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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 interactions with the HBS and up-regulation of TN-C. (A) EMSAs using FAK–wild-type (+) and - (−) fibroblast nuclear extracts, incubated with either a radiolabeled wild-type (W) or mutated (M) HBS probe. To determine the presence of Prx proteins in binding complexes, the wild-type probe and FAK–wild-type nuclear extracts were incubated with antibodies that recognize Prx1, Prx2, or a control IgG. In separate experiments, in vitro transcribed/translated, recombinant rPrx1 protein was incubated with the wild-type–HBS in the absence of nuclear extracts. These complexes were also treated with an anti–Prx1 antisera or a control IgG. (B) TN-C promoter/luciferase gene reporter assays in FAK–wild-type (+) or – (−) cells cotransfected with a TN-C promoter/luciferase gene reporter and HA epitope-tagged Prx expression vectors (i.e., HA-Prx1, HA-Prx2, or a combination of both). A β-galactosidase expression vector was included in each transfection to normalize for transfection efficiency. Shown is the relative fold increase in TN-C promoter activity ± SEM of triplicates from at least three independent experiments. *, P < 0.05 between all groups. #, P < 0.05 between HA vector alone transfected into FAK-wild versus - cells.
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fig4: Prx1 interactions with the HBS and up-regulation of TN-C. (A) EMSAs using FAK–wild-type (+) and - (−) fibroblast nuclear extracts, incubated with either a radiolabeled wild-type (W) or mutated (M) HBS probe. To determine the presence of Prx proteins in binding complexes, the wild-type probe and FAK–wild-type nuclear extracts were incubated with antibodies that recognize Prx1, Prx2, or a control IgG. In separate experiments, in vitro transcribed/translated, recombinant rPrx1 protein was incubated with the wild-type–HBS in the absence of nuclear extracts. These complexes were also treated with an anti–Prx1 antisera or a control IgG. (B) TN-C promoter/luciferase gene reporter assays in FAK–wild-type (+) or – (−) cells cotransfected with a TN-C promoter/luciferase gene reporter and HA epitope-tagged Prx expression vectors (i.e., HA-Prx1, HA-Prx2, or a combination of both). A β-galactosidase expression vector was included in each transfection to normalize for transfection efficiency. Shown is the relative fold increase in TN-C promoter activity ± SEM of triplicates from at least three independent experiments. *, P < 0.05 between all groups. #, P < 0.05 between HA vector alone transfected into FAK-wild versus - cells.

Mentions: FAK–wild-type fibroblast nuclear extracts incubated with a radiolabeled wild-type HBS probe formed three distinct DNA–protein complexes in EMSAs (Fig. 4 A, lane 2), whereas incubation of FAK–wild-type nuclear extracts with the mutated HBS probe failed to support complex formation (Fig. 4 A, lane 1). Similarly, DNA–protein complex formation did not occur when the wild-type or mutated HBS were incubated with nuclear extracts derived from FAK- cells (Fig. 4 A, lanes 6 and 7). To determine whether Prx proteins were present in DNA–protein complexes, the wild-type probe and FAK–wild-type nuclear extracts were incubated with antibodies that recognize either Prx1 or Prx2. Treatment with the Prx1 antibody resulted in an almost complete disruption of the DNA–protein complexes (Fig. 4 A, lane 3), whereas incubation with the anti-Prx2 antibody, or a control IgG, had no effect (Fig. 4 A, lanes 4 and 5). These experiments demonstrate that Prx1 protein binding to the HBS fails to occur in the absence of FAK, and indicate that Prx2 does not interact with the HBS in vitro, and is therefore unlikely to be a constituent of the FAK signaling pathway controlling TN-C expression in fibroblasts.


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 interactions with the HBS and up-regulation of TN-C. (A) EMSAs using FAK–wild-type (+) and - (−) fibroblast nuclear extracts, incubated with either a radiolabeled wild-type (W) or mutated (M) HBS probe. To determine the presence of Prx proteins in binding complexes, the wild-type probe and FAK–wild-type nuclear extracts were incubated with antibodies that recognize Prx1, Prx2, or a control IgG. In separate experiments, in vitro transcribed/translated, recombinant rPrx1 protein was incubated with the wild-type–HBS in the absence of nuclear extracts. These complexes were also treated with an anti–Prx1 antisera or a control IgG. (B) TN-C promoter/luciferase gene reporter assays in FAK–wild-type (+) or – (−) cells cotransfected with a TN-C promoter/luciferase gene reporter and HA epitope-tagged Prx expression vectors (i.e., HA-Prx1, HA-Prx2, or a combination of both). A β-galactosidase expression vector was included in each transfection to normalize for transfection efficiency. Shown is the relative fold increase in TN-C promoter activity ± SEM of triplicates from at least three independent experiments. *, P < 0.05 between all groups. #, P < 0.05 between HA vector alone transfected into FAK-wild versus - cells.
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Related In: Results  -  Collection

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fig4: Prx1 interactions with the HBS and up-regulation of TN-C. (A) EMSAs using FAK–wild-type (+) and - (−) fibroblast nuclear extracts, incubated with either a radiolabeled wild-type (W) or mutated (M) HBS probe. To determine the presence of Prx proteins in binding complexes, the wild-type probe and FAK–wild-type nuclear extracts were incubated with antibodies that recognize Prx1, Prx2, or a control IgG. In separate experiments, in vitro transcribed/translated, recombinant rPrx1 protein was incubated with the wild-type–HBS in the absence of nuclear extracts. These complexes were also treated with an anti–Prx1 antisera or a control IgG. (B) TN-C promoter/luciferase gene reporter assays in FAK–wild-type (+) or – (−) cells cotransfected with a TN-C promoter/luciferase gene reporter and HA epitope-tagged Prx expression vectors (i.e., HA-Prx1, HA-Prx2, or a combination of both). A β-galactosidase expression vector was included in each transfection to normalize for transfection efficiency. Shown is the relative fold increase in TN-C promoter activity ± SEM of triplicates from at least three independent experiments. *, P < 0.05 between all groups. #, P < 0.05 between HA vector alone transfected into FAK-wild versus - cells.
Mentions: FAK–wild-type fibroblast nuclear extracts incubated with a radiolabeled wild-type HBS probe formed three distinct DNA–protein complexes in EMSAs (Fig. 4 A, lane 2), whereas incubation of FAK–wild-type nuclear extracts with the mutated HBS probe failed to support complex formation (Fig. 4 A, lane 1). Similarly, DNA–protein complex formation did not occur when the wild-type or mutated HBS were incubated with nuclear extracts derived from FAK- cells (Fig. 4 A, lanes 6 and 7). To determine whether Prx proteins were present in DNA–protein complexes, the wild-type probe and FAK–wild-type nuclear extracts were incubated with antibodies that recognize either Prx1 or Prx2. Treatment with the Prx1 antibody resulted in an almost complete disruption of the DNA–protein complexes (Fig. 4 A, lane 3), whereas incubation with the anti-Prx2 antibody, or a control IgG, had no effect (Fig. 4 A, lanes 4 and 5). These experiments demonstrate that Prx1 protein binding to the HBS fails to occur in the absence of FAK, and indicate that Prx2 does not interact with the HBS in vitro, and is therefore unlikely to be a constituent of the FAK signaling pathway controlling TN-C expression in fibroblasts.

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