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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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Activated FAK is required for TN-C expression. (A) Immunostaining for wild-type FAK fusion protein (left, green), or a kinase-dead FAK mutant (Y397F; right, green), as well as TN-C (red), in transiently transfected FAK- (−) fibroblasts. Nuclei are stained blue with DAPI. Bar, 15 μm. (B) Semi-quantitative RT-PCR assays to assess the steady-state levels of TN-C and GAPDH mRNA levels in DA2 and control cells. The number of PCR cycles used to amplify TN-C mRNA is indicated. (C) Photomicrographs showing DA2 and control cells plated onto FN after immunostaining for TN-C (red). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm. (D) Photomicrographs showing FAK–wild-type (+) cells replated onto FN after transient transfection with FRNK. Cells in the left panel were stained for the c-myc/FRNK fusion protein (red) and activated FAK (Y397 phospho-FAK–specific antibody; green). Cells in the right panel were stained for the c-myc/FRNK fusion protein (red) and TN-C (green). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm. (E) Western immunoblotting for FRNK, total FAK, phosphorylated FAK and TN-C in cells transiently transfected with a control vector (−), or with a plasmid encoding a c-myc/FRNK fusion protein (+).
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fig2: Activated FAK is required for TN-C expression. (A) Immunostaining for wild-type FAK fusion protein (left, green), or a kinase-dead FAK mutant (Y397F; right, green), as well as TN-C (red), in transiently transfected FAK- (−) fibroblasts. Nuclei are stained blue with DAPI. Bar, 15 μm. (B) Semi-quantitative RT-PCR assays to assess the steady-state levels of TN-C and GAPDH mRNA levels in DA2 and control cells. The number of PCR cycles used to amplify TN-C mRNA is indicated. (C) Photomicrographs showing DA2 and control cells plated onto FN after immunostaining for TN-C (red). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm. (D) Photomicrographs showing FAK–wild-type (+) cells replated onto FN after transient transfection with FRNK. Cells in the left panel were stained for the c-myc/FRNK fusion protein (red) and activated FAK (Y397 phospho-FAK–specific antibody; green). Cells in the right panel were stained for the c-myc/FRNK fusion protein (red) and TN-C (green). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm. (E) Western immunoblotting for FRNK, total FAK, phosphorylated FAK and TN-C in cells transiently transfected with a control vector (−), or with a plasmid encoding a c-myc/FRNK fusion protein (+).

Mentions: Next, we determined whether FAK activity is required for TN-C expression. FAK- fibroblasts were replated onto FN after transient transfection with plasmids encoding either an IL2R-FAK fusion protein (i.e., constitutively active FAK), or a mutated IL2R-FAK protein (Y397F), in which the SH2 binding site tyrosine has been substituted for phenylalanine (Tamura et al., 1999). Immunostaining for IL2R-FAK and TN-C in transfected cells showed that constitutive expression of wild-type FAK, but not the mutated form, increased extracellular TN-C deposition (Fig. 2 A). In parallel, we examined TN-C mRNA and protein expression using a FAK- cell clone that was engineered to stably express FAK (i.e., DA2 cells; Sieg et al., 1999). The characteristics of DA2 cells have been described previously in detail (Sieg et al., 1999), including their restored ability to migrate toward FN. RT-PCR (Fig. 2 B) and immunofluorescence (Fig. 2 C) assays showed that TN-C expression is greater in DA2 fibroblasts than in controls. However, the basal levels of TN-C expression were greater in the control cells for DA2 when compared with FAK- cells (Fig. 1 B). A possible explanation for this difference may lie in the fact that DA2 and its control line represent hygromycin-selected clones. Because nonselected FAK- cells are heterogeneous in nature, and they do express some TN-C, it is possible that clonal selection enriched for cells expressing higher basal levels of TN-C.


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)

Activated FAK is required for TN-C expression. (A) Immunostaining for wild-type FAK fusion protein (left, green), or a kinase-dead FAK mutant (Y397F; right, green), as well as TN-C (red), in transiently transfected FAK- (−) fibroblasts. Nuclei are stained blue with DAPI. Bar, 15 μm. (B) Semi-quantitative RT-PCR assays to assess the steady-state levels of TN-C and GAPDH mRNA levels in DA2 and control cells. The number of PCR cycles used to amplify TN-C mRNA is indicated. (C) Photomicrographs showing DA2 and control cells plated onto FN after immunostaining for TN-C (red). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm. (D) Photomicrographs showing FAK–wild-type (+) cells replated onto FN after transient transfection with FRNK. Cells in the left panel were stained for the c-myc/FRNK fusion protein (red) and activated FAK (Y397 phospho-FAK–specific antibody; green). Cells in the right panel were stained for the c-myc/FRNK fusion protein (red) and TN-C (green). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm. (E) Western immunoblotting for FRNK, total FAK, phosphorylated FAK and TN-C in cells transiently transfected with a control vector (−), or with a plasmid encoding a c-myc/FRNK fusion protein (+).
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Related In: Results  -  Collection

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fig2: Activated FAK is required for TN-C expression. (A) Immunostaining for wild-type FAK fusion protein (left, green), or a kinase-dead FAK mutant (Y397F; right, green), as well as TN-C (red), in transiently transfected FAK- (−) fibroblasts. Nuclei are stained blue with DAPI. Bar, 15 μm. (B) Semi-quantitative RT-PCR assays to assess the steady-state levels of TN-C and GAPDH mRNA levels in DA2 and control cells. The number of PCR cycles used to amplify TN-C mRNA is indicated. (C) Photomicrographs showing DA2 and control cells plated onto FN after immunostaining for TN-C (red). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm. (D) Photomicrographs showing FAK–wild-type (+) cells replated onto FN after transient transfection with FRNK. Cells in the left panel were stained for the c-myc/FRNK fusion protein (red) and activated FAK (Y397 phospho-FAK–specific antibody; green). Cells in the right panel were stained for the c-myc/FRNK fusion protein (red) and TN-C (green). Cell nuclei were visualized with DAPI (blue). Bar, 30 μm. (E) Western immunoblotting for FRNK, total FAK, phosphorylated FAK and TN-C in cells transiently transfected with a control vector (−), or with a plasmid encoding a c-myc/FRNK fusion protein (+).
Mentions: Next, we determined whether FAK activity is required for TN-C expression. FAK- fibroblasts were replated onto FN after transient transfection with plasmids encoding either an IL2R-FAK fusion protein (i.e., constitutively active FAK), or a mutated IL2R-FAK protein (Y397F), in which the SH2 binding site tyrosine has been substituted for phenylalanine (Tamura et al., 1999). Immunostaining for IL2R-FAK and TN-C in transfected cells showed that constitutive expression of wild-type FAK, but not the mutated form, increased extracellular TN-C deposition (Fig. 2 A). In parallel, we examined TN-C mRNA and protein expression using a FAK- cell clone that was engineered to stably express FAK (i.e., DA2 cells; Sieg et al., 1999). The characteristics of DA2 cells have been described previously in detail (Sieg et al., 1999), including their restored ability to migrate toward FN. RT-PCR (Fig. 2 B) and immunofluorescence (Fig. 2 C) assays showed that TN-C expression is greater in DA2 fibroblasts than in controls. However, the basal levels of TN-C expression were greater in the control cells for DA2 when compared with FAK- cells (Fig. 1 B). A possible explanation for this difference may lie in the fact that DA2 and its control line represent hygromycin-selected clones. Because nonselected FAK- cells are heterogeneous in nature, and they do express some TN-C, it is possible that clonal selection enriched for cells expressing higher basal levels of TN-C.

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