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FAK induces expression of Prx1 to promote tenascin-C-dependent fibroblast migration.

McKean DM, Sisbarro L, Ilic D, Kaplan-Alburquerque N, Nemenoff R, Weiser-Evans M, Kern MJ, Jones PL - J. Cell Biol. (2003)

Bottom Line: Consistent with the idea that FAK regulates TN-C, migration-defective FAK- cells expressed reduced levels of TN-C.Furthermore, expression of FAK in FAK- fibroblasts induced TN-C, whereas inhibition of FAK activity in FAK-wild-type cells had the opposite effect.These results appear to be relevant in vivo because Prx1 and TN-C expression levels were reduced in FAK- embryos.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

ABSTRACT
Fibroblast migration depends, in part, on activation of FAK and cellular interactions with tenascin-C (TN-C). Consistent with the idea that FAK regulates TN-C, migration-defective FAK- cells expressed reduced levels of TN-C. Furthermore, expression of FAK in FAK- fibroblasts induced TN-C, whereas inhibition of FAK activity in FAK-wild-type cells had the opposite effect. Paired-related homeobox 1 (Prx1) encodes a homeobox transcription factor that induces TN-C by interacting with a binding site within the TN-C promoter, and it also promotes fibroblast migration. Therefore, we hypothesized that FAK regulates TN-C by controlling the DNA-binding activity of Prx1 and/or by inducing Prx1 expression. Prx1-homeodomain binding site complex formation observed with FAK-wild-type fibroblasts failed to occur in FAK- fibroblasts, yet expression of Prx1 in these cells induced TN-C promoter activity. Thus, FAK is not essential for Prx1 DNA-binding activity. However, activated FAK was essential for Prx1 expression. Functionally, Prx1 expression in FAK- fibroblasts restored their ability to migrate toward fibronectin, in a manner that depends on TN-C. These results appear to be relevant in vivo because Prx1 and TN-C expression levels were reduced in FAK- embryos. This paper suggests a model whereby FAK induces Prx1, and subsequently the formation of a TN-C-enriched ECM that contributes to fibroblast migration.

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Paired-related homeobox genes regulate TN-C and fibroblast migration. (A) Representative phase-contrast (top) and fluorescence (bottom) micrographs showing FAK–wild-type cells treated with FITC-labeled control or Prx1 and Prx2 antisense (AS) oligonucleotides. Bar, 30 μm. (B) Western immunoblotting for Prx1, Prx2, TN-C, and α-actin in control and AS oligonucleotide–treated FAK–wild-type fibroblasts. The anti–Prx1 antibody recognizes two isoforms designated Prx1a and Prx1b. An additional cross-reactive band was also detected in both samples, as reported previously (Chesterman et al., 2001). (C) Haptotactic migration assays were used to evaluate fibroblast migration toward FN in control and AS-treated cells. Shown is relative cell migration between groups ± SEM of triplicates from at least three independent experiments. *, P < 0.05.
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fig3: Paired-related homeobox genes regulate TN-C and fibroblast migration. (A) Representative phase-contrast (top) and fluorescence (bottom) micrographs showing FAK–wild-type cells treated with FITC-labeled control or Prx1 and Prx2 antisense (AS) oligonucleotides. Bar, 30 μm. (B) Western immunoblotting for Prx1, Prx2, TN-C, and α-actin in control and AS oligonucleotide–treated FAK–wild-type fibroblasts. The anti–Prx1 antibody recognizes two isoforms designated Prx1a and Prx1b. An additional cross-reactive band was also detected in both samples, as reported previously (Chesterman et al., 2001). (C) Haptotactic migration assays were used to evaluate fibroblast migration toward FN in control and AS-treated cells. Shown is relative cell migration between groups ± SEM of triplicates from at least three independent experiments. *, P < 0.05.

Mentions: Phase-contrast and fluorescence microscopic imaging of FAK–wild-type cells demonstrated uptake of control and antisense oligonucleotides in more than 70% of treated cells (Fig. 3 A), with no evidence of toxicity (unpublished data). Because morpholino oligonucleotides affect protein translation rather than mRNA stability, we next evaluated Prx1 and Prx2 protein levels by Western immunoblotting. Antisense morpholino-treated fibroblasts expressed reduced levels of Prx1 and Prx2 protein, when compared with controls (Fig. 3 B). It should also be noted that an additional cross-reactive band was detected in Western blots evaluating Prx1 expression, as reported previously (Chesterman et al., 2001). Targeted suppression of Prx1 and Prx2 also resulted in down-regulation of TN-C protein expression, whereas α-actin protein expression remained unchanged (Fig. 3 B). Having established a system in which expression of Prx1, Prx2, and TN-C are suppressed, we evaluated cell migration toward FN. Compared with control oligonucleotide-treated cells, haptotactic migration was significantly reduced in cultures treated with Prx antisense oligonucleotides (Fig. 3 C), despite the fact that cell adhesion to the transwell surface was unaffected (unpublished data). Thus, in addition to FAK and TN-C, Prx transcription factors are required for fibroblast migration toward FN.


FAK induces expression of Prx1 to promote tenascin-C-dependent fibroblast migration.

McKean DM, Sisbarro L, Ilic D, Kaplan-Alburquerque N, Nemenoff R, Weiser-Evans M, Kern MJ, Jones PL - J. Cell Biol. (2003)

Paired-related homeobox genes regulate TN-C and fibroblast migration. (A) Representative phase-contrast (top) and fluorescence (bottom) micrographs showing FAK–wild-type cells treated with FITC-labeled control or Prx1 and Prx2 antisense (AS) oligonucleotides. Bar, 30 μm. (B) Western immunoblotting for Prx1, Prx2, TN-C, and α-actin in control and AS oligonucleotide–treated FAK–wild-type fibroblasts. The anti–Prx1 antibody recognizes two isoforms designated Prx1a and Prx1b. An additional cross-reactive band was also detected in both samples, as reported previously (Chesterman et al., 2001). (C) Haptotactic migration assays were used to evaluate fibroblast migration toward FN in control and AS-treated cells. Shown is relative cell migration between groups ± SEM of triplicates from at least three independent experiments. *, P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172901&req=5

fig3: Paired-related homeobox genes regulate TN-C and fibroblast migration. (A) Representative phase-contrast (top) and fluorescence (bottom) micrographs showing FAK–wild-type cells treated with FITC-labeled control or Prx1 and Prx2 antisense (AS) oligonucleotides. Bar, 30 μm. (B) Western immunoblotting for Prx1, Prx2, TN-C, and α-actin in control and AS oligonucleotide–treated FAK–wild-type fibroblasts. The anti–Prx1 antibody recognizes two isoforms designated Prx1a and Prx1b. An additional cross-reactive band was also detected in both samples, as reported previously (Chesterman et al., 2001). (C) Haptotactic migration assays were used to evaluate fibroblast migration toward FN in control and AS-treated cells. Shown is relative cell migration between groups ± SEM of triplicates from at least three independent experiments. *, P < 0.05.
Mentions: Phase-contrast and fluorescence microscopic imaging of FAK–wild-type cells demonstrated uptake of control and antisense oligonucleotides in more than 70% of treated cells (Fig. 3 A), with no evidence of toxicity (unpublished data). Because morpholino oligonucleotides affect protein translation rather than mRNA stability, we next evaluated Prx1 and Prx2 protein levels by Western immunoblotting. Antisense morpholino-treated fibroblasts expressed reduced levels of Prx1 and Prx2 protein, when compared with controls (Fig. 3 B). It should also be noted that an additional cross-reactive band was detected in Western blots evaluating Prx1 expression, as reported previously (Chesterman et al., 2001). Targeted suppression of Prx1 and Prx2 also resulted in down-regulation of TN-C protein expression, whereas α-actin protein expression remained unchanged (Fig. 3 B). Having established a system in which expression of Prx1, Prx2, and TN-C are suppressed, we evaluated cell migration toward FN. Compared with control oligonucleotide-treated cells, haptotactic migration was significantly reduced in cultures treated with Prx antisense oligonucleotides (Fig. 3 C), despite the fact that cell adhesion to the transwell surface was unaffected (unpublished data). Thus, in addition to FAK and TN-C, Prx transcription factors are required for fibroblast migration toward FN.

Bottom Line: Consistent with the idea that FAK regulates TN-C, migration-defective FAK- cells expressed reduced levels of TN-C.Furthermore, expression of FAK in FAK- fibroblasts induced TN-C, whereas inhibition of FAK activity in FAK-wild-type cells had the opposite effect.These results appear to be relevant in vivo because Prx1 and TN-C expression levels were reduced in FAK- embryos.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

ABSTRACT
Fibroblast migration depends, in part, on activation of FAK and cellular interactions with tenascin-C (TN-C). Consistent with the idea that FAK regulates TN-C, migration-defective FAK- cells expressed reduced levels of TN-C. Furthermore, expression of FAK in FAK- fibroblasts induced TN-C, whereas inhibition of FAK activity in FAK-wild-type cells had the opposite effect. Paired-related homeobox 1 (Prx1) encodes a homeobox transcription factor that induces TN-C by interacting with a binding site within the TN-C promoter, and it also promotes fibroblast migration. Therefore, we hypothesized that FAK regulates TN-C by controlling the DNA-binding activity of Prx1 and/or by inducing Prx1 expression. Prx1-homeodomain binding site complex formation observed with FAK-wild-type fibroblasts failed to occur in FAK- fibroblasts, yet expression of Prx1 in these cells induced TN-C promoter activity. Thus, FAK is not essential for Prx1 DNA-binding activity. However, activated FAK was essential for Prx1 expression. Functionally, Prx1 expression in FAK- fibroblasts restored their ability to migrate toward fibronectin, in a manner that depends on TN-C. These results appear to be relevant in vivo because Prx1 and TN-C expression levels were reduced in FAK- embryos. This paper suggests a model whereby FAK induces Prx1, and subsequently the formation of a TN-C-enriched ECM that contributes to fibroblast migration.

Show MeSH
Related in: MedlinePlus