Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

Transforming growth factor-beta1 expression in cultured corneal fibroblasts in response to injury

The mechanisms underlying TGF-beta regulation in response to injury are not fully understood. We have developed an in vitro wound model to evaluate the expression and localization of transforming growth factor-beta1 in rabbit corneal fibroblasts in response to injury. Experiments were conducted in the presence or absence of serum so that the effect of the injury could be distinguished from exogenous wound mediators. Cultures were wounded and evaluations conducted over a number of time points. Expression of TGF-beta1 RNA was determined using Northern blot analysis and in situ hybridization, while the TGF-beta receptors were identified by affinity cross-linking. Injury increased the expression of TGF-beta1 mRNA in cells at the wound edge after 30 min; this response was amplified by the addition of serum. TGF-beta1 mRNA expression was observed in a number of cells distal from the wound. After wound closure, TGF-beta1 mRNA was negligible and resembled unwounded cultures. The half-life of TGF-beta1 mRNA was two times greater in the wounded cultures, indicating that the injury itself maintained the expression, while cell migration was present. Analogous to these findings, we found that binding of TGF-beta to its receptors was maximal at the wound edge, decreasing with time and distance from the wound. These results indicate that injury increases the level of expression of TGF-beta1 mRNA and maintains a higher level of receptor binding during events in wound repair and that these might facilitate the migratory and synthetic response of stromal fibroblasts.     

Selective increase in cytokeratin synthesis in cultured rat hepatocytes in response to hormonal stimulation

Addition of a combination of insulin, dexamethasone and EGF at seeding time to cultured rat hepatocytes in serum-free medium caused a selective increase in the biosynthesis of particular cytokeratin components. This increase was prominent during the first day in culture. No significant increases were detected in the absence of hormones or in the presence of either hormones added alone or in pairs, except in the case of insulin plus dexamethasone, which yielded an effect close to that obtained with the three factors. Interestingly, the latter condition also maintained a high level of albumin production over a 6-day period in culture.     

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.     

Characterization of proteoglycans synthesized by cultured corneal fibroblasts in response to transforming growth factor beta and fetal calf serum

A culture system was developed to analyze the relationship between proteoglycans and growth factors during corneal injury. Specifically, the effects of transforming growth factor beta-1 (TGF-beta1) and fetal calf serum on proteoglycan synthesis in corneal fibroblasts were examined. Glycosaminoglycan synthesis and sulfation were determined using selective polysaccharidases. Proteoglycan core proteins were analyzed using gel electrophoresis and Western blotting. Cells cultured in 10% dialyzed fetal calf serum exhibited decreased synthesis of more highly sulfated chondroitin sulfate and heparan sulfate compared with cells cultured in 1% dialyzed fetal calf serum. The amount and sulfation of the glycosaminoglycans was not significantly influenced by TGF-beta1. The major proteoglycan species secreted into the media were decorin and perlecan. Decorin was glycanated with chondroitin sulfate. Perlecan was linked to either chondroitin sulfate, heparan sulfate, or both chondroitin sulfate and heparan sulfate. Decorin synthesis was reduced by either TGF-beta1 or serum. At early time points, both TGF-beta1 and serum induced substantial increases in perlecan bearing chondroitin sulfate and/or heparan sulfate chains. In contrast, after extended periods in culture, the amount of perlecan bearing heparan sulfate chains was unaffected by TGF-beta1 and decreased by serum. The levels of perlecan bearing chondroitin sulfate chains were elevated with TGF-beta1 treatment and were decreased with serum. Because both decorin and perlecan bind growth factors and are proposed to modulate their activity, changes in the expression of either of these proteoglycans could substantially affect the cellular response to injury.     

Regulation of ovarian progestin production by epidermal growth factor in cultured rat granulosa cells

The modulation of ovarian steroidogenesis by epidermal growth factor (EGF) was investigated in cultured rat granulosa cells. Granulosa cells, obtained from ovaries of immature, hypophysectomized, estrogen-treated rats, were incubated for 2 days with EGF, follicle-stimulating hormone (FSH), or EGF plus FSH. Treatment with EGF did not affect estrogen production, but stimulated progestin (i.e. progesterone and 20 alpha-hydroxy-pregn-4-en-3-one) production in a dose-dependent manner. Stimulation of progestin production by EGF appears to be the result of an increase in pregnenolone biosynthesis as well as increases in the activities of 20 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase/isomerase. Treatment with FSH increased both estrogen and progestin production by cultured granulosa cells. When cells were treated concomitantly with EGF, FSH-stimulated estrogen production was inhibited, while progestin production was further enhanced. The EGF enhancement of FSH-stimulated progestin production appears to be the result of synergistic increases in pregnenolone biosynthesis and 20 alpha-hydroxysteroid dehydrogenase activity, resulting in substantial increases in 20 alpha-hydroxypregn-4-en-3-one but not progesterone production. The effects of EGF were shown to be time-dependent. The concept of a direct action of EGF on rat granulosa cells is reinforced by the demonstration of high affinity (Kd approximately 3 X 10(-10) M), low capacity (approximately 5,000 sites/cell) EGF binding sites in these cells. Thus, EGF interacts with specific granulosa cell receptors to stimulate progestin but to inhibit estrogen biosynthesis.     

The glucose transporter in human fibroblasts is phosphorylated in response to phorbol ester but not in response to growth factors

The possibility that the stimulation of hexose transport in human fibroblasts by phorbol myristate acetate (PMA), insulin, platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) is associated with phosphorylation of the glucose transporter has been investigated. The time and concentration dependencies of the stimulation of transport by these agents under conditions identical to those used for phosphorylation were determined. Each agent, when used at the concentration that resulted in the maximal increase in transport rate, elicited this effect within 30 min of exposure. The extent of stimulation ranged from 15 to 70%. For determination of phosphorylation of the glucose transporter, fibroblasts were incubated for 16 h with [32P]Pi and exposed to the agonist for 30 min; the transporter was then isolated from a detergent lysate of the cells by immunoprecipitation with a monoclonal antibody. Under these conditions, there was no phosphorylation of transporter in basal cells and only PMA caused detectable incorporation of phosphate into the transporter. Thus, it is unlikely that the stimulation of glucose transport by insulin, PDGF and EGF involve transporter phosphorylation.     

Interleukin-6 induces thermotolerance in cultured Caco-2 cells independent of the heat shock response

In recent studies, induction of the heat shock response increased IL-6 production in gut mucosa in vivo and in cultured Caco-2 cells in vitro. The heat shock response is associated with increased survival of cells exposed to otherwise lethal hyperthermia, so called thermotolerance, but the role of IL-6 in the induction of thermotolerance is not known. We tested the hypothesis that treatment of cultured Caco-2 cells with IL-6 results in the development of thermotolerance. Cells were treated with human recombinant IL-6 for 1h followed by 3 h recovery in cytokine-free medium whereafter cells were exposed to heat stress (48 degrees C for 2 h). In untreated cells, the heat stress resulted in an approximately 80% cell death. In cells treated with IL-6, cell viability after heat stress was significantly improved and was doubled at an IL-6 concentration of 20 ng/ml. Treatment of the cells with other cytokines (IL-4, IL-10, IL-1beta, or TNFalpha) did not induce thermotolerance, suggesting that the effect of IL-6 may be specific for this cytokine. The induction of thermotolerance by IL-6 was blocked by an IL-6 receptor antibody, suggesting that the development of thermotolerance was receptor-mediated. Treatment of cells with IL-6 did not induce an heat shock response as suggested by unaltered heat shock protein 70 and 90 levels and unaffected heat shock factor DNA binding activity. In addition, the IL-6-induced thermotolerance was not inhibited by quercetin. The present study provides the first evidence of IL-6-induced thermotolerance and suggests that this effect of IL-6 is independent of the heat shock response.     

Factors affecting heat production of rat 3Y1 fibroblasts in flow microcalorimetry

Quiescent 3Y1 cells in monolayer cultures were dispersed with trypsin-EDTA, suspended in various media, and the cellular heat production was measured in a flow-type microcalorimeter set at 37 degrees C. A linear relationship was found to exist between the number of cells applied to the microcalorimeter and the heat output. Increasing concentrations of bovine serum albumin (BSA) and of fetal calf serum (FCS) added in Dulbecco's modified Eagle's medium (DEM) enhanced the heat output to the same saturation level. Trypsin inhibitor added in DEM enhanced the heat output, but to a lower saturation level than FCS or BSA did, indicating that BSA has an activity to enhance cellular heat production by a mechanism other than neutralizing residual trypsin. The heat output was found to gradually decrease in the microcalorimeter. This reduction was not enhanced by a two-fold dilution of the medium (DEM plus FCS) with phosphate-buffered saline, indicating that this reduction is not caused by the depletion of nutrients and serum factors in the medium. Similarly, when cells were incubated for 155 or 220 min in suspension in DEM plus BSA at 37 degrees C and applied to the microcalorimeter, the heat output decreased. However, no significant reduction of the heat output was observed after holding the cells at 0 degree C in suspension for the same period. This and other facts suggest that depletion of O2 dissolved in the medium is involved in the gradual decrease in heat output.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen concentration regulates the proliferative response of human fibroblasts to serum and growth factors

This report demonstrates that oxygen concentration within the physiologic range of 2.5 to 20% controls the pattern of proliferation of human diploid fibroblasts by modulating their response to serum and purified growth factors. Reducing oxygen concentration from 20 to 2.5% increased the division rate and final density of fibroblasts cultured in serum-containing medium. DNA synthesis in response to serum, as well as to EGF and PDGF, was enhanced significantly. Exposing quiescent cells to reduced oxygen enhanced serum-induced DNA synthesis in a time-dependent manner. The stimulatory effect persisted when the oxygen concentration was raised to ambient levels before the addition of serum. These results suggest that oxygen concentration within the physiologic range may control proliferation indirectly by altering the activity of a stable intermediate that regulates the cellular response to growth factors.     

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture

Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.     

Regulation of fibronectin by platelet-derived growth factors in cultured rat thoracic aortic smooth muscle cells

Induction of Fibronectin (FN) gene expression by platelet-derived growth factor (PDGF) isoforms in rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances FN levels in SMC cultures in a time- and concentration-response fashion. PDGF-AA and PDGF-AB show no effect on FN levels. The effects of insulin and insulin-like growth factor-I (IGF-I) on PDGF-BB-induced FN levels were examined. No additivity of FN levels is observed between PDGF-BB and insulin and/or IGF-I. Experiments also show that PDGF-BB enhances FN mRNA levels, implying that acquisition of additional FN mRNA units accounts for the increase in FN levels. Induction of FN and FN mRNA levels by PDGF-BB could be one of the initial events in vascular SMC proliferation and extracellular matrix expansion, leading to atherosclerosis and hypertension.     

Regulation of NaK-ATPase by platelet-derived growth factors in cultured rat thoracic aortic smooth muscle cells

Regulation of (Na⁺ + K⁺)-adenosine triphosphatase (NaK-ATPase) by platelet-derived growth factor (PDGF) in cultured rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances SMC proliferation and NaK-ATPase activity. Ouabain, an inhibitor of NaK-ATPase activity, prevents PDGF-BB-induced SMC proliferation. As shown by Western blot and immunochemiluminescence analysis, PDGF-BB also enhances ₁, truncated ₁, and ₁ NaK-ATPase subunit levels. PDGF-AA and PDGF-AB show no effect on ₁ and truncated ₁ levels in slot blot analysis. Induction of NaK-ATPase subunit levels by PDGF-BB could be one of the initial events in vascular SMC proliferation.     

Differences in growth response to hydrocortisone and ascorbic acid by human diploid fibroblasts

Summary The effects of hydrocortisone and ascorbic acid on growth parameters were measured in human diploid skin fibroblasts from fetal and adult donors. In the presence of culture medium containing 10% fetal bovine serum, 0.3 μM hydrocortisone produced a 20% increase in the population growth rate and a 50 to 70% increase in the confluent density of fibroblasts from adult donors. Daily addition of 28 μM ascorbic acid also stimulated the population growth rate and cell density at confluency. The effects of hydrocortisone and ascorbic acid on the final cell density were additive. The action of hydrocortisone was restricted to cells in log-phase growth, whereas ascorbic acid affected cells in both the log and the postconfluent phases of the growth cycle. In fibroblasts from fetal donors, ascorbic acid was stimulative but hydrocortisone was not. The data suggest that whereas both compounds stimulate cell growth in an additive manner, they do so by different cellular mechanisms. This investigation was supported in part by USPHS Grants AM 02456, AM 05020 and AM 15312, and by the Kroc Foundation, No. UW 63-2986. Dr. Rowe is a fellow of the Helen Hay Whitney Foundation. Dr. Fujimoto is a recipient of a Research Career Development Award, AM 47142, from NIAMDD.     

Ca2+]i independent mitogenesis in cultured human fibroblasts revealed by single cell microfluorimetry

A transient rise in intracellular free Ca2+ concentration ([Ca2+]i) has been implicated in mitogenic induction of cell division. Individual human foreskin fibroblasts in confluent cultures examined with the Ca2+ indicator Fura-2 and a fluorescence microscope-imaging system had a basal [Ca2+]i which varied markedly from cell-to-cell. A transient serum-induced rise in [Ca2+]i was demonstrated the magnitude of which was directly correlated with the basal [Ca2+]i level. In contrast to serum-induced increase in [Ca2+]i, exposure to an elevated level of extracellular Ca2+, which is at least equally mitogenic for fibroblasts, did not alter the basal [Ca2+]i of single subconfluent cells or confluent cells. Elevated extracellular Ca2+ does not exert its mitogenicity via a transient rise in [Ca2+]i.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

Obligatory action of polypeptide growth factors for the IL-1-mediated prostaglandin E2 production in fibroblasts. Potential role of growth factors in modulation of tissue response to IL-1

Our studies show that in connective tissue cells, induction of PGE2 synthesis in response to IL-1 requires costimulation with platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). In cells incubated in medium containing fresh serum, IL-1 induced a dose-dependent synthesis of PGE2. However, when the cells were incubated in medium containing low serum or platelet poor plasma (lacking PDGF), IL-1 alone failed to induce PGE2 synthesis. PGE2 synthesis was restored when platelet poor plasma was supplemented with PDGF. Addition of PDGF or FGF together with IL-1 resulted in a 14- and 66-fold stimulation of PGE2 synthesis, respectively. Stimulation was dependent on the concentration of both IL-1 and the growth factor. PGE2 synthesis was also dependent on the synthesis of new proteins. In cells simultaneously treated with IL-1 and PDGF, PGE2 synthesis was initiated after a lag of 2 to 3 h, proceeded first with a rapid rate for 6 h, and then with a slower rate through 24 h. PGE2 synthesis during the latter, slower phase was greatly enhanced by pretreatment with PDGF, but not by pretreatment with IL-1. PDGF pretreatment also resulted in maintenance of 10- to 12-fold higher cell surface IL-1-binding during this phase. These data provide evidence for potentially novel interactions between PDGF and IL-1 activities, one of which is the modulation of IL-1 receptors by PDGF. Furthermore, these studies suggest that by virtue of their effect on IL-1 activities, PDGF and FGF may play additional roles in connective tissues, including an indirect role in inflammatory processes.