Noncollagen Protein Synthesis Research Articles

Inhibition of pleural mesothelial cell collagen synthesis by nitric oxide

The pleural mesothelial cell has a critical role in repairing the mesothelium after injury via its ability to produce connective tissue macromolecules. We have recently shown that proinflammatory cytokines and lipopoly-saccharide induce pleural mesothelial cells to produce nitric oxide. The present study examined the effect of nitric oxide on pleural mesothelial cell protein synthesis. Rat pleural mesothelial cells were exposed to various combinations of tumor necrosis factor, interleukin-1, interferon-γ, and lipopolysaccharide or to the nitric oxide donors: 6-morpholino-sydnonimine, S-nitroso-N-acetyl- d,l-penicillamine, sodium nitroprusside, and spermine-NO adduct for 24–48 h. Nitrate and nitrite (an index of nitric oxide production) and net collagen and noncollagen protein production (uptake of 3H-proline into collagenase-sensitive protein) were then determined. Net collagen production was significantly inhibited by the cytokine-lipopolysaccharide combinations tested. Collagen inhibition paralleled the time course of increased nitric oxide production. The inhibition of collagen production was also significantly reversed by the addition of N G-nitro-L-arginine methyl ester, and was reproduced by the addition of a 5:1 molar excess of Larginine to N G-nitro-L-arginine methyl ester. Additionally, nitric oxide-generating compounds significantly inhibited collagen production in a dose-dependent manner compared to unexposed control cells. Net collagen production was inhibited to a greater degree than noncollagen protein synthesis. These results suggest that nitric oxide may be a significant mediator of PMC collagen production during conditions of significant pleural inflammation.

Advanced age alone does not suppress anastomotic healing in the intestine

Because retrospective clinical studies yield conflicting results and experimental data are completely absent, this study was performed to determine whether anastomotic repair in the intestine deteriorates with age. Ileal and colonic anastomoses were constructed in two groups of healthy rats, ages 2 to 3 months and 27 to 30 months, respectively. Healing was assessed, both 3 and 7 days after operation, by measuring anastomotic bursting pressure, breaking strength, and collagen content, the latter both biochemically (hydroxyproline) and morphometrically. In addition, the ex vivo collagen synthetic capacities were compared. The development of anastomotic strength was similar in young and old rats; average strength increased from 3 to 7 days and was never lower in the older animals. This was true for both bursting pressure and breaking strength. The collagen production capacity was suppressed in the old rats, particularly in the ileum (p < 0.05), whereas the synthesis of noncollagenous protein remained unaltered. However, this did not result in a reduced accumulation of collagen in the anastomotic area--both anastomotic hydroxyproline content and the volume percentage of collagen in the actual wound area were unchanged. Advanced age per se does not affect development of strength or deposition of collagen during early repair of intestinal anastomoses.

Cerium stimulates protein biosynthesis in rat heart in vivo.

The aim of the study was to ascertain whether Ce, a lanthanide that has been implicated in the pathogenesis of tropical endomyocardial fibrosis, interferes with the biosynthetic repertoire of the cardiac muscle in vivo. Female Sprague-Dawley rats received Ce chloride iv at 1.3 mg/kg body wt.; controls received an equal volume of physiological saline. Rates of protein synthesis and transcription in cardiac muscle, measured in terms of incorporation of (3H)-phenylalanine and (3H)-uridine, respectively, into trichloroacetic acid-insoluble material were found to be significantly higher in Ce-treated animals. As low levels of Ce were earlier shown to stimulate collagen as well as noncollagen protein synthesis in cardiac fibroblasts in vitro, the stimulatory effect of the element in vivo reported here supports the speculation that it may influence the expression of proteins like collagen in the heart and contribute to their accumulation as in endomyocardial fibrosis.

Glutamine increases collagen gene transcription in cultured human fibroblasts

We have previously shown that glutamine stimulates the synthesis of collagen in human dermal confluent fibroblast cultures (Bellon, G. et al. [1987] Biochim. Biophys. Acta, 930, 39–47). In this paper, we examine the effects of glutamine on collagen gene expression. A dose-dependent effect of glutamine on collagen synthesis was demonstrated from 0 to 0.25 mM followed by a plateau up to 10 mM glutamine. Depending on the cell population, collagen synthesis was increased by 1.3-to 2.3-fold. The mean increase in collagen and non-collagen protein synthesis was 63% and 18% respectively. Steady-state levels of α1(I) and α1(III) mRNAs, were measured by hybridizing total RNA to specific cDNA probes at high stringency. Glutamine increased the steady-state level of collagen α1(I) and α1(III) mRNAs in a dose-dependent manner. At 0.15 mM glutamine, collagen mRNAs were increased by 1.7-and 2.3-fold respectively. Nuclear run-off experiments at this concentration of glutamine indicated that the transcriptional activity was increased by 3.4-fold for the pro α1(I) collagen gene. The effect of glutamine on gene transcription was also supported by the measurement of pro α1(I) collagen mRNA half-life since glutamine did not affect its stability. Protein synthesis seemed to be required for the glutamine-dependent induction of collagen gene expression since cycloheximide suppressed the activation. The effect of glutamine appeared specific because analogues and/or derivatives of glutamine, such as acivicin, 6-diazo-5-oxo- l-norlleucine, homoglutamine, ammonium chloride and glutamate did not replace glutamine. The influence of amino acid transport systems through plasma membrane was assessed by the use of 2(methylamino)-isobutyric acid and β2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. The glutamine-dependent induction of collagen gene expression was found to be independent of transport system A but dependent on transport system L whose inhibition induced a decrease in pro α1 (I) collagen gene transcription by an unknown mechanism. Thus, glutamine, at physiological concentrations, indirectly regulates collagen gene expression.

Response of human gingival fibroblasts to prostaglandins.

The effects of various prostaglandins (PGs) on the functions of human gingival fibroblasts (Gin-1 cells; ATCC CRL 1292) were examined by phase-contrast microscopy, cell-counting and radioautographic experiments. Tested PGs were PGA1, PGA2, PGB1, PGB2, PGD2, PGE1, PGE2, PGF1 alpha, PGF2 alpha, PGI2, 6-keto-PGF1 alpha, 9 alpha-11 alpha-methanoepoxy-PGF2 alpha, and thromboxane (TX) B2. PGA1 and PGD2 at 30 microM caused morphological deformation of Gin-1 cells. All the PGs tested at 30 microM suppressed the proliferation of Gin-1 cells in the logarithmic growth phase. Furthermore, all the PGs tested at 10 microM suppressed DNA synthesis, collagen synthesis, and noncollagenous protein synthesis in confluent Gin-1 cells, while exerting no effect on GAG synthesis. The concentrations of PGs used are beyond those found in healthy gingiva. However, in periodontitis the local concentrations of some PGs within the gingiva are expected to be extremely elevated beyond the physiological level. These results suggest that PGs may play an important role as a negative regulator in metabolism and some pathologic gingival conditions by suppressing the functions of gingival fibroblasts.

Fibroblast responses to cytokines are maintained during aging.

Impaired wound healing in older individuals may result from a global deficit of fibroblast activity or a decreased response of aged fibroblasts to stimulation by inflammatory cytokines. Twenty-eight fibroblast cell strains were developed from normal human skin aged 3 days to 84 years. Mitogenic response to cytokines, epidermal growth factor, tumor necrosis factor-alpha, platelet-derived growth factor or fetal bovine serum was determined using a 4-hour 3H-thymidine incorporation assay. Synthesis of collagen and noncollagen proteins was determined basally and in response to transforming growth factor-beta using a 6-hour 3H-proline incorporation assay. Neither the mitogenic response to cytokine stimulation (p > 0.3) nor the synthesis of collagen and noncollagen protein after transforming growth factor-beta stimulation (p > 0.4) varied with the age of the cell donor. Individual cell lines' response to cytokine stimulation varied widely, reflecting differences commonly seen in patients' wound-healing abilities. Cellular responses to wound-healing cytokines are preserved as people age. Abnormalities in wound healing in older patients may be the result of altered immune initiation of healing or the cumulative result of concomitant disease.

Endothelin Receptors, Second Messengers, and Actions in Bone

Endothelins are a class of peptides that are produced by and elicit responses in many tissues. A growing literature documents the presence and effects of endothelins in bone. Both endothelinA and endothelinB receptors have been demonstrated in osteoblastic cells by ligand binding. Major signal transduction pathways for endothelin in bone cells appear to be stimulation of phospholipid turnover, by activation of A, C and D phospholipases, stimulation of calcium flux from intracellular and extracellular stores and activation of tyrosine kinases. Endothelins also modulate calcium signaling elicited by other agents in osteoblastic cells. The parathyroid hormone-stimulated calcium transient in UMR-106 cells is enhanced by endothelins, acting through an endothelinB receptor, whereas the parathyroid hormone-stimulated increase in cyclic AMP is inhibited by endothelins. Phenotypic responses to endothelin-1 include changes in alkaline phosphatase activity, stimulation of osteocalcin and osteopontin message, stimulation of collagen and noncollagenous protein synthesis, inhibition of osteoclast motility and stimulation of prostaglandin-dependent resorption. Endothelin-1 also enhances the interleukin-1-induced increase in interleukin-6. Endothelins can also potentially affect calcium metabolism through their actions to inhibit the secretion of parathyroid hormone.

Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells In vitro

To characterize the mineralized nodules produced by rat periodontal ligament (PDL) cells in vitro, we have studied the synthesis and distribution of mineralized tissue proteins at various stages of nodule formation. PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of ascorbic acid (50 micrograms/ml), dexamethasone (5 microM), and beta-glycerophosphate (10 mM) for 3 weeks. Four stages showing distinct morphological characteristics during development of mineralized nodules were identified. Protein synthesis and deposition of proteins into the matrix were studied during these stages by metabolic labeling with [35S]methionine for 24 hours. Large quantities of SPARC (secreted protein, acidic and rich in cysteine) were synthesized by confluent cells but decreased during the progress of mineralized nodule formation. Two forms of osteopontin (OPN) (67 kDa and 61 kDa) were synthesized in comparable quantities by confluent cells; OPN and bone sialoprotein (BSP) were induced by dexamethasone and represented the major proteins in the mineralized matrix. The 67 kDa form of OPN was the predominant species in the mineralized matrix. Both OPN and BSP were localized by immunogold electron microscopy on globular as well as fused electron-dense structures at sites of tissue mineralization.

Changes in Biocompatibility of Dialysis Fluid during Its Dwell in the Peritoneal Cavity

To evaluate the changes in biocompatibility of peritoneal dialysis solutions during intraperitoneal dwell. We studied the effect of the drained dialysates at time 0 and after 30, 60, 120, 240, and 360 minutes of intraperitoneal dwell on the growth of peritoneal mesothelial cells and fibroblasts and the synthesis of proteins by these cells. On one day the patients were dialyzed with glucose-based Dianeal and on alternate days with an amino acid-containing solution based on Travasol. Dialysates were collected from 4 patients during continuous ambulatory peritoneal dialysis (CAPD) training. Unused dialysis solutions containing glucose or amino acids inhibit growth of mesothelial cells and fibroblasts. Dialysates obtained after 30 or 60 minutes of intraperitoneal dwell support the growth of these cells in a way similar to 10% fetal calf serum, but dialysates drained after a longer dwell of 120-360 minutes had a stronger effect on growth of these cells than did serum. All glucose-based dialysates stimulate the synthesis of collagen in mesothelial cells, whereas they reduce the synthesis of non-collagen proteins. All glucose-based dialysates reduce the synthesis of collagen and non-collagen proteins in fibroblasts compared with the production of these proteins in the presence of serum. Changes in the properties of the dialysis solutions during their intraperitoneal dwells do not seem to increase their biocompatibility. Indeed, excessive mitogenic effect and the stimulation of collagen synthesis of the dialysates may induce pathological changes in the peritoneum.

Cyclosporin A and FK-506 in inhibition of rat ito cell activation in vitro

Ito cells are the primary matrix-producing cells in the liver. In hepatic fibrosis in vivo or culture on plastic, these cells undergo activation, a process characterized by cell proliferation, fibrogenesis, and smooth muscle alpha-actin expression. The cytosolic-binding proteins of cyclosporin A (CsA) and FK506 accelerate folding of various proteins including collagen and become inactivated by binding to those agents. CsA is shown to inhibit collagen synthesis in cultured fibroblasts. These findings prompted us to examine the effect of cyclosporin A and FK506 on Ito cell activation. CsA and FK506 reduced DNA synthesis in a dose-related manner, to 26% and 45% of controls at 5 mumol/L, respectively, without affecting total protein synthesis. CsA reduced collagen synthesis in a dose-related manner, to 70% of controls at 5 mumol/L without affecting noncollagenous protein synthesis, whereas FK506 changed neither collagen synthesis nor noncollagenous protein synthesis. Moreover, smooth muscle alpha-actin expression was reduced by 0.5 mumol/L CsA, but not by FK506. CsA merits consideration for the therapy of hepatic fibrosis. FK506 may also be a candidate for such therapy through inhibitory action on Ito cell proliferation.

Cyclosporin a and fk-506 in inhibition of rat ito cell activation in vitro

Ito cells are the primary matrix-producing cells in the liver. In hepatic fibrosis in vivo or culture on plastic, these cells undergo activation, a process characterized by cell proliferation, fibrogenesis, and smooth muscle α-actin expression. The cytosolic-binding proteins of cyclosporin A (CsA) and FK506 accelerate folding of various proteins including collagen and become inactivated by binding to those agents. CsA is shown to inhibit collagen synthesis in cultured fibroblasts. These findings prompted us to examine the effect of cyclosporin A and FK506 on Ito cell activation. CsA and FK506 reduced DNA synthesis in a dose-related manner, to 26% and 45% of controls at 5 μmol/L, respectively, without affecting total protein synthesis. CsA reduced collagen synthesis in a dose-related manner, to 70% of controls at 5 μmol/ L without affecting noncollagenous protein synthesis, whereas FK506 changed neither collagen synthesis nor noncollagenous protein synthesis. Moreover, smooth muscle α-actin expression was reduced by 0.5 μmol/L CsA, but not by FK506. CsA merits consideration for the therapy of hepatic fibrosis. FK506 may also be a candidate for such therapy through inhibitory action on Ito cell proliferation.

Autogenous flexor tendon grafts: Fibroblast activity and matrix remodeling in dogs

To investigate rates of cellular proliferation and matrix turnover in autogenous flexor tendon grafts, hindlimb intrasynovial (flexor digitorum profundus) and extrasynovial (peroneus longus) tendons were placed within the synovial sheaths of the medial and lateral forepaw digits of 18 dogs and treated with controlled early passive motion. After the dogs had been killed, short-term culture and labeling in vitro were utilized to determine rates of DNA, proteoglycan, collagen, and noncollagen protein synthesis. Schiff base covalent collagen crosslink concentrations and total collagen and protein content also were evaluated at intervals through 6 weeks. Tendon grafts of extrasynovial origin showed greater rates of DNA synthesis and significantly elevated levels of proteoglycan, collagen, and noncollagen protein synthesis and Schiff base covalent collagen crosslink concentrations (dihydroxylysinonorleucine) compared with intrasynovial tendon grafts. It was not clear to what extent the increased activity in the extrasynovial graft was due to actual differences between the intrasynovial and extrasynovial tendons or to the responses of the connective tissue surrounding the extrasynovial tendon graft. Since both types of grafts demonstrated similar unaltered levels of collagen and protein content over time, these data suggest greater rates of matrix turnover in tendon grafts of extrasynovial origin than in those of intrasynovial origin. Coupled with previous findings showing increased cellular proliferation in extrasynovial tendon grafts, these data indicate that the process of translation to an intrasynovial environment necessitates a more active process of soft-tissue repair and remodeling when extrasynovial donor tendons are used.

Oncostatin M Stimulates Collagen and Glycosaminoglycan Production by Cultured Normal Dermal Fibroblasts: Insensitivity of Sclerodermal and Keloidal Fibroblasts

It is thought that normal fibrotic repair progresses to dermal fibrosis when fibroblasts are activated persistently by chronic exposure to cytokines such as transforming growth factor-beta. However, additional cytokines and mechanisms may play a role in the development of fibrosis. Thus, we examined a recently described T-lymphocyte/macrophage-derived cytokine, oncostatin M, for its effect on the production of collagen and glycosaminoglycan by microcultures of normal dermal, sclerodermal, and keloidal fibroblasts. Treatment with oncostatin M for 48 h induced dose-dependent (1-100 ng/ml) increases in the collagen and glycosaminoglycan production of nine normal fibroblast strains, which in the absence of fetal bovine serum at 100 ng/ml averaged 196% and 244%, respectively. Oncostatin M treatment increased both types I and III procollagens and their mRNA transcripts, as well as levels of hyaluronic acid, chondroitin-4/6 sulfates, and dermatan sulfate, but not fibronectin or general noncollagenous protein synthesis. In contrast, the collagen production of six of eight sclerodermal and keloidal fibroblast strains was essentially unresponsive to oncostatin M treatment, with 100 ng/ml inducing an average increase of only 34% for the eight fibrotic strains. Oncostatin M stimulation of fibrotic fibroblast glycosaminoglycan production was also hyporesponsive, as 100 ng/ml of oncostatin M induced an average increase of only 101%. These results indicate that oncostatin M could function as a stimulator of normal fibrotic repair via activation of fibroblast collagen and glycosaminoglycan synthesis and that the persistent activation of sclerodermal and keloidal fibroblasts is accompanied by a loss of sensitivity to oncostatin M stimulation.

Tranilast, a selective inhibitor of collagen synthesis in human skin fibroblasts.

Effects of tranilast, N-(3,4-dimethoxycinnamoyl)anthranilic acid, on collagen synthesis in cultured human skin fibroblasts were studied. Tranilast was found to inhibit collagen synthesis in a dose-dependent manner to a maximum of 55% at 300 microM during 48 h of treatment; the synthesis of type I and type III collagens was equally affected. Administered simultaneously or subsequently, tranilast reduced the stimulatory effect of transforming growth factor beta 1 (2.5 ng/ml) on collagen synthesis without affecting the accompanying stimulation of noncollagen protein synthesis. It did not affect prolyl or lysyl hydroxylase activity in vitro and in cells. The content of pro alpha 1(I) collagen mRNA was decreased 60% by tranilast. Tranilast prevented the TGF beta 1-mediated increase in pro alpha 1(I) collagen mRNA. These results indicate that tranilast specifically inhibits collagen production at a pretranslational level by interfering with TGF beta 1 effects. Tranilast also inhibited collagen synthesis in scleroderma fibroblasts to the same extent and in keloid fibroblasts to a greater extent than in normal fibroblasts, attesting to its therapeutic potential as an antifibrotic drug.

Cadmium selectively inhibits fibroblast procollagen production and proliferation.

Chronic inhalation of cadmium fumes has been associated with the development of emphysema, a disease characterized by extensive disruption of lung connective tissue. Cadmium is also an important contaminant of tobacco and may play a role in cigarette smoking-related emphysema. In this paper we investigated the effect of nontoxic doses of cadmium chloride (CdCl2) on fibroblast procollagen production and proliferation, key features of connective tissue repair following injury. CdCl2 inhibited fibroblast procollagen production in a dose-dependent manner in two different cell lines. For fetal rat fibroblasts, maximal effects were observed at 10 microM CdCl2, with values reduced by 82 +/- 6% (mean +/- SE, n = 6, P < 0.01) relative to control cells. In contrast, noncollagen protein synthesis by these cells was enhanced in the presence of CdCl2. In human fetal lung fibroblasts (HFL1), maximal inhibition of procollagen production (83 +/- 2%, P < 0.01) was observed at 40 microM CdCl2, whereas noncollagen protein synthesis was unaffected. In both cell lines the inhibition of procollagen production was shown to be due to decreased procollagen synthesis and an increase in the proportion of newly synthesized procollagen degraded. Cadmium also affected fibroblast proliferation in response to 2% serum, with values for fetal rat cells depressed by 17 +/- 4, 72 +/- 2, and 86 +/- 4% (all P < 0.01) compared with controls at 1, 5, and 10 microM CdCl2, respectively. These data show that cadmium selectively inhibits fibroblast procollagen production and also attenuates their mitogenic response to serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone cells from spinal hyperostotic mouse (twy/twy) maintain elevated levels of collagen productionin vitro

The twy (tiptoe-walking-Yoshimura) mouse is a mutant having a systemic hyperostotic disposition. Osseous overgrowth occurs not only in the spine but also in other skeletal tissues including the calvarium. To clarify the pathogenic mechanism of hyperostotic lesions, bone cells were cultured from twy and normal mouse calvariae to analyze the growth and extracellular matrix production in both genotypes. The cultures of osteoblast-enriched cells were established by explant procedure. The cells of the twy genotype showed accelerated growth and elevated levels of collagen synthetic activity compared to normal cells. However, no significant difference was noted in noncollagenous protein synthesis between the two genotypes. Furthermore, the mutant cells maintained these abnormal functions during several cell passagesin vitro. These results indicated that an unusual phenotype of twy bone cells distinguished by accelerated growth and selective stimulation of collagen production may play a major role in the development of hyperostotic lesions in twy micein vivo.

Activation of fibroblast proliferation by Werner's syndrome fibroblast-conditioned medium

The effects of Werner's syndrome (WS) fibroblast-conditioned medium on cell proliferation and collagen production of normal fibroblasts were studied using four WS fibroblast strains. The conditioned medium from the WS fibroblasts brought about activation of normal fibroblast proliferation, whereas, that from late passage normal fibroblasts and fibroblasts from aged donors' skin did not. Collagen and non-collagenous protein synthesis in normal fibroblasts were increased by the addition of conditioned medium from the WS fibroblasts, but the relative rates of collagen synthesis and non-collagenous protein synthesis were unaltered. These results suggest that activation of fibroblast proliferation by conditioned medium is one of the characteristics of WS fibroblasts.

Cell stage-dependent effects of ascorbic acid on cultured porcine bone cells

Pig bone cells were isolated from fetuses or young animals. In culture, they proliferated for 6 days, became confluent and began differentiating. The effects of ascorbic acid (AsA) on cell proliferation, alkaline phosphatase (ALP) activity, non-collagenous protein (NCP) and collagen synthesis, were studied by adding AsA to the medium at different times during culture. AsA affected fetal and post-natal cells similarly: ALP activity, NCP and collagen synthesis were markedly reduced in cells treated before confluence, but were strongly and dose-dependently stimulated in cells treated after confluence. AsA also stimulated cell proliferation. The cell stage-dependent action of AsA suggests that it may interfere with differentiation. The effects of AsA on ALP activity and DNA content were not coupled to its effect on collagen synthesis, raising the question of whether AsA action is matrix-mediated.

Inhibition of collagen hydroxylation by lithospermic acid magnesium salt, a novel compound isolated from Salviae miltiorrhizae Radix

We have screened several chinese medicinal herbs for the presence of antifibrotic agents. An aqueous extract of Salviae miltorrhizea Radix was found to inhibit collagen secretion by human skin fibroblasts without affecting DNA or noncollagen protein synthesis. We have subsequently purified the material exhibiting the inhibitory activity and identified it as magnesium lithospermate. From its chemical structure this compound was predicted to be an inhibitor of the post-translational modifying enzymes prolyl and lysyl hydroxylases in collagen biosynthesis. Accordingly, it decreased the extent of prolyl and lysyl hydroxylations in collagen by approx. 50%. Added to cell extracts it inhibited both prolyl and lysyl hydroxylase activities, but only lysyl hydroxylase activity when added to intact cells. Oral administration of this compound to mice led to a significant reduction of prolyl hydroxylation in newly-synthesized skin collagen. This naturally-occurring compound thus offers a potential means for treating fibrotic diseases, such as systemic scleroderma and keloid.

Effects of transforming growth factor-beta 1 and fibroblast growth factor on DNA synthesis in growth plate chondrocytes are enhanced by insulin-like growth factor-I.

The local tissue metabolism is controlled through the complex interaction between systemic and local growth factors. In recent years, an increasing number of autocrine or paracrine growth regulators have been identified in physeal cartilage. While these factors act to alter chondrocytes phenotypically and presumably are important mediators in the process of endochondral ossification, the manner in which they interact with the systemically regulated growth factor insulin-like growth factor-I is unknown. In the present study, the interactive effects of insulin-like growth factor-I with transforming growth factor-beta 1 or basic fibroblast growth factor were examined in short-term monolayer cultures of chick growth plate chondrocytes. [3H]thymidine incorporation was maximally stimulated 11-fold by fibroblast growth factor (10 ng/ml) and 3.5-fold by transforming growth factor-beta 1 following a 24-hour exposure in serum-containing cultures. The effects of transforming growth factor-beta 1 and fibroblast growth factor at both high and low concentrations were enhanced in a dose-dependent manner by insulin-like growth factor-I, with a 40-50% increase in DNA synthesis in the presence of 100 ng/ml of insulin-like growth factor-I. Since insulin-like growth factor-I increased [3H]thymidine incorporation after 48 hours (50% increase) but not after 24 hours of exposure, these observations represent a synergistic interaction. Total DNA in cultures treated for 5 days confirmed the modulating effect of insulin-like growth factor-I with transforming growth factor-beta 1 and fibroblast growth factor. The growth factors were further examined for their effects on markers of chondrocyte differentiation. While all three caused a dose-dependent inhibition of alkaline phosphatase activity, the effects of insulin-like growth factor-I were additive only to those of transforming growth factor-beta 1 and fibroblast growth factor. Similarly, insulin-like growth factor-I did not affect the sulfate incorporation stimulated by fibroblast growth factor or transforming growth factor-beta 1. Insulin-like growth factor-I had no effect on total protein synthesis after 24 hours and, although type-II collagen mRNA levels were stimulated, it had no effect on type-X collagen mRNA, as determined by quantitative in situ hybridization. Finally, insulin-like growth factor-I did not alter the dose-dependent stimulation of noncollagen protein synthesis and the inhibition of collagen synthesis caused by fibroblast growth factor and transforming growth factor-beta 1 in 24-hour cultures. Thus, the data suggest that insulin-like growth factor-I may have a role in augmenting the effects of other growth factors found in cartilage.(ABSTRACT TRUNCATED AT 400 WORDS)