF). The tumor promoter phorbol 12-myristate 13-acetate, an activator of protein kinase C and a partial mitogen, shares this property with the competence growth factors. Removal of these factors from the medium causes cells to enter Go and nucleoids to sediment at a slower rate. Protein synthesis is required for growth factor induction of change in nucleoid sedimentation, but inhibition of either DNA synthesis or DNA repair does not antagonize the effect of growth factors. Titration of nucleoids with ethidium bromide indicates that one possible mechanism for the nucleoid change is the unwinding of DNA in supercoiled loops. The results indicated that the nucleoid change constitutes a cell response to competence factors that might have an important role in cell proliferation.     

Regulation of synovial cell growth: basic fibroblast growth factor synergizes with interleukin 1 beta stimulating phospholipase A2 enzyme activity, phospholipase A2 activating protein production and release of prostaglandin E2 by rheumatoid arthritis synovial cells in culture.

Cytokines have been implicated in the regulation of eicosanoid synthesis and synovial cell proliferation. To further define these mechanisms, we have compared the effects of basic fibroblast growth factor and platelet-derived growth factor on cell growth, prostaglandin E2 (PGE2) production and phospholipase A2 enzyme activity in long-term cultures of synovial cells from rheumatoid arthritis (RA) patients capable of proliferating in serum-free medium. Compared with serum-free medium alone, RA synovial cell growth was significantly enhanced by adding either basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF) to the culture medium. Growing RA synovial cells for 14 days in serum-free medium plus bFGF caused them to spontaneously release significant amounts of PGE2, an effect not seen if cells were grown in serum-free medium alone, or serum-free medium plus PDGF. Enhanced release of PGE2 occurred when arachidonic acid was added to bFGF but not PDGF-treated RA synovial cells, suggesting that bFGF increased cyclooxygenase enzyme activity in these cells. Moreover, phospholipase A2 (PLA2) enzyme activity was found to be significantly greater in RA synovial cells grown for 14 days in serum-free medium containing bFGF alone, or bFGF plus interleukin 1 beta (IL-1 beta) compared with cells grown in either serum-free medium alone, or serum-free medium plus PDGF. Similarly, bFGF plus IL-1 beta-stimulated release of PLA2 activating protein, a novel mammalian phospholipase stimulator found in high concentrations in RA synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine fibroblasts after exposure to a variety of growth-promoting factors. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal growth factor, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for 16 h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent fibroblasts in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.     

Mitochondrial protein p26 BCL2 reduces growth factor requirements of NIH3T3 fibroblasts

The BCL2 (B cell lymphoma/leukemia-2) proto-oncogene encodes a 26-kDa protein that has been localized to the inner mitochondrial membrane and that has been shown to enhance the survival of some types of hematopoietic cells. Here we show that NIH3T3 fibroblasts stably transfected with a BCL2 expression plasmid exhibit reduced dependence on competence-inducing growth factors (platelet-derived growth factor, PDGF; epidermal growth factor, EGF) for initiation of DNA synthesis. The importance of BCL2 for growth factor-induced proliferation of these cells was further confirmed by the useage of BCL2 antisense oligodeoxynucleotides. The mechanisms by which overexpression of p26 BCL2 contributes to fibroblast proliferation are unknown, but do not involve alterations in: (a) the production of inositol triphosphates (IP3), (b) PDGF-induced transient elevations in cytosolic Ca2+ ions, or (c) the activity of protein kinase C enzymes in these transfected cells. The results imply that changes in mitochondrial functions play an important role in the early stages of the cell cycle that render 3T3 cells competent to respond to the serum progression factors that stimulate entry into S-phase.     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described.     

Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium

The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.     

Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth

Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of platelet-derived growth factor and fibroblast growth factor on free intracellular calcium and mitogenesis

Although increased free intracellular calcium (Cai) may be one of the main regulators of cell growth and differentiation, studies in cell populations have implied that not all growth factors produce Cai increases. In order to examine in more detail whether Cai increases were related to mitogenesis, we used digital image analysis of intracellular Fura-2 fluorescence to measure Cai in individual BALB/c 3T3 cells stimulated with either platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). We found that PDGF induced larger and more prolonged Cai increases than FGF did, but that both growth factors induced an initial rapid increase in Cai (less than 2 min) followed by a later sustained increase (greater than 20 min). Only the prolonged Cai increase required extracellular calcium. Following PDGF treatment (1-8 units/ml), the percentage of cells with a large peak Cai increase (greater than twofold) correlated with the percentage of cells made competent (subsequent growth in 1% platelet-poor-plasma). In contrast, purified bovine basic FGF (200-800 pg/ml) and recombinant human acidic FGF (10-300 ng/ml) produced peak Cai increases that were not directly correlated with mitogenesis. In addition, concentrations of intracellular Quin 2 that inhibited Cai transients also inhibited PDGF stimulation but not FGF stimulation of mitogenesis. Thus, Cai increases are necessary for mitogenesis in BALB/c 3T3 cells stimulated by PDGF, but not that stimulated by FGF.     

Overexpression of the type II transforming growth factor-beta receptor inhibits fibroblasts proliferation and activates extracellular signal regulated kinase and c-Jun N-terminal kinase

Transforming growth factor-beta (TGF-beta) is a bimodal regulator of cellular growth. The cellular effects of TGF-beta depend on the intensity of signals emanating from TGF-beta receptors. Low levels of receptor activity are sufficient to stimulate cell proliferation, while higher degrees of receptor activation are associated with growth inhibition. To study the mechanisms of these effects, a tetracycline-inducible expression system was used to overexpress type II TGF-beta receptors in NIH 3T3 fibroblasts. Overexpressed type II TGF-beta receptors suppressed fibroblast proliferation elicited by TGF-beta1, fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF). Accompanying these anti-proliferative effects, increases in extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activity were detected. Furthermore, PDGF alpha-, but not PDGF beta-receptor protein levels were reduced by type II TGF-beta receptor overexpression. In conclusion, our system is an excellent tool to study the molecular mechanisms of growth inhibition by TGF-beta in fibroblasts. Activation of JNK and ERK, or modulation of PDGF receptor expression may be involved in this process.     

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.     

Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with various combinations of transforming growth factor (TGF)-beta, fibroblast growth factor (FGF), and insulin-like growth factor I (IGF-I). In serum-free defined medium the following observations were made: TGF-beta depressed proliferation and inhibited differentiation; FGF stimulated proliferation and depressed differentiation; IGF-I stimulated proliferation to a small degree but demonstrated a more pronounced stimulation of differentiation. In evaluating combinations of these three factors, the differentiation inhibiting effect of TGF-beta could not be counteracted by any combination of IGF-I or FGF. The proliferation-depressing activity of TGF-beta, however, could not inhibit the mitogenic activity of FGF. Maximum stimulation of proliferation was observed in the presence of both FGF and IGF-I. The highest percentage fusion was also observed under these conditions, but differentiation with minimal proliferation resulted from treatment with IGF-I, alone. By altering the concentrations of TGF-beta, FGF, and IGF-I, satellite cells can be induced to proliferate, differentiate, or to remain quiescent.