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)     

Autocrine regulation of rheumatoid arthritis synovial cell growth in vitro

Rheumatoid arthritis (RA), and not osteoarthritis (OA) synovial cells proliferate in serum-free medium, a finding that suggests that, in vitro, RA synovial cells may be stimulated to grow by the continuous autocrine production of at least one polypeptide growth factor. Adding monoclonal antibody 1D11.16, or rabbit polyclonal anti-tumor growth factor beta (anti-TGF-beta) antibodies (both neutralizing antibodies to TGF-beta 1 and TGF-beta 2) to RA synovial cells, in culture, caused a significant reduction in cell growth, an effect not seen when other growth factor antibodies (platelet-derived growth factor [PDGF], epidermal growth factor [EGF], or EGF receptor) were added to the culture medium. Taken together, these data are consistent with the concept that RA synovial cell growth in vitro is driven endogenous TGF-beta. Moreover, when EGF was added to the culture medium, this caused the numbers of RA, and not OA, synovial cells to increase significantly. This finding suggests that RA synovial cells are in G1 phase of the cell cycle; an effect that could be mediated by endogenous TGF-beta.     

Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.

We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-beta blocked the cellular network formation. Conversely, addition of TGF-beta 1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF, EGF, and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-beta. Some TGF-beta remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior, suggesting caution in the interpretation of experiments on cellular activity related to Matrigel, collagen type IV, and possibly other extracellular matrix components.     

Platelet-derived growth factor or basic fibroblast growth factor induce anchorage-independent growth of human fibroblasts

Anchorage-independent growth, i.e., growth in semi-solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage-independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage-independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage-independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet-derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage-independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF-beta) decreased the growth induced by PDGF. EGF combined with TGF-beta induced colony formation in semi-solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage-independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF-beta can stimulate anchorage-independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.     

Differential effects of growth factors and cytokines on the synthesis of SPARC, DNA, fibronectin and alkaline phosphatase activity in human periodontal ligament cells

Growth factors and cytokines play an important role in tissue development and repair. However, it remains unknown how they act on proliferation and differentiation of periodontal ligament cells. In this study, we investigated the effects of several growth factors and cytokines on the synthesis of DNA, alkaline phosphatase (ALPase), fibronectin, and secreted protein acidic and rich in cysteine (SPARC) in human periodontal ligament (HPL) cells. Transforming growth factor-beta (TGF-beta) increased the synthesis of DNA, fibronectin and SPARC, whereas it decreased ALPase activity. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and tumor necrosis factor-alpha (TNF-alpha) decreased SPARC and ALPase levels, whereas these peptides increased DNA synthesis and did not affect fibronectin synthesis. Epidermal growth factor (EGF) up-regulated the synthesis of DNA and fibronectin and inhibited SPARC and ALPase levels. Interleukin-1beta (IL-1beta) decreased the synthesis of DNA, ALPase, fibronectin and SPARC. These findings demonstrate that TGF-beta, bFGF, EGF, PDGF, TNF-alpha and IL-1beta have characteristically different patterns of action on DNA, SPARC, fibronectin and ALPase synthesis by HPL cells. The differences in regulation of function of periodontal ligament cells by these peptides may be involved in the regeneration and repair of periodontal tissue.     

Transforming growth factor type beta 1 modulates the effects of basic fibroblast growth factor on growth and phenotypic expression of rat astroblasts in vitro

In a search of the growth factors possibly involved in brain ontogenesis we have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth and phenotypic expression of rat astroblasts in primary culture. Along TGF-beta 1 elicited only a slight negative effect on the growth of these cells. However, this factor was found to modulate the mitogenic effects of other growth factors. On quiescent cells it potentiates the mitogenic effect of basic fibroblast growth factor (bFGF) but not that of other growth factors, namely, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and thrombin. TGF-beta 1 did not modulate significantly the stimulatory effect of these growth factors on the activity of the enzyme glutamine synthetase (GS); but kinetic studies showed that TGF-beta 1 delays the stimulation of GS activity. DNA synthesis monitored by the incorporation of [125I]iododeoxyuridine (125I-dUrd) was maximum after 24-30 h of treatment with bFGF. With bFGF plus TGF-beta 1 the maximum was shifted to 30-36 h. This shift is compatible with the idea that TGF-beta 1 induces responsiveness in some cells which are otherwise unresponsive to the mitogenic action of bFGF, and that this induction requires some time. This hypothesis is sustained by the observation that in cells treated for only 12 h with bFGF, the treatment with TGF-beta 1 for the same 12 h or for longer time did not stimulate significantly the cell growth. Stimulation occurred only when the bFGF treatment was continued after 12 h. Potentiation of the mitogenic effect of bFGF and shift of the maximum 125I-dUrd incorporation towards 24 h was seen with cells pretreated with TGF-beta 1. This potentiation effect decreased with increasing time between the two treatments. The potentiation effect of TGF-beta 1 is not mediated by an induction of new bFGF membrane receptors as seen by binding studies.     

p38 MAP kinase regulates IL-1 beta responses in cultured airway smooth muscle cells

We have previously reported that interleukin (IL)-1 beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2 expression. The purpose of this study was to determine whether p38 mitogen-activated protein (MAP) kinase is involved in these events. IL-1 beta (2 ng/ml for 15 min) increased p38 phosphorylation fourfold. The p38 inhibitor SB-203580 (3 microM) decreased IL-1 beta-induced COX-2 by 70 +/- 7% (P < 0.01). SB-203580 had no effect on PGE(2) release in control cells but caused a significant (70-80%) reduction in PGE(2) release in IL-1 beta-treated cells. IL-1 beta increased the binding of nuclear proteins to the oligonucleotides encoding the consensus sequences for activator protein (AP)-1 and nuclear factor (NF)-kappa B, but SB-203580 did not affect this binding, suggesting that the mechanism of action of p38 was not through AP-1 or NF-kappa B activation. The NF-kappa B inhibitor MG-132 did not alter IL-1 beta-induced COX-2 expression, indicating that NF-kappa B activation is not required for IL-1 beta-induced COX-2 expression in HASM cells. IL-1 beta attenuated isoproterenol-induced decreases in HASM stiffness as measured by magnetic twisting cytometry, and SB-203580 abolished this effect. These results are consistent with the hypothesis that p38 is involved in the signal transduction pathway through which IL-1 beta induces COX-2 expression, PGE(2) release, and beta-adrenergic hyporesponsiveness.     

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Altered response to growth factors or retinoic acid in phenotypic transformation of normal rat kidney cells expressing human c-fos gene.

Anchorage-independent growth of normal rat kidney (NRK) fibroblast in soft agar depends on both transforming growth factor beta (TGF beta) and epidermal growth factor (EGF). To examine whether c-fos protein is involved in phenotypic transformation of NRK cells, we have transfected and isolated several NRK cell lines that carry the human c-fos gene fused to the metallothionein IIA promoter. A transfectant, Nf-1, had constitutive levels of the human c-fos expression. Anchorage-independent growth of Nf-1 was already stimulated by EGF alone, and the colony sizes of Nf-1 were comparable to those of the parental NRK in the presence of both EGF and TGF beta. Anchorage-independent growth of NRK could be observed in the presence of TGF beta or retinoic acid or platelet derived growth factor (PDGF) and EGF. No growth of NRK in soft agar appeared when basic fibroblast growth factor (bFGF) and EGF were present. By contrast, anchorage-independent growth of Nf-1 was surprisingly enhanced by EGF and TGF beta or retinoic acid or PDGF or bFGF. Expression of the human c-fos gene may compensate the signal to phenotypic transformation induced by TGF beta as well as retinoic acid or PDGF or bFGF.     

Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells

Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.     

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.     

Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts.

The effect of various growth factors on the synthesis of hyaluronan in human fibroblasts was investigated. When tested in medium containing 0.5% fetal calf serum, platelet-derived growth factor (PDGF)-BB was found to stimulate hyaluronan synthesis; the maximal response was equal to or higher than that obtained with 10% fetal calf serum. PDGF-AA gave only a limited effect, indicating that the stimulatory effect of PDGF on hyaluronan synthesis was mainly transduced via the B-type PDGF receptor. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 1 also stimulated hyaluronan synthesis; their effects were less than that of PDGF-BB, but combinations of factors produced potent stimulatory effects on hyaluronan synthesis. All factors stimulated hyaluronan synthesis in sparse as well as dense cultures. The effects of the factors on hyaluronan synthesis did not correlate with their mitogenic activities; PDGF-BB, EGF and bFGF are equipotent mitogens, but PDGF-BB had a much more potent effect on hyaluronan synthesis, and TGF-beta actually inhibits the growth of fibroblasts under the conditions of the assay.     

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.     

Possible role of prostaglandins as negative regulators in growth stimulation by tumor necrosis factor and epidermal growth factor in human fibroblasts

Recombinant tumor necrosis factor (TNF), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) stimulated growth of confluent human diploid fibroblasts (FS-4 cells) in the presence of fetal calf serum. TGF-beta synergistically enhanced both the TNF- and EGF-stimulated cell growth, whereas synergism between the mitogenic action of EGF and that of TNF was not observed. When indomethacin or acetylsalicylic acid, an inhibitor of prostaglandin production, was added to FS-4 cells, cell growth stimulated by EGF or TNF was increased, suggesting that prostaglandins induced by these mitogens antagonize their growth stimulatory actions. In contrast, neither indomethacin nor acetylsalicylic acid had a significant effect on the TGF-beta-induced growth of FS-4 cells. Mitogenic responses of indomethacin-treated cells to EGF, TNF, and TGF-beta were similarly suppressed by the addition of exogenous prostaglandin D2 (PGD2). Other prostaglandins such as PGE2 and PGF2 produced less inhibition of the cell growth.     

Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor. Differences to the activation by IL-1

The pronounced synovial hyperplasia often found in the joints of patients with rheumatoid arthritis could be explained partially by the action of monocyte-macrophage polypeptides (monokines). This report demonstrates that two cytokines which may be derived from monocyte-macrophage populations, namely platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), stimulate the DNA synthesis and proliferation of human synovial fibroblast-like cells cultured in low (i.e., 1%) fetal bovine serum. Epidermal growth factor, insulin-like growth factor-I, insulin-like growth factor-II (multiplication stimulating activity) and substance P were inactive. Unlike IL-1, PDGF and FGF do not also stimulate PGE2, plasminogen activator, and hyaluronic acid levels. Thus PDGF and FGF, arising from stimulated monocyte-macrophages, may play a role in the stimulation of mesenchymal cell proliferation that often accompanies chronic inflammatory arthritic disease. The synovial cells respond to a variety of cytokines in different ways suggesting multiple-signaling pathways.