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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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Prx1 promotes TN-C–dependent migration. (A) FAK- fibroblasts transfected with an HA-control or HA-Prx1 expression vector were allowed to migrate for 3 h toward FN in a transwell migration assay, in the presence of control IgG or an anti–TN-C antibody. Transfection efficiency was the same for all cells. Shown is the relative cell migration from at least three independent experiments; *, P < 0.05. (B) Representative fluorescence micrograph showing Prx1 immunoreactivity in FAK- fibroblasts transiently transfected with the HA-Prx1 expression vector. Cells were stained for Prx1 via HA (red) and TN-C (green). Nuclei are visualized by DAPI staining. Bar, 30 μm. (C) Immunofluorescence photomicrographs showing FAK- (−) fibroblasts plated onto either FN or TN-C, and then 24 h later immunostained with antibodies recognizing α-actin or vinculin (both in green). Cell nuclei were visualized with DAPI (blue). Bar, 20 μm. (D) Haptotactic migration assays were used to evaluate fibroblast migration toward TN-C or FN in FAK–wild-type (+) and - (−) fibroblasts. Shown is the relative cell migration from at least three independent experiments; *, P < 0.05.
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fig6: Prx1 promotes TN-C–dependent migration. (A) FAK- fibroblasts transfected with an HA-control or HA-Prx1 expression vector were allowed to migrate for 3 h toward FN in a transwell migration assay, in the presence of control IgG or an anti–TN-C antibody. Transfection efficiency was the same for all cells. Shown is the relative cell migration from at least three independent experiments; *, P < 0.05. (B) Representative fluorescence micrograph showing Prx1 immunoreactivity in FAK- fibroblasts transiently transfected with the HA-Prx1 expression vector. Cells were stained for Prx1 via HA (red) and TN-C (green). Nuclei are visualized by DAPI staining. Bar, 30 μm. (C) Immunofluorescence photomicrographs showing FAK- (−) fibroblasts plated onto either FN or TN-C, and then 24 h later immunostained with antibodies recognizing α-actin or vinculin (both in green). Cell nuclei were visualized with DAPI (blue). Bar, 20 μm. (D) Haptotactic migration assays were used to evaluate fibroblast migration toward TN-C or FN in FAK–wild-type (+) and - (−) fibroblasts. Shown is the relative cell migration from at least three independent experiments; *, P < 0.05.

Mentions: The aforementioned studies indicate that FAK-dependent induction of Prx1 activates TN-C gene transcription, and subsequently the formation of a TN-C–enriched ECM that conceivably supports cell migration on FN. To determine whether Prx1 acts downstream from FAK in promoting TN-C–dependent migration toward FN, we transiently transfected FAK- fibroblasts with either a control HA vector or the HA-tagged Prx1 plasmid, together with a β-galactosidase expression construct. Transfection efficiency was the same in all cells (unpublished data). Thereafter, transfected cells were replated onto transwells undercoated with FN, either in the presence of a control IgG, or an anti–TN-C antibody. Antibody treatment had no effect on cell adhesion to the transwell surface. In IgG-treated cultures, Prx1 expression in FAK- cells promoted cell migration toward FN compared with control-transfected cells (Fig. 6 A), whereas the anti–TN-C antibody significantly inhibited this effect (Fig. 6 A).


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)

Prx1 promotes TN-C–dependent migration. (A) FAK- fibroblasts transfected with an HA-control or HA-Prx1 expression vector were allowed to migrate for 3 h toward FN in a transwell migration assay, in the presence of control IgG or an anti–TN-C antibody. Transfection efficiency was the same for all cells. Shown is the relative cell migration from at least three independent experiments; *, P < 0.05. (B) Representative fluorescence micrograph showing Prx1 immunoreactivity in FAK- fibroblasts transiently transfected with the HA-Prx1 expression vector. Cells were stained for Prx1 via HA (red) and TN-C (green). Nuclei are visualized by DAPI staining. Bar, 30 μm. (C) Immunofluorescence photomicrographs showing FAK- (−) fibroblasts plated onto either FN or TN-C, and then 24 h later immunostained with antibodies recognizing α-actin or vinculin (both in green). Cell nuclei were visualized with DAPI (blue). Bar, 20 μm. (D) Haptotactic migration assays were used to evaluate fibroblast migration toward TN-C or FN in FAK–wild-type (+) and - (−) fibroblasts. Shown is the relative cell migration from at least three independent experiments; *, P < 0.05.
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Related In: Results  -  Collection

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fig6: Prx1 promotes TN-C–dependent migration. (A) FAK- fibroblasts transfected with an HA-control or HA-Prx1 expression vector were allowed to migrate for 3 h toward FN in a transwell migration assay, in the presence of control IgG or an anti–TN-C antibody. Transfection efficiency was the same for all cells. Shown is the relative cell migration from at least three independent experiments; *, P < 0.05. (B) Representative fluorescence micrograph showing Prx1 immunoreactivity in FAK- fibroblasts transiently transfected with the HA-Prx1 expression vector. Cells were stained for Prx1 via HA (red) and TN-C (green). Nuclei are visualized by DAPI staining. Bar, 30 μm. (C) Immunofluorescence photomicrographs showing FAK- (−) fibroblasts plated onto either FN or TN-C, and then 24 h later immunostained with antibodies recognizing α-actin or vinculin (both in green). Cell nuclei were visualized with DAPI (blue). Bar, 20 μm. (D) Haptotactic migration assays were used to evaluate fibroblast migration toward TN-C or FN in FAK–wild-type (+) and - (−) fibroblasts. Shown is the relative cell migration from at least three independent experiments; *, P < 0.05.
Mentions: The aforementioned studies indicate that FAK-dependent induction of Prx1 activates TN-C gene transcription, and subsequently the formation of a TN-C–enriched ECM that conceivably supports cell migration on FN. To determine whether Prx1 acts downstream from FAK in promoting TN-C–dependent migration toward FN, we transiently transfected FAK- fibroblasts with either a control HA vector or the HA-tagged Prx1 plasmid, together with a β-galactosidase expression construct. Transfection efficiency was the same in all cells (unpublished data). Thereafter, transfected cells were replated onto transwells undercoated with FN, either in the presence of a control IgG, or an anti–TN-C antibody. Antibody treatment had no effect on cell adhesion to the transwell surface. In IgG-treated cultures, Prx1 expression in FAK- cells promoted cell migration toward FN compared with control-transfected cells (Fig. 6 A), whereas the anti–TN-C antibody significantly inhibited this effect (Fig. 6 A).

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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