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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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TN-C expression correlates with FAK. (A) FAK–wild-type and - fibroblasts were allowed to migrate toward FN for 3 h (left); *, P < 0.05. FAK–wild-type cells were allowed to migrate toward FN for 3 h in the presence of a control IgG or an anti–TN-C antibody (right); *, P < 0.01. (B) Semi-quantitative RT-PCR assays to assess the steady-state levels of TN-C and GAPDH mRNA levels in FAK–wild-type (+) and - fibroblasts (−). The number of PCR cycles used to amplify TN-C mRNA is indicated. (C) Western immunoblots for TN-C (top) and smooth muscle α-actin (bottom) in FAK–wild-type (+) and - (−) fibroblasts. (D) Photomicrographs showing FAK–wild-type (+) and - (−) fibroblasts plated onto FN for 24 h after immunostaining for TN-C, α-actin, or vinculin (green). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm.
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fig1: TN-C expression correlates with FAK. (A) FAK–wild-type and - fibroblasts were allowed to migrate toward FN for 3 h (left); *, P < 0.05. FAK–wild-type cells were allowed to migrate toward FN for 3 h in the presence of a control IgG or an anti–TN-C antibody (right); *, P < 0.01. (B) Semi-quantitative RT-PCR assays to assess the steady-state levels of TN-C and GAPDH mRNA levels in FAK–wild-type (+) and - fibroblasts (−). The number of PCR cycles used to amplify TN-C mRNA is indicated. (C) Western immunoblots for TN-C (top) and smooth muscle α-actin (bottom) in FAK–wild-type (+) and - (−) fibroblasts. (D) Photomicrographs showing FAK–wild-type (+) and - (−) fibroblasts plated onto FN for 24 h after immunostaining for TN-C, α-actin, or vinculin (green). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm.

Mentions: To determine whether FAK-dependent fibroblast migration toward FN relies on cellular interactions with TN-C, haptotactic migration assays were performed. Consistent with previous studies (Sieg et al., 1999), migration of FAK–wild-type cells through transwells undercoated with FN was significantly greater than that of FAK- cells (Fig. 1 A, left). To determine whether TN-C plays a role in this process, FAK–wild-type fibroblasts were plated onto transwells either in the presence of an anti–TN-C antibody, or a control IgG. Although antibody treatment did not affect cell adhesion to the transwell surface (unpublished data), blocking cellular interactions with TN-C significantly reduced fibroblast migration toward FN (Fig. 1 A, right). It should be noted that the TN-C antibody did not reduce the relative rate of cell migration to the levels observed with FAK- cells, thereby indicating that FAK-dependent fibroblast migration relies on other molecules besides TN-C. Nevertheless, these experiments allowed us to hypothesize that FAK regulates fibroblast migration, at least in part, via its effects on TN-C expression.


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)

TN-C expression correlates with FAK. (A) FAK–wild-type and - fibroblasts were allowed to migrate toward FN for 3 h (left); *, P < 0.05. FAK–wild-type cells were allowed to migrate toward FN for 3 h in the presence of a control IgG or an anti–TN-C antibody (right); *, P < 0.01. (B) Semi-quantitative RT-PCR assays to assess the steady-state levels of TN-C and GAPDH mRNA levels in FAK–wild-type (+) and - fibroblasts (−). The number of PCR cycles used to amplify TN-C mRNA is indicated. (C) Western immunoblots for TN-C (top) and smooth muscle α-actin (bottom) in FAK–wild-type (+) and - (−) fibroblasts. (D) Photomicrographs showing FAK–wild-type (+) and - (−) fibroblasts plated onto FN for 24 h after immunostaining for TN-C, α-actin, or vinculin (green). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172901&req=5

fig1: TN-C expression correlates with FAK. (A) FAK–wild-type and - fibroblasts were allowed to migrate toward FN for 3 h (left); *, P < 0.05. FAK–wild-type cells were allowed to migrate toward FN for 3 h in the presence of a control IgG or an anti–TN-C antibody (right); *, P < 0.01. (B) Semi-quantitative RT-PCR assays to assess the steady-state levels of TN-C and GAPDH mRNA levels in FAK–wild-type (+) and - fibroblasts (−). The number of PCR cycles used to amplify TN-C mRNA is indicated. (C) Western immunoblots for TN-C (top) and smooth muscle α-actin (bottom) in FAK–wild-type (+) and - (−) fibroblasts. (D) Photomicrographs showing FAK–wild-type (+) and - (−) fibroblasts plated onto FN for 24 h after immunostaining for TN-C, α-actin, or vinculin (green). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm.
Mentions: To determine whether FAK-dependent fibroblast migration toward FN relies on cellular interactions with TN-C, haptotactic migration assays were performed. Consistent with previous studies (Sieg et al., 1999), migration of FAK–wild-type cells through transwells undercoated with FN was significantly greater than that of FAK- cells (Fig. 1 A, left). To determine whether TN-C plays a role in this process, FAK–wild-type fibroblasts were plated onto transwells either in the presence of an anti–TN-C antibody, or a control IgG. Although antibody treatment did not affect cell adhesion to the transwell surface (unpublished data), blocking cellular interactions with TN-C significantly reduced fibroblast migration toward FN (Fig. 1 A, right). It should be noted that the TN-C antibody did not reduce the relative rate of cell migration to the levels observed with FAK- cells, thereby indicating that FAK-dependent fibroblast migration relies on other molecules besides TN-C. Nevertheless, these experiments allowed us to hypothesize that FAK regulates fibroblast migration, at least in part, via its effects on TN-C expression.

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