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The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue.

Feugate JE, Wong L, Li QJ, Martins-Green M - BMC Cell Biol. (2002)

Bottom Line: In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression.Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels.In addition, cCAF is chemotactic for fibroblasts and accelerates their migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of California, Riverside, California, USA. feugate@citrus.ucr.edu

ABSTRACT

Background: During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF), is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM) is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules.

Results: Here we show that this chemokine can stimulate precocious deposition of tenascin, fibronectin and collagen I, but not collagen III. Studies in culture and in vivo show that tenascin stimulation can also be achieved by the N-terminal 15 aas of the protein and occurs at the level of gene expression. In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression. Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels. In addition, cCAF is chemotactic for fibroblasts and accelerates their migration.

Conclusions: These previously unknown functions for chemokines suggest that cCAF, the chicken orthologue of human IL-8, enhances healing by rapidly chemoattracting fibroblasts into the wound site and stimulating them to produce ECM molecules, leading to precocious development of granulation tissue. This acceleration of the repair process may have important application to healing of impaired wounds.

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Potential mecahnisms for cCAF-induced FN accumulation.(A) Analysis of plasminogen activator (PA) activity. Using a synthetic chromogenic substrate (Boc-Val-Gly-Arg-β NA.AcOH) there was an increase in fluorescence detected in supernatant from Rous sarcoma virus transformed fibroblasts which secrete large amounts of PA but not with the control or cCAF-treated supernatants. (B) Casein zymography to detect MMP-3, MMP-7, and MMP-10. No detectable caseinolytic activity in the supernatant of control or cCAF-treated fibroblast was found. The general protease trypsin was used as a positive control. Because this enzyme is not pure, multiple sites of protein digestion were seen. (C) Blocking TN expression using antisense oligonucleotides. Immunoblot analysis of TN and of FN; equal amounts of protein, as determined by using the DC protein assay kit (Bio-Rad),were loaded in each lane. Use of antisense TN oligos blocked the cCAF-induced expression of both FN and TN, whereas the use of sense oligos had no effect on TN or FN levels.
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Figure 5: Potential mecahnisms for cCAF-induced FN accumulation.(A) Analysis of plasminogen activator (PA) activity. Using a synthetic chromogenic substrate (Boc-Val-Gly-Arg-β NA.AcOH) there was an increase in fluorescence detected in supernatant from Rous sarcoma virus transformed fibroblasts which secrete large amounts of PA but not with the control or cCAF-treated supernatants. (B) Casein zymography to detect MMP-3, MMP-7, and MMP-10. No detectable caseinolytic activity in the supernatant of control or cCAF-treated fibroblast was found. The general protease trypsin was used as a positive control. Because this enzyme is not pure, multiple sites of protein digestion were seen. (C) Blocking TN expression using antisense oligonucleotides. Immunoblot analysis of TN and of FN; equal amounts of protein, as determined by using the DC protein assay kit (Bio-Rad),were loaded in each lane. Use of antisense TN oligos blocked the cCAF-induced expression of both FN and TN, whereas the use of sense oligos had no effect on TN or FN levels.

Mentions: Taken together the results in culture and in vivo suggest that the chemokine does not stimulate FN gene expression but rather may directly or indirectly affect stability of this protein. Therefore, we tested the possibility that cCAF treatment leads to stability of the protein by stimulating decrease in matrix degrading enzymes. In chickens, plasmin generated from plasminogen by plasminogen activator (PA) degrades much of the FN. To investigate whether activity of PA might be decreased with cCAF treatment leading to accumulation of FN, we examined the levels of PA activity. Our results showed that there is no detectable PA activity in untreated or treated fibroblasts whereas the activity is high in RSV-transformed CEFs (tCEFs; Fig. 5A) which are known to produce high levels of PA [31]. Because PA does not appear to be activated by cCAF, we tested the possibility that the stability of FN is due to a decrease in MMP production/activation. In humans, MMP-3, MMP-7 and MMP-10 (stromelysins) are the most active proteases for FN, but these molecules have not been isolated in chickens. However, we can test for general activity of these MMPs in the chicken system by zymography using casein as substrate (Fig. 5B). Whereas the positive control (trypsin) was able to digest the substrate at specific sites in the gel, the supernatants of our treated fibroblasts did not. This suggests that the stromelysins are not active in this system or that our test is not sensitive enough. To further investigate whether MMPs are involved in the cCAF induced high levels of FN will require evaluation of matrix-degrading enzymes specific for chickens and is an ongoing project in our laboratory as we obtain/develop probes for theses MMPs. However, another possible mechanism for the cCAF-induced FN accumulation could be linked to an increase in TN. TN-C knockout mice show decreased accumulation of FN in wounded tissue and this decrease has been hypothesized to be due to disrupted incorporation of FN into the matrix in the absence of TN-C [32,33]. To determine whether cCAF-induced TN production is involved in the increase in FN observed after cCAF treatment, we used antisense phosphothioated oligonucleotides to inhibit TN production. Sense oligonucleotides did not have an effect on cCAF-induced TN or FN production. However, the antisense oligonucleotides were able to significantly decrease the production of both TN and FN (Fig. 5C).


The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue.

Feugate JE, Wong L, Li QJ, Martins-Green M - BMC Cell Biol. (2002)

Potential mecahnisms for cCAF-induced FN accumulation.(A) Analysis of plasminogen activator (PA) activity. Using a synthetic chromogenic substrate (Boc-Val-Gly-Arg-β NA.AcOH) there was an increase in fluorescence detected in supernatant from Rous sarcoma virus transformed fibroblasts which secrete large amounts of PA but not with the control or cCAF-treated supernatants. (B) Casein zymography to detect MMP-3, MMP-7, and MMP-10. No detectable caseinolytic activity in the supernatant of control or cCAF-treated fibroblast was found. The general protease trypsin was used as a positive control. Because this enzyme is not pure, multiple sites of protein digestion were seen. (C) Blocking TN expression using antisense oligonucleotides. Immunoblot analysis of TN and of FN; equal amounts of protein, as determined by using the DC protein assay kit (Bio-Rad),were loaded in each lane. Use of antisense TN oligos blocked the cCAF-induced expression of both FN and TN, whereas the use of sense oligos had no effect on TN or FN levels.
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Related In: Results  -  Collection

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Figure 5: Potential mecahnisms for cCAF-induced FN accumulation.(A) Analysis of plasminogen activator (PA) activity. Using a synthetic chromogenic substrate (Boc-Val-Gly-Arg-β NA.AcOH) there was an increase in fluorescence detected in supernatant from Rous sarcoma virus transformed fibroblasts which secrete large amounts of PA but not with the control or cCAF-treated supernatants. (B) Casein zymography to detect MMP-3, MMP-7, and MMP-10. No detectable caseinolytic activity in the supernatant of control or cCAF-treated fibroblast was found. The general protease trypsin was used as a positive control. Because this enzyme is not pure, multiple sites of protein digestion were seen. (C) Blocking TN expression using antisense oligonucleotides. Immunoblot analysis of TN and of FN; equal amounts of protein, as determined by using the DC protein assay kit (Bio-Rad),were loaded in each lane. Use of antisense TN oligos blocked the cCAF-induced expression of both FN and TN, whereas the use of sense oligos had no effect on TN or FN levels.
Mentions: Taken together the results in culture and in vivo suggest that the chemokine does not stimulate FN gene expression but rather may directly or indirectly affect stability of this protein. Therefore, we tested the possibility that cCAF treatment leads to stability of the protein by stimulating decrease in matrix degrading enzymes. In chickens, plasmin generated from plasminogen by plasminogen activator (PA) degrades much of the FN. To investigate whether activity of PA might be decreased with cCAF treatment leading to accumulation of FN, we examined the levels of PA activity. Our results showed that there is no detectable PA activity in untreated or treated fibroblasts whereas the activity is high in RSV-transformed CEFs (tCEFs; Fig. 5A) which are known to produce high levels of PA [31]. Because PA does not appear to be activated by cCAF, we tested the possibility that the stability of FN is due to a decrease in MMP production/activation. In humans, MMP-3, MMP-7 and MMP-10 (stromelysins) are the most active proteases for FN, but these molecules have not been isolated in chickens. However, we can test for general activity of these MMPs in the chicken system by zymography using casein as substrate (Fig. 5B). Whereas the positive control (trypsin) was able to digest the substrate at specific sites in the gel, the supernatants of our treated fibroblasts did not. This suggests that the stromelysins are not active in this system or that our test is not sensitive enough. To further investigate whether MMPs are involved in the cCAF induced high levels of FN will require evaluation of matrix-degrading enzymes specific for chickens and is an ongoing project in our laboratory as we obtain/develop probes for theses MMPs. However, another possible mechanism for the cCAF-induced FN accumulation could be linked to an increase in TN. TN-C knockout mice show decreased accumulation of FN in wounded tissue and this decrease has been hypothesized to be due to disrupted incorporation of FN into the matrix in the absence of TN-C [32,33]. To determine whether cCAF-induced TN production is involved in the increase in FN observed after cCAF treatment, we used antisense phosphothioated oligonucleotides to inhibit TN production. Sense oligonucleotides did not have an effect on cCAF-induced TN or FN production. However, the antisense oligonucleotides were able to significantly decrease the production of both TN and FN (Fig. 5C).

Bottom Line: In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression.Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels.In addition, cCAF is chemotactic for fibroblasts and accelerates their migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of California, Riverside, California, USA. feugate@citrus.ucr.edu

ABSTRACT

Background: During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF), is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM) is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules.

Results: Here we show that this chemokine can stimulate precocious deposition of tenascin, fibronectin and collagen I, but not collagen III. Studies in culture and in vivo show that tenascin stimulation can also be achieved by the N-terminal 15 aas of the protein and occurs at the level of gene expression. In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression. Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels. In addition, cCAF is chemotactic for fibroblasts and accelerates their migration.

Conclusions: These previously unknown functions for chemokines suggest that cCAF, the chicken orthologue of human IL-8, enhances healing by rapidly chemoattracting fibroblasts into the wound site and stimulating them to produce ECM molecules, leading to precocious development of granulation tissue. This acceleration of the repair process may have important application to healing of impaired wounds.

Show MeSH
Related in: MedlinePlus