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Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte.

Klooster AR, Bernier SM - Arthritis Res. Ther. (2004)

Bottom Line: EGF alone did not activate NF-kappaB or alter NF-kappaB activation by TNF-alpha.TNF-alpha attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-alpha or basal activity.TNF-alpha and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIHR Group in Skeletal Development and Remodeling, Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada. aaron.klooster@pharma.novartis.com

ABSTRACT
The failure of chondrocytes to replace the lost extracellular matrix contributes to the progression of degenerative disorders of cartilage. Inflammatory mediators present in the joint regulate the breakdown of the established matrix and the synthesis of new extracellular matrix molecules. In the present study, we investigated the effects of tumor necrosis factor alpha (TNF-alpha) and epidermal growth factor (EGF) on chondrocyte morphology and matrix gene expression. Chondrocytes were isolated from distal femoral condyles of neonatal rats. Cells in primary culture displayed a cobblestone appearance. EGF, but not TNF-alpha, increased the number of cells exhibiting an elongated morphology. TNF-alpha potentiated the effect of EGF on chondrocyte morphology. Individually, TNF-alpha and EGF diminished levels of aggrecan and type II collagen mRNA. In combination, the effects of TNF-alpha and EGF were additive, indicating the involvement of discrete signaling pathways. Cell viability was not compromised by TNF-alpha or by EGF, alone or in combination. EGF alone did not activate NF-kappaB or alter NF-kappaB activation by TNF-alpha. Pharmacologic studies indicated that the effects of TNF-alpha and EGF alone or in combination were independent of protein kinase C signaling, but were dependent on MEK1/2 activity. Finally, we analyzed the involvement of Sox-9 using a reporter construct of the 48 base pair minimal enhancer of type II collagen. TNF-alpha attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-alpha or basal activity. TNF-alpha and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function. These findings provide critical insights into the control of chondrocytes through the integration of multiple extracellular signals.

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Apoptosis is not observed following tumor necrosis factor alpha (TNF-α) and/or epidermal growth factor (EGF) treatment. Confluent monolayers of chondrocytes were treated with vehicle, TNF-α (30 ng/ml), EGF (10 ng/ml) or TNF-α + EGF for 24 hours. (a) Early stages of apoptosis were assayed by immunoblot with an antibody specific for intact and cleaved forms of poly(ADP ribose) polymerase (PARP). No cleavage of PARP (i.e. appearance of a band at 89 kDa) was detected following any of the treatments. Blot shown is representative of three independent experiments. (b) Apoptosis-induced DNA strand breaks were examined by in situ labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL]) and imaged using confocal microscopy. No TUNEL labeling was detected with any of the treatments. Cells treated with DNAse I to induce DNA breaks served as a positive control. Bar = 50 μm. Images are representative of three independent experiments.
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Figure 3: Apoptosis is not observed following tumor necrosis factor alpha (TNF-α) and/or epidermal growth factor (EGF) treatment. Confluent monolayers of chondrocytes were treated with vehicle, TNF-α (30 ng/ml), EGF (10 ng/ml) or TNF-α + EGF for 24 hours. (a) Early stages of apoptosis were assayed by immunoblot with an antibody specific for intact and cleaved forms of poly(ADP ribose) polymerase (PARP). No cleavage of PARP (i.e. appearance of a band at 89 kDa) was detected following any of the treatments. Blot shown is representative of three independent experiments. (b) Apoptosis-induced DNA strand breaks were examined by in situ labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL]) and imaged using confocal microscopy. No TUNEL labeling was detected with any of the treatments. Cells treated with DNAse I to induce DNA breaks served as a positive control. Bar = 50 μm. Images are representative of three independent experiments.

Mentions: Cultures treated with TNF-α, with EGF or with TNF-α + EGF were assessed for evidence of apoptosis using an early marker, PARP (Fig. 3a). PARP is a 116 kDa protein involved in DNA repair [23] that is cleaved as part of the caspase cascade initiated in cells undergoing apoptosis. Cell extracts were immunoblotted for the presence of intact and cleaved forms of PARP. Neither loss of intact PARP nor the appearance of cleaved moieties (85 kDa) was detected following 24 hours of treatment with TNF-α, with EGF or with TNF-α + EGF. Interestingly, TNF-α + EGF increased the amount of PARP present in the chondrocytes. To confirm the lack of apoptosis in factor-treated cultures, the presence of DNA strand breaks was evaluated by in situ labeling (TUNEL) (Fig. 3b). TUNEL labeling was not detected following any of the treatments.


Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte.

Klooster AR, Bernier SM - Arthritis Res. Ther. (2004)

Apoptosis is not observed following tumor necrosis factor alpha (TNF-α) and/or epidermal growth factor (EGF) treatment. Confluent monolayers of chondrocytes were treated with vehicle, TNF-α (30 ng/ml), EGF (10 ng/ml) or TNF-α + EGF for 24 hours. (a) Early stages of apoptosis were assayed by immunoblot with an antibody specific for intact and cleaved forms of poly(ADP ribose) polymerase (PARP). No cleavage of PARP (i.e. appearance of a band at 89 kDa) was detected following any of the treatments. Blot shown is representative of three independent experiments. (b) Apoptosis-induced DNA strand breaks were examined by in situ labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL]) and imaged using confocal microscopy. No TUNEL labeling was detected with any of the treatments. Cells treated with DNAse I to induce DNA breaks served as a positive control. Bar = 50 μm. Images are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: Apoptosis is not observed following tumor necrosis factor alpha (TNF-α) and/or epidermal growth factor (EGF) treatment. Confluent monolayers of chondrocytes were treated with vehicle, TNF-α (30 ng/ml), EGF (10 ng/ml) or TNF-α + EGF for 24 hours. (a) Early stages of apoptosis were assayed by immunoblot with an antibody specific for intact and cleaved forms of poly(ADP ribose) polymerase (PARP). No cleavage of PARP (i.e. appearance of a band at 89 kDa) was detected following any of the treatments. Blot shown is representative of three independent experiments. (b) Apoptosis-induced DNA strand breaks were examined by in situ labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL]) and imaged using confocal microscopy. No TUNEL labeling was detected with any of the treatments. Cells treated with DNAse I to induce DNA breaks served as a positive control. Bar = 50 μm. Images are representative of three independent experiments.
Mentions: Cultures treated with TNF-α, with EGF or with TNF-α + EGF were assessed for evidence of apoptosis using an early marker, PARP (Fig. 3a). PARP is a 116 kDa protein involved in DNA repair [23] that is cleaved as part of the caspase cascade initiated in cells undergoing apoptosis. Cell extracts were immunoblotted for the presence of intact and cleaved forms of PARP. Neither loss of intact PARP nor the appearance of cleaved moieties (85 kDa) was detected following 24 hours of treatment with TNF-α, with EGF or with TNF-α + EGF. Interestingly, TNF-α + EGF increased the amount of PARP present in the chondrocytes. To confirm the lack of apoptosis in factor-treated cultures, the presence of DNA strand breaks was evaluated by in situ labeling (TUNEL) (Fig. 3b). TUNEL labeling was not detected following any of the treatments.

Bottom Line: EGF alone did not activate NF-kappaB or alter NF-kappaB activation by TNF-alpha.TNF-alpha attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-alpha or basal activity.TNF-alpha and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIHR Group in Skeletal Development and Remodeling, Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada. aaron.klooster@pharma.novartis.com

ABSTRACT
The failure of chondrocytes to replace the lost extracellular matrix contributes to the progression of degenerative disorders of cartilage. Inflammatory mediators present in the joint regulate the breakdown of the established matrix and the synthesis of new extracellular matrix molecules. In the present study, we investigated the effects of tumor necrosis factor alpha (TNF-alpha) and epidermal growth factor (EGF) on chondrocyte morphology and matrix gene expression. Chondrocytes were isolated from distal femoral condyles of neonatal rats. Cells in primary culture displayed a cobblestone appearance. EGF, but not TNF-alpha, increased the number of cells exhibiting an elongated morphology. TNF-alpha potentiated the effect of EGF on chondrocyte morphology. Individually, TNF-alpha and EGF diminished levels of aggrecan and type II collagen mRNA. In combination, the effects of TNF-alpha and EGF were additive, indicating the involvement of discrete signaling pathways. Cell viability was not compromised by TNF-alpha or by EGF, alone or in combination. EGF alone did not activate NF-kappaB or alter NF-kappaB activation by TNF-alpha. Pharmacologic studies indicated that the effects of TNF-alpha and EGF alone or in combination were independent of protein kinase C signaling, but were dependent on MEK1/2 activity. Finally, we analyzed the involvement of Sox-9 using a reporter construct of the 48 base pair minimal enhancer of type II collagen. TNF-alpha attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-alpha or basal activity. TNF-alpha and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function. These findings provide critical insights into the control of chondrocytes through the integration of multiple extracellular signals.

Show MeSH
Related in: MedlinePlus