Limits...
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.

Show MeSH

Related in: MedlinePlus

Tumor necrosis factor alpha (TNF-α) enhances elongated cell morphology induced by epidermal growth factor (EGF). 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) Cell morphology was observed by phase contrast microscopy. Arrowheads indicate spinous processes that appear following incubation with EGF or TNF-α + EGF. An elongated cell is defined as having a predominant axis with a length exceeding three times the maximum width of the cell. Digital images of live cultures were captured at 20 × objective magnification. Bar = 100 μm. Images shown are representative of three independent experiments. (b) The total number of elongated cells per field (1.376 mm2) were counted, averaged for at least three independent experiments (n = 3–5), and analyzed by analysis of variance. a Significant difference from control (P < 0.01), b significant difference from control (P < 0.001) and significant difference from EGF-treated cells (P < 0.01).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1064891&req=5

Figure 1: Tumor necrosis factor alpha (TNF-α) enhances elongated cell morphology induced by epidermal growth factor (EGF). 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) Cell morphology was observed by phase contrast microscopy. Arrowheads indicate spinous processes that appear following incubation with EGF or TNF-α + EGF. An elongated cell is defined as having a predominant axis with a length exceeding three times the maximum width of the cell. Digital images of live cultures were captured at 20 × objective magnification. Bar = 100 μm. Images shown are representative of three independent experiments. (b) The total number of elongated cells per field (1.376 mm2) were counted, averaged for at least three independent experiments (n = 3–5), and analyzed by analysis of variance. a Significant difference from control (P < 0.01), b significant difference from control (P < 0.001) and significant difference from EGF-treated cells (P < 0.01).

Mentions: The cellular morphology reflects the differentiation status of cells such as chondrocytes. For example, a change from a rounded to a more elongated morphology in response to EGF by CFK2 chondrocytic cells is associated with a diminished onset of expression of aggrecan and link protein gene [2]. To determine whether the morphology of primary chondrocytes expressing the matrix was affected by TNF-α or EGF, live cultures were examined by phase-contrast microscopy (Fig. 1a) and the number of elongated cells per field was quantified (Fig. 1b).


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)

Tumor necrosis factor alpha (TNF-α) enhances elongated cell morphology induced by epidermal growth factor (EGF). 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) Cell morphology was observed by phase contrast microscopy. Arrowheads indicate spinous processes that appear following incubation with EGF or TNF-α + EGF. An elongated cell is defined as having a predominant axis with a length exceeding three times the maximum width of the cell. Digital images of live cultures were captured at 20 × objective magnification. Bar = 100 μm. Images shown are representative of three independent experiments. (b) The total number of elongated cells per field (1.376 mm2) were counted, averaged for at least three independent experiments (n = 3–5), and analyzed by analysis of variance. a Significant difference from control (P < 0.01), b significant difference from control (P < 0.001) and significant difference from EGF-treated cells (P < 0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064891&req=5

Figure 1: Tumor necrosis factor alpha (TNF-α) enhances elongated cell morphology induced by epidermal growth factor (EGF). 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) Cell morphology was observed by phase contrast microscopy. Arrowheads indicate spinous processes that appear following incubation with EGF or TNF-α + EGF. An elongated cell is defined as having a predominant axis with a length exceeding three times the maximum width of the cell. Digital images of live cultures were captured at 20 × objective magnification. Bar = 100 μm. Images shown are representative of three independent experiments. (b) The total number of elongated cells per field (1.376 mm2) were counted, averaged for at least three independent experiments (n = 3–5), and analyzed by analysis of variance. a Significant difference from control (P < 0.01), b significant difference from control (P < 0.001) and significant difference from EGF-treated cells (P < 0.01).
Mentions: The cellular morphology reflects the differentiation status of cells such as chondrocytes. For example, a change from a rounded to a more elongated morphology in response to EGF by CFK2 chondrocytic cells is associated with a diminished onset of expression of aggrecan and link protein gene [2]. To determine whether the morphology of primary chondrocytes expressing the matrix was affected by TNF-α or EGF, live cultures were examined by phase-contrast microscopy (Fig. 1a) and the number of elongated cells per field was quantified (Fig. 1b).

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