Connective tissue activation in guinea pig lung fibroblast cultures: regulatory effects of glucocorticoids

Earlier studies showed that guinea pig lung fibroblasts in cell culture could be "activated" by naturally occurring peptides with a resultant increase in glycolysis and glycosaminoglycan formation. Such connective tissue activation (CTA) in human cell systems (synovial, cartilage, dermal) has proved a useful tool for studying the mechanisms of inflammation and dissecting the efficacy and actions of anti-inflammatory drugs. The present study examined the consequences of treating basal and activated guinea pig lung fibroblasts with glucocorticoids. The data indicate that glucocorticoids minimally suppress glycosaminoglycan (GAG) synthesis in nonactivated cultures. Further, CTA was inhibited to only a minor degree in activated lung fibroblast cultures by steroids, and even markedly supraphysiologic concentrations of glucocorticoids were not notably inhibitory. It was of interest that thiols enhanced suppression of incremental GAG synthesis by some glucocorticoids in activated lung fibroblast cultures.     

Lipid metabolism in cultured cells. Activators of endogenous thromboxane A2 synthesis in cultured lung fibroblasts

Thromboxane A2 (rabbit aorta-contracting substance) is a proaggregatory vasoconstrictive, oxygenated metabolite of arachidonic acid which was originally discovered in guinea pig lung perfusates during antigen-induced anaphylaxis. The specific stimuli which activate synthesis and the cellular source in the lung remain undefined. In order to study pulmonary thromboxane A2 (TXA2) synthesis, a cultured lung cell model has been used. Monolayer cultures of human diploid embryonic lung fibroblast (WI-38) metabolized exogenously supplied [14C]arachidonic acid to TXA2 as well as prostaglandin E2. Both were unequivocally identified by gas chromatography/mass spectrometry. Cellular phospholipids were labeled by preincubating cultures overnight with [14C]arachidonic acid. Release of thromboxane A2 into the culture fluid from these prelabeled cultures was stimulated by two phospholipase activating agents, mellitin and the calcium ionophore A23187. The lung cells also released TXA2 and prostaglandin in a dose-dependent fashion when treated with thrombin but not when exposed to trypsin. Bradykinin, an anaphylactic mediator in vivo, was a potent TXA2 releasing agent in this in vitro system whereas histamine was inactive. In addition, anaphylactic shock perfusates from guinea pig lung were shown to contain a factor (other than bradykinin) which activates fibroblasts TXA2 synthesis in these cultured lung cells. These experiments indicate that the lung fibroblast is probably a source of pulmonary thromboxane in vivo and that the cultured lung cell system described here is a useful model for defining the complex interactions of mediators of anaphylaxis and asthma.     

Abrogation of apoptosis through PDGF-BB-induced sulfated glycosaminoglycan synthesis and secretion

Platelet-derived growth factor (PDGF)-BB-stimulated glycosaminoglycan (GAG) synthesis/secretion in fetal lung fibroblasts is dependent on sequential activation of the PDGF beta-receptor, phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt-1,2, and the GTPase Rab3D. Because the Akt pathway has been implicated in cell survival mechanisms, we investigated whether the pathway regulating GAG synthesis/secretion was antiapoptotic. PDGF-BB treatment protected fetal lung fibroblasts against serum starvation-induced apoptosis, whereas wortmannin, an inhibitor of PI3K, abrogated this protective effect. Transfection of constitutively active Akt into fetal lung fibroblasts also safeguarded the cells from apoptosis induced by serum starvation. To determine whether the antiapoptotic response was due, at least in part, to GAGs, we treated lung fibroblasts with beta-D-xyloside as well as with topically applied GAGs, specifically those produced by fetal lung fibroblasts. beta-D-xyloside increased GAG synthesis/secretion and diminished apoptosis. Application of sulfated GAGs, chondroitin sulfate, and heparan sulfate, but not nonsulfated hyaluronan, also resulted in diminished apoptosis. Moreover, topically applied sulfated GAGs increased Bcl-associated death promoter phosphorylation and diminished caspase-3 and -7 cleavage, indicating an antiapototic response. These data are compatible with the PDGF-BB-GAG signaling pathway regulating programmed fibroblast death in the fetal lung.     

Stimulation of glycosaminoglycan production in murine tumors

Three types of murine tumors, B-16 melanoma, A-10 carcinoma, and S-180 sarcoma, were shown to contain elevated glycosaminoglycan (GAG) concentrations in vivo as compared to normal muscle or subcutaneous tissue. Hyaluronate was especially concentrated in the A-10 carcinoma, which contained approximately six times more hyaluronate than subcutaneous tissue and 18 times more than muscle. In all three tumors, chondroitin sulfates, especially chondroitin-4-sulfate, were present in higher concentrations than in the normal tissues. In culture, however, all three tumor cell lines produced less than 5% as much GAG as mouse fibroblasts, when measured by incorporation of [3H] acetate or by chemical analysis. Varying the culture passage number or the medium composition, ie, glucose, serum, and insulin concentrations, had little effect on GAG synthesis by the tumor cells. The low GAG levels in the tumor cell cultures were not due to hyaluronidase activity in their media. In an attempt to mimic possible host-tumor cell interactions that could account for the elevated GAG levels in vivo, tumor cells were cocultured with fibroblasts, but no stimulation above the amount made by the tumor cells alone plus that by the fibroblasts alone was observed. Conditioned media from the tumor cells, either dialyzed or not against fresh complete medium, had no effect on fibroblast GAG synthesis. Tumor extracts, however, were found to stimulate synthesis of hyaluronate by fibroblasts. Stimulation by extracts of A-10 carcinoma was greater than and additive to that of serum. The above results strongly suggest that GAG production in these tumors is in part regulated by host-tumor interactions.     

Stimulation by glucocorticoids of the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture

Hydrocortisone stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype, of rabbit costal chondrocytes in confluent quiescent culture, as judged by the incorporations of [35S]sulfate and [3H]glucosamine. Hydrocortisone also stimulated incorporation of [3H]serine into proteoglycan. The stimulation of GAG synthesis by hydrocortisone was dose-dependent and maximal at a physiological concentration of 10(-7) M. Hydrocortisone also stimulated GAG synthesis in cultures in the log-phase of growth. In this case, its maximal effect was observed at a concentration of 10(-6) M. The magnitude of the increase of GAG synthesis in response to hydrocortisone was larger in confluent culture than in log-phase cultures. Hydrocortisone stimulated DNA synthesis dose-dependently, and its effect was observable at a physiological concentration. However, no stimulation of DNA synthesis by hydrocortisone was observed in serum-free medium, in contrast to that of GAG synthesis. Hydrocortisone also increased protein synthesis and the cell number. Dexamethasone also stimulated the syntheses of both GAG and DNA. These results show that glucocorticoids stimulated both the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture. Moreover, the effect of glucocorticoids was primarily on the differentiated phenotype of chondrocytes and its effect on proliferation was permissive.     

Hyaluronate-cell interaction : Effects of exogenous hyaluronate on muscle fibroblast cell surface composition

Evidence that exogenous hyaluronate (HA) binds to the surface of muscle fibroblast cultures was obtained by incubating confluent fibroblasts with ¹⁴C-HA purified from fibroblast cell surfaces. Surface-bound ¹⁴C-HA was operationally defined as material resistant to six saline washes and solubilized by brief trypsinization. All of the surface-bound radioactivity remains as authentic HA. Exposure of fibroblasts to 100 μg/ml cold HA caused a nearly 3-fold ‘reduction’ in incorporation of isotopic precursors into glycosaminoglycan (GAG); but when intracellular ¹⁴C precursors to GAG were quantitated, the entire ‘reduction’ could be accounted for by decreased precursor uptake. Exposure to exogenous HA altered the distribution of newly synthesized GAG by stimulating an increase in total GAG secreted to the medium at the expense of that bound to the culture surface. Qualitatively, the cell surface ratio of ¹⁴C-HA: ¹⁴C-sulfated GAG (SGAG) of HA-treated cells is about 2.5 times greater than that of untreated cells and the medium ratio is correspondingly reversed. This is primarily the result of stimulated ¹⁴C-SGAG release to the culture medium. Addition of cold HA to prelabeled cultures also stimulates the selective turnover of SGAG from the culture surface. Thus, exposure to HA alters the fibroblast surface by accumulation of exogenous HA as well as by stimulation of SGAG turnover.     

Influence of cyclosporine A on growth and extracellular matrix synthesis of human fibroblasts.

Cyclosporine A (CyA) is a powerful nonsteroidal immunosuppressive agent used to prevent graft rejection of organ and bone marrow transplants. A major side effect observed can be attributed to the fibroblast and its functions: proliferation of fibroblasts and formation of fibrotic tissue in the gingiva (fibrous hyperplasia) and in the kidney are induced. The mechanism of both is still obscure. In order to elucidate whether these side effects are due to the drug acting on human fibroblasts itself or whether they are indirect ones mediated by factors released by lymphocytes, cultures of human gingiva fibroblasts were exposed to CyA under defined in vitro conditions. Incubation with CyA for 72 hours resulted in a dose-dependent stimulation of DNA synthesis, whereas glycosaminoglycan (GAG) synthesis was slightly suppressed. Long-term incubation (6 weeks) with 1 micrograms/ml CyA resulted again in stimulation of growth parameters: compared to the drug-free control, cell number increased to 168%, incorporation of 3H-thymidine into DNA to 143%, and overall protein content to 159%. Collagen and GAG synthesis were elevated to approximately 120%. When corrected for cell number or cell protein content, this represents a decline in matrix synthesis, comparable to short-term incubations. These results indicate that a direct effect of CyA on proliferation of human gingival fibroblasts is responsible for some of the observed hyperplasia.     

Glucose deficiency inhibits glycosaminoglycans synthesis in fibroblast cultures

We decided to study the effect of glucose deprivation on glycosaminoglycan (GAG) synthesis and degradation in fibroblast cultures, vitality of these cells and a correlation of these processes with the expression of oxygen/glucose-regulated proteins (ORP150/GRP170). The incorporation of [³H]-glucosamine into both newly synthesised hyaluronic acid and sulphated GAGs and [³⁵S]-sulphate into GAGs was used as an index of glycosaminoglycan synthesis. Quantitative evaluation of newly synthesised GAGs degradation was determined by pulse-chase experiments. We demonstrated that fibroblasts incubated in high glucose medium synthesised significant amounts of GAGs. Most of them were secreted into the culture medium. The shortage of glucose resulted in about 40% reduction in synthesis of GAGs, both those secreted into culture medium and remaining in the cell layer. The pulse-chase experiments demonstrated that the reduced amount of newly synthesised glycosaminoglycans was protected against intracellular degradation. Proportionally less GAGs were degraded in cultures incubated in low glucose than in high glucose media. These phenomena were accompanied by an increase in the expression of chaperon – ORP150 in cultures growing in low glucose medium. We suggest that the increased expression of ORP150 is a factor which prolongs the cell vitality and protects glycosaminoglycans against intracellular degradation induced by glucose deprivation.     

Effects of human carbonic anhydrase III (CA III) on synovial and muscle fibroblast glycosaminoglycan metabolism

We investigated the ability of CA III, isolated from adult human skeletal muscle, to regulate cell growth and glycosaminoglycan (GAG) formation in connective tissue cells derived from various human tissues. Unlike muscle, dermal, and cartilage fibroblasts, synovial connective tissue cells were substantially activated by CA III and showed enhanced hyaluronic acid (HA) synthesis. Cell culture experiments showed that CA III induced a 2- to 11-fold increase in [14C]HA synthesis by human synovial fibroblasts (SF) in a dose-dependent manner (P less than 0.001); erythrocyte CA I and CA II were inactive. Exposure of SF and muscle fibroblasts to CA III also resulted in a 20-45% and 16-70% increased 35S incorporation into proteoglycans, respectively. When adult human skin and cartilage fibroblasts were studied in the presence of CA III, no differences in the level of DNA and GAG formation were noted. These latter cell types were clearly activated by a platelet (CTAP-III) growth factor. The potential physiological implications of these observations are discussed.     

Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level

Glucocorticoids accelerate fetal lung maturation by acting on the fetal lung fibroblast to induce the synthesis of fibroblast-pneumonocyte factor which in turn stimulates pulmonary surfactant synthesis by the alveolar type II cell. We have studied the site of glucocorticoid regulation of fibroblast-pneumonocyte factor synthesis in primary cultures of fetal rat lung fibroblasts. Conditioned media from fetal rat lung fibroblasts exposed to cortisol stimulate [Me-3H]choline incorporation into saturated phosphatidylcholine by primary cultures of fetal rat lung alveolar type II cells. This effect is blocked by the presence of actinomycin D during the first, but not the second, 24 h of incubation of the fibroblasts with cortisol. Cycloheximide blocks this effect if present during either the first or second 24 h of incubation. We fractionated mRNA from fetal rat lung fibroblasts incubated in the presence or absence of dexamethasone and observed that cell-free translation products from a fraction of approximately 500 bases possess biological activity in the bioassay. Such activity is only present in cell-free translation products of mRNA isolated from fibroblasts treated with dexamethasone. These results suggest that glucocorticoids act at a pretranslational level to induce production of fibroblast-pneumonocyte factor and that the primary translation products are biologically active.     

Characterization of fibroblast proliferation factors elaborated by antigen- and mitogen-stimulated guinea pig lymph node cells: Differentiation from lymphocyte-derived chemotactic factor for fibroblasts,lymphocyte mitogenic factor,and interleukin 1

Guinea pig lymph node cells stimulated in culture by T-cell mitogens or sensitizing antigens release ~60,000- and ~16,000-mol wt proteins that induce normal guinea pig fibroblasts to proliferate in vitro. These fibroblast proliferation factors can be separated from lymphocytederived chemotactic factor for fibroblasts and from lymphocyte mitogenic factor by gel filtration employing Sephadex G-100. The 16,000-mol wt fibroblast proliferation factor was found to coelute with interleukin 1 (IL 1) from gel filtration columns. When the 16,000 molecular weight factor was further analyzed by anion exchange-high-performance liquid chromatography five major peaks containing IL 1 activity were obtained, only one contained fibroblast proliferation activity, suggesting forms of IL 1 exist that are not mitogenic for fibroblast. Occasionally, a large-molecular-weight inhibitor of fibroblast proliferation was detectable in void volume fractions from gel filtration of supernatant from antigen-stimulated lymph node cell cultures. This inhibition was accompanied by gross aggregation of fibroblasts. These studies suggest that fibroblast accumulation at sites of certain cell-mediated immune reactions in vivo may in part be attributable to the release of mediators by lymphocytes and, or macrophages that induce fibroblast growth.     

Temporal and spatial differences in glycosaminoglycan synthesis by fetal lung fibroblasts

In studies of the ontogeny of fibroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [3H]glucosamine and Na2 35SO4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts (t1/2 = 12 h) than from that of epithelial cells (t1/2 = 48 h). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation.     

Cell surface changes correlated with density-dependent growth inhibition. Glycosaminoglycan metabolism in 3T3, SV3T3, and con A selected revertant cells.

A 35SO4-labeling/chromatography technique has been developed which facilitates quantitation of sulfated glycosaminoglycan (GAG) synthesis in mammalian cell cultures. The technique has been used to compare sulfated GAG biosynthesis, degradation, and turnover in three related cell lines with differing degrees of density-dependent inhibition of growth in vitro (Balb/c 3T3, SV3T3, and SV3T3 revertant cells). Viral transformation of Balb 3T3 cells is accompanied by a 2-5-fold decrease in cell associated sulfated GAG. SV3T3 revertant cells, which show partial reversion to low saturation density in vitro, show a 2.5-8-fold increase in cell-associated sulfated GAG compared to the parental SV3T3 cells from which they were selected. In addition, the distribution of 35SO4 and [3H]glucosamine among the different GAG species produced by SV3T3 revertant cells reverts so that it is similar to the distribution characteristic of untransformed 3T3 cells rather than SV3T3 cells. Mild trypsin treatment of 35SO4-labeled cells removed 68-84% of the cellular sulfated GAG, suggesting that at least this proportion of the total cellular sulfated GAG was located at the cell periphery. Removal of 35SO4-labeled cells from the Petri dish with a Ca2+ selective chelating agent revealed a fraction of the sulfated GAG that remained tightly bound to the Petri dish. A higher proportion of the total cell-associated sulfated GAG remained attached to the Petri dish in cultures of untransformed and revertant cells compared to that present in cultures of transformed cells. A role for sulfated GAG in density-dependent growth inhibition of fibroblast cultures is proposed and discussed in the light of the data obtained.     

Hypoxia modifies the effect of PDGF on glycosaminoglycan synthesis by primary human lung cells

Hypoxia, a consequence of interstitial lung diseases, may lead to secondary pulmonary hypertension and pulmonary vascular remodeling. Hypoxia induces activation and proliferation of lung cells and enhances the deposition of extracellular matrix including glycosaminoglycans (GAGs). To elucidate the cell biological mechanisms underlying the development of secondary pulmonary hypertension, we studied the effect of hypoxia on GAG synthesis by human lung cells. GAG synthesis was measured by incorporation of [(3)H]glucosamine; GAGs were isolated, purified, and characterized with GAG-degrading enzymes. Fibroblasts and vascular smooth muscle cells (VSMCs) synthesized hyaluronic acid, heparan sulfate, and chondroitin sulfates, whereas dermatan sulfate was found only in fibroblasts. Hypoxia did not influence the size or charge of the individual GAGs. However, hypoxia inhibited platelet-derived growth factor-induced [(3)H]glucosamine incorporation in secreted GAGs, especially hyaluronic acid, in VSMCs. In contrast, it stimulated GAG secretion, specifically heparan sulfate, by fibroblasts. Our results indicate that hypoxia induces modifications in GAG synthesis by human lung VSMCs and fibroblasts that may be correlated to pathophysiological manifestations in lung diseases causing hypoxia.     

The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.     

Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum.

Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo.     

Accelerated fetal lung maturation by estrogen is associated with an epithelial-fibroblast interaction

Summary The role of epithelial-mesenchymal interactions in the stimulation of lung development by estrogen is now investigated using organ cultures of lung from male and female fetal rats taken from Days 17 to 21 of gestation. Estradiol at 1 μg/ml was found to reduce cell proliferation in explants taken during a rapid growth phase (Day 18) and to stimulate surfactant synthesis in both males and females only in Day 20 explants when cell division is much slower. At this time more epithelial cells from estrogen-treated explants contained lamellar bodies, which were also secreted to fill the air sacs. These cultures also showed a significant increase in the frequency of cell-to-cell contacts between epithelial cells and fibroblasts. Uptake of tritiated estradiol by explants increased from Day 18 onward, and by autoradiography, labeling was located predominantly over fibroblasts. Using pure cultures of fetal and adult cells, uptake of labeled estradiol was significantly higher in fibroblasts than in corresponding epithelial cells, and estradiol did not directly enhance palmitate incorporation into epithelial cells. The results suggest that the earlier maturation and increased surfactant synthesis in female fetal lung is related at least in part to enhanced binding of estrogen by the fibroblast with subsequent transfer of a maturation factor to the fetal epithelium. This research project was supported by grants from the Medical Research Council of Canada and the Council for Tobacco Research, U.S.A., Inc.     

Hyaluronic acid synthesis is absent in normal human endothelial cells irrespective of hyaluronic acid synthetase inhibitor activity, but is significantly high in transformed cells

The characteristics of glycosaminoglycan (GAG) synthesis in normal and transformed human endothelial cells were analyzed by the incorporation of [3H]glucosamine and by the activities of GAG synthetases. The GAG synthesized by normal endothelial cells consisted of mainly heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate but little hyaluronic acid (HA) (less than 1%). The characteristics of GAG synthesis by normal cells reflected the synthetic enzyme activities for each individual GAG: the activity of HA synthetase was very low. In spite of this, the activity of HA synthetase inhibitor, induced in growth-retarded fibroblasts with low HA synthetase activity (Matuoka et al. (1987 J. Cell Biol., 104, 1105-1115), was very low in endothelial cells. In contrast to normal cells, transformed endothelial (ECV304) cells synthesized mainly HA (62% of total GAGs). These findings suggest that the regulatory system of GAG metabolism is cell type specific, and that transformation is accompanied by high levels of HA synthesis in endothelial cells.     

Incorporation of radioactive precursors into glycosaminoglycans by rat muscle fibroblasts exposed to a solubilized rat bone matrix fraction

Confluent cultures of rat muscle fibroblastic cells respond by increased glycosaminoglycan (GAG) synthesis when cultured in medium containing a solubilized bone matrix fraction (SBM) at a concentration of 100 micrograms/ml. The metabolism of the GAG associated with the cell pellet, the cell surface and the tissue culture medium fractions was studied, in the presence and absence of SBM, by measuring the incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the isolated GAG. Net synthesis of hyaluronic acid and of chondroitin sulfate in the medium fraction increased more rapidly in cultures containing SBM compared to controls, and the accumulation of labelled GAG in the medium of the treated cultures was approximately linear with respect to the length of incubation. The addition of SBM also resulted in increased incorporation of 3H and of 35S into the GAG of the cell surface and cell pellet fractions. In these fractions, stimulation of incorporation of radioactivity occurred in two waves: an early, relatively minor increase and a later relatively major increase. The relatively major stimulation of radioactivity into the GAG of the cell surface fraction occurred between 24 and 48 h and was independent of any apparent effect of serum.     

Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells

Recent investigations credited important roles to C-type natriuretic peptide (CNP) signaling during chondrogenesis. This study investigated the putative role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10^?8 and 10^?7 M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10^?6 M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect.