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proteoglycan and glycosaminoglycan synthesis ..

A simple and reliable method to measure proteoglycan synthesis by chondrocytes in culture is described

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Chondrocyte Culture and Assay-Manual | Cartilage | …

AB - Background. Hepatocyte growth factor (HGF) is a clinically important growth factor with therapeutic potential for the treatment of interstitial fibrosis and chronic renal failure. Proteoglycans are components of the renal interstitium, which have multiple actions, including growth regulation. In this study, we examined the effects of HGF on proteoglycan synthesis in interstitial fibroblasts, and the mechanisms of these effects. Methods and Results. Expression and agonist-induced activation of the HGF receptor c-Met was detected in rat renal interstitial fibroblasts (NRK-49F) by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immune complex/immunoblot assay. Moreover, stimulation of the cells with HGF resulted in a marked increase (five- to tenfold) in phosphorylation of extracellular signal-related protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), but not of c-Jun NH2 terminal kinase (JNK). Treatment with HGF resulted in a time- and dose-dependent increase (P > 0.01) in both cell-associated and secreted proteoglycan synthesis to two- to fourfold of control levels. This effect was attenuated by the MAPK/ERK kinase (MEK) inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Ion-exchange chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were up-regulated after HGF treatment. Northern blot, RT-PCR, Western blot, and promoter activity assays revealed that HGF caused a significant increase in decorin mRNA and protein, as well as in biglycan mRNA, protein, and promoter activity, suggesting transcriptional control of gene expression. Since the effects of biglycan on fibroblast proliferation are still unclear, the effects of biglycan were examined by thymidine assay, and biglycan was found to attenuate transforming growth factor-β (TGF-β)-induced changes in cell proliferation. Conclusion. These results suggest that HGF causes an increase in the small leucine-rich proteoglycans biglycan and decorin by ERK1/2- and p38 MAPK-mediated pathways in fibroblasts. These findings may be relevant for understanding potential mechanisms by which HGF can exert TGF-β inhibitory actions in the kidney.

08/02/2010 · Brazilian Journal of Medical and Biological Research ..

AB - The insulin-like growth factors were discovered on the basis of their ability to stimulate cartilage sulfation and to replace the “sulfation factor activity” of GH, as determined using an in vivo assay, in an in vitro test system (1). The biological significance of this finding was quickly expanded beyond the study of cartilage sulfation to include stimulation of DNA synthesis (2), proteoglycan synthesis (3), glycosaminoglycan synthesis (4), and protein synthesis (5). Most of these studies used tissue preparations such as isolated diaphragm, cartilage, or epididymal fat pads to study biologicalactivity. In recognition of its generalized pleiotypic actions, in the early 1970’s sulfation factor was renamed somatomedin (mediator of the effects of somatotropin) and was included in the emerging classification of broad spectrum growth factors along with platelet derived growth factor, fibroblast growth factor, and epidermal growth factor (6). During the period in which the biological actions of sulfation factor were being characterized, parallel studies were initiated that attempted to define factors in serum that could stimulate insulin-like effects. Because these factors were known to be distinct from immunoreactive insulin, their bioassay was based on insulin-like actions that could not be abolished by the simultaneous addition of anti-insulin antibody, and the factors were termed nonsuppressible insulin-like activity (NSILA) (7). Initial biological studies of both NSILA and somatomedin were based on the use of crude serum extracts. Early attempts to extract these factors from tissues were generally not successful, and by 1970 it had been concluded that no concentrated organ source for them existed (8). However, highly purified preparations of both factors were prepared from serum extracts.

Glucocorticoid regulation of proteoglycan synthesis in ..

Chondrocytes are the cells solely responsible for the synthesis and maintenance of cartilage

Background. Hepatocyte growth factor (HGF) is a clinically important growth factor with therapeutic potential for the treatment of interstitial fibrosis and chronic renal failure. Proteoglycans are components of the renal interstitium, which have multiple actions, including growth regulation. In this study, we examined the effects of HGF on proteoglycan synthesis in interstitial fibroblasts, and the mechanisms of these effects. Methods and Results. Expression and agonist-induced activation of the HGF receptor c-Met was detected in rat renal interstitial fibroblasts (NRK-49F) by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immune complex/immunoblot assay. Moreover, stimulation of the cells with HGF resulted in a marked increase (five- to tenfold) in phosphorylation of extracellular signal-related protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), but not of c-Jun NH2 terminal kinase (JNK). Treatment with HGF resulted in a time- and dose-dependent increase (P > 0.01) in both cell-associated and secreted proteoglycan synthesis to two- to fourfold of control levels. This effect was attenuated by the MAPK/ERK kinase (MEK) inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Ion-exchange chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were up-regulated after HGF treatment. Northern blot, RT-PCR, Western blot, and promoter activity assays revealed that HGF caused a significant increase in decorin mRNA and protein, as well as in biglycan mRNA, protein, and promoter activity, suggesting transcriptional control of gene expression. Since the effects of biglycan on fibroblast proliferation are still unclear, the effects of biglycan were examined by thymidine assay, and biglycan was found to attenuate transforming growth factor-β (TGF-β)-induced changes in cell proliferation. Conclusion. These results suggest that HGF causes an increase in the small leucine-rich proteoglycans biglycan and decorin by ERK1/2- and p38 MAPK-mediated pathways in fibroblasts. These findings may be relevant for understanding potential mechanisms by which HGF can exert TGF-β inhibitory actions in the kidney.

We offer assay kits for measuring nitric oxide and proteoglycan. Easily measure cartilage synthesis and monitor collagen-induced arthritis with reagents that are tested for stability and function. .

Effects of glucocorticoids on proteoglycan synthesis in rat ..

N2 - The insulin-like growth factors were discovered on the basis of their ability to stimulate cartilage sulfation and to replace the “sulfation factor activity” of GH, as determined using an in vivo assay, in an in vitro test system (1). The biological significance of this finding was quickly expanded beyond the study of cartilage sulfation to include stimulation of DNA synthesis (2), proteoglycan synthesis (3), glycosaminoglycan synthesis (4), and protein synthesis (5). Most of these studies used tissue preparations such as isolated diaphragm, cartilage, or epididymal fat pads to study biologicalactivity. In recognition of its generalized pleiotypic actions, in the early 1970’s sulfation factor was renamed somatomedin (mediator of the effects of somatotropin) and was included in the emerging classification of broad spectrum growth factors along with platelet derived growth factor, fibroblast growth factor, and epidermal growth factor (6). During the period in which the biological actions of sulfation factor were being characterized, parallel studies were initiated that attempted to define factors in serum that could stimulate insulin-like effects. Because these factors were known to be distinct from immunoreactive insulin, their bioassay was based on insulin-like actions that could not be abolished by the simultaneous addition of anti-insulin antibody, and the factors were termed nonsuppressible insulin-like activity (NSILA) (7). Initial biological studies of both NSILA and somatomedin were based on the use of crude serum extracts. Early attempts to extract these factors from tissues were generally not successful, and by 1970 it had been concluded that no concentrated organ source for them existed (8). However, highly purified preparations of both factors were prepared from serum extracts.

N2 - Background. Hepatocyte growth factor (HGF) is a clinically important growth factor with therapeutic potential for the treatment of interstitial fibrosis and chronic renal failure. Proteoglycans are components of the renal interstitium, which have multiple actions, including growth regulation. In this study, we examined the effects of HGF on proteoglycan synthesis in interstitial fibroblasts, and the mechanisms of these effects. Methods and Results. Expression and agonist-induced activation of the HGF receptor c-Met was detected in rat renal interstitial fibroblasts (NRK-49F) by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immune complex/immunoblot assay. Moreover, stimulation of the cells with HGF resulted in a marked increase (five- to tenfold) in phosphorylation of extracellular signal-related protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), but not of c-Jun NH2 terminal kinase (JNK). Treatment with HGF resulted in a time- and dose-dependent increase (P > 0.01) in both cell-associated and secreted proteoglycan synthesis to two- to fourfold of control levels. This effect was attenuated by the MAPK/ERK kinase (MEK) inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Ion-exchange chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were up-regulated after HGF treatment. Northern blot, RT-PCR, Western blot, and promoter activity assays revealed that HGF caused a significant increase in decorin mRNA and protein, as well as in biglycan mRNA, protein, and promoter activity, suggesting transcriptional control of gene expression. Since the effects of biglycan on fibroblast proliferation are still unclear, the effects of biglycan were examined by thymidine assay, and biglycan was found to attenuate transforming growth factor-β (TGF-β)-induced changes in cell proliferation. Conclusion. These results suggest that HGF causes an increase in the small leucine-rich proteoglycans biglycan and decorin by ERK1/2- and p38 MAPK-mediated pathways in fibroblasts. These findings may be relevant for understanding potential mechanisms by which HGF can exert TGF-β inhibitory actions in the kidney.

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  • Mechanical regulation of proteoglycan synthesis in …

    Proteoglycan - Wikipedia

  • How to measure Glycosaminoglycans and Proteoglycans?

    Synthesis The protein ..

  • Assay of proteoglycan and collagen degradation in …

    The completed proteoglycan is then exported in secretory vesicles to the extracellular matrix of the tissue

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on proteoglycan and collagen degradation were ..

The insulin-like growth factors were discovered on the basis of their ability to stimulate cartilage sulfation and to replace the “sulfation factor activity” of GH, as determined using an in vivo assay, in an in vitro test system (1). The biological significance of this finding was quickly expanded beyond the study of cartilage sulfation to include stimulation of DNA synthesis (2), proteoglycan synthesis (3), glycosaminoglycan synthesis (4), and protein synthesis (5). Most of these studies used tissue preparations such as isolated diaphragm, cartilage, or epididymal fat pads to study biologicalactivity. In recognition of its generalized pleiotypic actions, in the early 1970’s sulfation factor was renamed somatomedin (mediator of the effects of somatotropin) and was included in the emerging classification of broad spectrum growth factors along with platelet derived growth factor, fibroblast growth factor, and epidermal growth factor (6). During the period in which the biological actions of sulfation factor were being characterized, parallel studies were initiated that attempted to define factors in serum that could stimulate insulin-like effects. Because these factors were known to be distinct from immunoreactive insulin, their bioassay was based on insulin-like actions that could not be abolished by the simultaneous addition of anti-insulin antibody, and the factors were termed nonsuppressible insulin-like activity (NSILA) (7). Initial biological studies of both NSILA and somatomedin were based on the use of crude serum extracts. Early attempts to extract these factors from tissues were generally not successful, and by 1970 it had been concluded that no concentrated organ source for them existed (8). However, highly purified preparations of both factors were prepared from serum extracts.

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