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1.
Inhibitors of p38 mitogen-activated protein kinase (MAPK) diminish inflammatory arthritis in experimental animals. This may be effected by diminishing the production of inflammatory mediators, but this kinase is also part of the IL-1 signal pathway in articular chondrocytes. We determined the effect of p38 MAPK inhibition on proliferative and synthetic responses of lapine chondrocytes, cartilage, and synovial fibroblasts under basal and IL-1-activated conditions.Basal and growth factor-stimulated proliferation and proteoglycan synthesis were determined in primary cultures of rabbit articular chondrocytes, first-passage synovial fibroblasts, and cartilage organ cultures. Studies were performed with or without p38 MAPK inhibitors, in IL-1-activated and control cultures. Media nitric oxide and prostaglandin E2 were assayed.p38 MAPK inhibitors blunt chondrocyte and cartilage proteoglycan synthesis in response to transforming growth factor beta; responses to insulin-like growth factor 1 (IGF-1) and fetal calf serum (FCS) are unaffected. p38 MAPK inhibitors significantly reverse inhibition of cartilage organ culture proteoglycan synthesis by IL-1. p38 MAPK inhibition potentiated basal, IGF-1-stimulated and FCS-stimulated chondrocyte proliferation, and reversed IL-1 inhibition of IGF-1-stimulated and FCS-stimulated DNA synthesis. Decreases in nitric oxide but not prostaglandin E2 synthesis in IL-1-activated chondrocytes treated with p38 MAPK inhibitors are partly responsible for this restoration of response. Synovial fibroblast proliferation is minimally affected by p38 MAPK inhibition.p38 MAPK activity modulates chondrocyte proliferation under basal and IL-1-activated conditions. Inhibition of p38 MAPK enhances the ability of growth factors to overcome the inhibitory actions of IL-1 on proliferation, and thus could facilitate restoration and repair of diseased and damaged cartilage. 相似文献
2.
T I Morales 《Archives of biochemistry and biophysics》1991,286(1):99-106
Previous work showed that transforming growth factor-beta 1 (TGF-beta 1), added alone to bovine cartilage organ cultures, stimulated [35S]sulfate incorporation into macromolecular material but did not investigate the fidelity of the stimulated system to maintain synthesis of cartilage-type proteoglycans. This paper provides evidence that chondrocytes synthesize the appropriate proteoglycan matrix under TGF-beta 1 stimulation: (i) there is a coordinated increase in hyaluronic acid and proteoglycan monomer synthesis, (ii) link-stable proteoglycan aggregates are assembled, (ii) the hybrid chondroitin sulfate/keratan sulfate monomeric species is synthesized, and (iv) there is an increase in protein core synthesis. Some variation in glycosylation patterns was observed when proteoglycans synthesized under TGF-beta 1 stimulation were compared to those synthesized under basal conditions. Thus comparing TGF-beta 1 to basal samples respectively, the monomers were larger (Kav on Sepharose CL-2B = 0.29 vs 0.41), the chondroitin sulfate chains were longer by approximately 3.5 kDa, the percentage of total glycosaminoglycan in keratan sulfate increased slightly from approximately 4% (basal) to approximately 6%, and the unsulfated disaccharide decreased from 28% (basal) to 12%. All of these variations are in the direction of a more anionic proteoglycan. Since the ability of proteoglycans to confer resiliency to the cartilage matrix is directly related to their anionic nature, these changes would presumably have a beneficial effect on tissue function. 相似文献
3.
In calf articular cartilage organ cultures, retinoic acid depressed proteoglycan anabolism to levels approximately 10% of control values and increased their catabolism approximately 14-fold at concentrations of 1 x 10(-8) and 1 x 10(-6) M, respectively, leading to a severe depletion of this component from the extracellular matrix (95% loss in 3 weeks). These effects were powerfully antagonized by maximal levels of transforming growth factors-beta (TGF-beta s) 1, 2, and 3, leading to preservation of matrix components. At a concentration of 1 x 10(-8) M retinoic acid, the TGF-beta s restored anabolism to control levels and lowered catabolic rates greater than 3-fold. While the TGF-beta s increased protein synthesis 2- to 3-fold over controls, retinoic acid alone did not change protein synthesis, as determined by incorporation of [3H]serine. Nevertheless, retinoic acid effectively antagonized the stimulation of protein synthesis by TGF-beta and restored control levels of synthesis at 1 x 10(-7) M. Analysis of proteins, labeled using [3H]serine and [35S]sulfate as precursors, by SDS-PAGE revealed that large molecular weight proteins (greater than 100 kDa) were not detectable in retinoic-acid-treated cultures, but treatment with the TGF-beta s restored these components in coincubation cultures, again supporting the antagonistic role of the polypeptide effectors on retinoid action. Treatment of the cultures with retinoic acid elevated levels of TGF-beta 2 synthesis, but not TGF-beta 1. While the role of the newly synthesized TGF-beta 2 in the set of events elicited by retinoic acid in articular cartilage is unclear, the results establish an intrinsic metabolic link between the isoprenoid and TGF-beta in articular cartilage. We propose that the retinoids and TGF-beta s are integral parts of a regulatory network that controls homeostasis, resorption, or growth, depending on their relative contributions. 相似文献
4.
Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage. 总被引:6,自引:1,他引:5
D J McQuillan C J Handley M A Campbell S Bolis V E Milway A C Herington 《The Biochemical journal》1986,240(2):423-430
The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days. 相似文献
5.
The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures 总被引:2,自引:0,他引:2
The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains. 相似文献
6.
Brown CT Nugent MA Lau FW Trinkaus-Randall V 《The Journal of biological chemistry》1999,274(11):7111-7119
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. 相似文献
7.
Proteoglycans synthesized by chondrocytes of human nonarthritic and osteoarthritic cartilage 总被引:2,自引:0,他引:2
R Shuckett C J Malemud 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1989,190(3):275-281
Chondrocyte cultures were developed from the cell outgrowths of explanted human nonarthritic and osteoarthritic human cartilage. Two significant differences in sulfated proteoglycan synthesis were demonstrated between the chondrocytes obtained in this manner. With 35SO4 to measure newly synthesized proteoglycan, we found that chondrocytes derived from osteoarthritic cartilage secreted significantly less (P less than 0.05) high density proteoglycan into the culture medium than did chondrocytes from nonarthritic cartilage after 20 hr of radiolabeling. This reduced amount of high density proteoglycan was sustained when chondrocytes were maintained in unlabeled culture medium ("chase" medium). In addition, the osteoarthritic chondrocytes secreted an increased amount of low density proteoglycan when compared with their nonarthritic counterparts. The elution profile of secreted high density proteoglycan isolated from the osteoarthritic chondrocyte culture medium was assessed by gel filtration on Sepharose CL-2B and revealed the presence of two proteoglycan subpopulations (Kav, 0.25, 0.58), whereas only one proteoglycan series (Kav, 0.37) was seen in the high density fraction of nonarthritic chondrocyte culture medium. Similar gel filtration profiles were also obtained when chondrocytes were maintained in chase medium. The results of this study demonstrated that stable differences in proteoglycan synthesis, but not in intracellular processing, exist between nonarthritic and osteoarthritic chondrocytes. The findings are noteworthy in that these differences were not previously apparent when organ-cultured cartilage was used to assess putative alterations in proteoglycans between the two groups. 相似文献
8.
Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture 总被引:8,自引:0,他引:8
The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism. 相似文献
9.
Canine cartilage explants were maintained in a basal medium supplemented with a commercially available supplement (ITSCR+) which includes insulin for up to 12 days in culture. During this time it was found that proteoglycan synthesis, as measured by 35SO4 incorporation into high molecular weight proteoglycans, was maintained at levels comparable to those at Day O. This is in substantial agreement with the results of McQuillan et al. (1) for bovine cartilage explants. Since the basal medium which we used, Ham's F12, is low in calcium, we found that supplementation with additional calcium also was needed for maintenance of proteoglycan synthesis. This defined medium was not adequate to prevent a decrease in fibronectin, total protein, and collagen synthesis relative to Day O levels. The addition of transforming growth factor-beta (TGF-beta) at 2 and 10 ng/ml to the defined medium not only prevented the decline in fibronectin synthesis but progressively increased the rate of fibronectin synthesis until the Day O levels were exceeded by an average of fourfold. This TGF-beta-induced increase in fibronectin synthesis was contrasted with the increase in fibronectin synthesis previously reported for degenerated cartilage of osteoarthritic joints (2,3), and possible implications for understanding the disease were discussed. 相似文献
10.
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. 相似文献
11.
Cell surface adhesion and extracellular matrix proteins are known to play a key role in the formation of cell condensations during skeletal development, and their formation is crucial for the expression of cartilage-specific genes. However, little is known about the relationship between adhesion molecules (N-cadherin and N-CAM), extracellular matrix proteins (fibronectin and tenascin) and TGF-beta1, TGF-beta2 and TGF-beta3 during in vitro precartilage condensations in mouse chondrogenesis. On these bases, we determined the participation of mammalian TGF-beta1, TGF-beta2 and TFG-beta3 and Xenopus TGF-beta5 on the expression of cell surface adhesion and extracellular matrix proteins during the formation of precartilage condensations. Also, we characterized the effects of TGF-betas on proteoglycan metabolism at different cellular densities in mouse embryonic limb bud mesenchymal cells. In TGF-beta1 and TGF-beta5-treated cultures, proteoglycan biosynthesis was higher than in controls, while there were no differences in proteoglycan catabolism, which caused the accumulation of cartilage extracellular matrix. When mesenchymal cells were seeded at three different cellular densities in the presence of TGF-betas, only high density cultures presented increased stimulation of proteoglycan biosynthesis, compared to low and intermediate densities. To determine whether the effect of TGF-betas on precartilage condensations is mediated through the expression of N-cadherin, N-CAM, fibronectin and tenascin, we evaluated their expression. Results showed that TGF-beta1, TGF-beta2, TGF-beta3, and TGF-beta5 differentially enhanced the expression of N-cadherin, N-CAM, fibronectin and tenascin in precartilage condensations, suggesting that TGF-beta isoforms play an important role in the establishment of cell-cell and cell-extracellular matrix interactions during precartilage condensations. 相似文献
12.
Transforming growth factor beta regulates the metabolism of proteoglycans in bovine cartilage organ cultures 总被引:7,自引:0,他引:7
The effect of transforming growth factor beta (TGF-beta) has been studied in a bovine articular cartilage organ culture. The peptide stimulates synthesis of proteoglycans in a dose-dependent manner, reaching saturation at 10 ng/ml. This dose gave an approximate 7-fold increase in synthesis over basal controls. In addition, the peptide decreased the rates of catabolism of proteoglycans with an approximately 2-fold maximal effect seen at 5 ng/ml. At the latter concentration, TGF-beta prevented the 4-fold loss of proteoglycans which occurred in cultures maintained under basal conditions over the course of 3 weeks. There was no increase in cell (DNA) content of the cartilage explants under these conditions of TGF-beta treatment, and the net collagen content of the explants remained constant. 相似文献
13.
Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta 总被引:10,自引:1,他引:9
This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation. 相似文献
14.
The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures 总被引:1,自引:0,他引:1
This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures. 相似文献
15.
The in vitro cell culture age and cell density of articular chondrocytes alter sulfated-proteoglycan biosynthesis 总被引:1,自引:0,他引:1
The effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated-proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied. Low density (LD, 2 d), middle density (MD, 5-7 d), and high density (HD, 12-15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis. DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate [3H]-thymidine. LD cultures incorporated 35SO4 into sulfated-proteoglycans at a higher rate than MD or LD cultures. The qualitative nature of the sulfated-proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCl density gradients and by elution profiles on Sepharose CL-2B. Chondrocytes deposited into the extracellular matrix (cell-associated fraction) 35SO4-labeled proteoglycan aggregate. More aggregated proteoglycan was found in the MD and HD cultures than at LD. A 35SO4-labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell-associated fraction was secreted into the culture medium at each culture age. The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities. The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains. A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured. The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures. These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits. These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid. Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length. Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined. 相似文献
16.
J R Merwin J M Anderson O Kocher C M Van Itallie J A Madri 《Journal of cellular physiology》1990,142(1):117-128
Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with anti-ZO-1 label a 220 kd band which co-migrates with the bonafide ZO-1 protein. These data confirm and support the hypothesis that TGF-beta 1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube-like structures with apparent tight junctions and abluminal basal lamina deposition in three-dimensional cultures. 相似文献
17.
P Chen J L Carrington V M Paralkar G F Pierce A H Reddi 《Experimental cell research》1992,200(1):110-117
Platelet-derived growth factor (PDGF) influences the proliferation and differentiation of a variety of cells. In this study, we have investigated the effect of PDGF isoforms on chondrogenesis by stage 24 chick limb bud mesoderm cells in culture. Synthesis of sulfated proteoglycans, an index of chondrogenesis, was inhibited by all three PDGF isoforms (PDGF-AA, PDGF-AB, and PDGF-BB). Application of PDGF isoforms during the first 2 days of culture, before the cells were overtly differentiating, resulted in decreased synthesis of sulfated proteoglycans. This was similar to when PDGF isoforms were present throughout the culture period. However, application of PDGF isoform during only the last 2 days of culture, did not inhibit cartilage matrix production. When chondrogenic and nonchondrogenic cells were separated from the cultures and replated, PDGF-AB and PDGF-BB inhibited the incorporation of sulfate by the chondrogenic cells. Recombinant bone morphogenetic protein 2B reversed the inhibitory effects of PDGF on sulfated proteoglycan synthesis and DNA synthesis. PDGF receptor binding analysis indicated that beta-receptors were predominant receptors present on the chondrogenic and nonchondrogenic cells of the stage 24 mesoderm. PDGF isoforms increased thymidine incorporation by 48 h in both high and low density cultures. However, at later periods, cell proliferation was inhibited by PDGF-AA and PDGF-AB but not by PDGF-BB. PDGF acted as a bifunctional modulator of mesodermal cell proliferation and thus may regulate chondrogenesis during limb differentiation and morphogenesis. 相似文献
18.
Crochiere ML Kubilus JK Linsenmayer TF 《The International journal of developmental biology》2008,52(1):63-70
We previously observed using cultured tibiotarsal long-bone rudiments from which the perichondrium (PC) and periosteum (PO) was removed that the PC regulates cartilage growth by the secretion of soluble negative regulatory factors. This regulation is "precise" in that it compensates exactly for removal of the endogenous PC and is mediated through at least three independent mechanisms, one of which involves a response to TGF-beta. PC cell cultures treated with 2 ng/ml TGF-beta1 produced a conditioned medium which when added to PC/PO-free organ cultures effected precise regulation of cartilage growth. In the present study, we have investigated the possibility that TGF-beta itself might be the negative regulator which is produced by the PC cells in response to their treatment with TGF-beta1. Using a TGF-beta responsive reporter assay, we determined that PC cell cultures, when treated with 2 ng/ml or greater exogenous TGF-beta1, produce 300 pg/ml of active TGF-beta. Then we observed that this concentration (300 pg/ml) of active TGF-beta1, when added to PC/PO-free tibiotarsal organ cultures, effected precise regulation of cartilage growth, whereas concentrations of TGF-beta1 either greater or less than 300 pg/ml produced abnormally small cartilages. These results suggest that one mechanism by which the PC effects normal cartilage growth is through the production of a precisely regulated amount of TGF-beta which the PC produces in response to treatment with exogenous TGF-beta itself. 相似文献
19.
Stimulation of sulfated-proteoglycan synthesis by forskolin in monolayer cultures of rabbit articular chondrocytes 总被引:1,自引:0,他引:1
Forskolin, a plant cardiotonic diterpene, stimulated proteoglycan biosynthesis by chondrocytes in monolayer culture. The quantitative increase in proteoglycans was dependent on the concentration of forskolin, but was relatively independent of the presence of serum. At forskolin concentrations that stimulated proteoglycan synthesis, a significant stimulation of adenylate cyclase and cAMP was also measured. The quantitative increase in proteoglycans was characterized, qualitatively, by an increased deposition of newly synthesized proteoglycan in the cell-associated fraction. An analysis of the most dense proteoglycans (fraction dA1) in the cell-associated fraction showed that more of the proteoglycans eluted in the void volume of a Sepharose CL-2B column, indicating that an increased amount of proteoglycan aggregate was synthesized in forskolin-treated cultures. The proteoglycan monomer dA1D1 secreted into the culture medium of forskolin-stimulated cultures overlapped in hydrodynamic size with that of control cultures, although cultures stimulated with forskolin and phosphodiesterase inhibitors produced even larger proteoglycans. The hydrodynamic size of 35SO4 and 3H-glucosamine-labelled glycosaminoglycans isolated from the dA1D1 fraction of the culture medium was greater in forskolin-treated chondrocytes, especially from those in which phosphodiesterase inhibitors had been added. These results indicated that forskolin, a direct activator of chondrocyte adenylate cyclase mimicked the effects of cAMP analogues on chondrocyte proteoglycan synthesis previously reported. These results implicate activation of adenylate cyclase as a regulatory event in the biosynthesis of cartilage proteoglycans, and more specifically in the production of hydrodynamically larger glycosaminoglycans. 相似文献
20.
M A Campbell C J Handley V C Hascall R A Campbell D A Lowther 《Archives of biochemistry and biophysics》1984,234(1):275-289
Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix. 相似文献