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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 expression depends on activated FAK. (A) Semi-quantitative RT-PCR assays to assess steady-state levels of Prx1 and GAPDH in FAK–wild-type (+) and - (−) fibroblasts. The number of PCR cycles used to amplify Prx1 mRNA is indicated. (B) Western immunoblots for Prx1a and Prx1b (top) and α-actin (bottom) in FAK–wild-type (+) and - (−) fibroblasts. An additional cross-reactive band was also detected in both samples. (C) Representative fluorescence micrographs assessing Prx1 immunoreactivity in FAK- (−) fibroblasts (left), or in FAK- fibroblasts transiently transfected with the IL2R-FAK wild-type fusion protein (right). FAK is shown in green, whereas Prx1 is shown in red. Nuclei are visualized by DAPI staining. Bar, 15 μm. (D) Semi-quantitative RT-PCR assays were used to assess the steady-state levels of Prx1 and GAPDH mRNA levels in DA2 cells (i.e., FAK- cells stably transfected with FAK) and its control line (i.e., FAK- cells stably transfected with pcDNA3.1). The number of PCR cycles used to amplify Prx1 mRNA is indicated. (E) Photomicrographs showing DA2 cells and its control line plated onto FN after immunostaining for Prx1 protein (red). Bar, 30 μm. (F) Photomicrographs showing FAK–wild-type (+) cells replated onto FN followed by immunostaining for Prx1 (left). FAK–wild-type cells were transiently transfected with the c-myc/FRNK fusion protein (right), and then stained for FRNK using c-myc antibody (green) and for Prx1 protein (red). Cell nuclei were visualized with DAPI (blue). Bar, 15 μm.
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fig5: Prx1 expression depends on activated FAK. (A) Semi-quantitative RT-PCR assays to assess steady-state levels of Prx1 and GAPDH in FAK–wild-type (+) and - (−) fibroblasts. The number of PCR cycles used to amplify Prx1 mRNA is indicated. (B) Western immunoblots for Prx1a and Prx1b (top) and α-actin (bottom) in FAK–wild-type (+) and - (−) fibroblasts. An additional cross-reactive band was also detected in both samples. (C) Representative fluorescence micrographs assessing Prx1 immunoreactivity in FAK- (−) fibroblasts (left), or in FAK- fibroblasts transiently transfected with the IL2R-FAK wild-type fusion protein (right). FAK is shown in green, whereas Prx1 is shown in red. Nuclei are visualized by DAPI staining. Bar, 15 μm. (D) Semi-quantitative RT-PCR assays were used to assess the steady-state levels of Prx1 and GAPDH mRNA levels in DA2 cells (i.e., FAK- cells stably transfected with FAK) and its control line (i.e., FAK- cells stably transfected with pcDNA3.1). The number of PCR cycles used to amplify Prx1 mRNA is indicated. (E) Photomicrographs showing DA2 cells and its control line plated onto FN after immunostaining for Prx1 protein (red). Bar, 30 μm. (F) Photomicrographs showing FAK–wild-type (+) cells replated onto FN followed by immunostaining for Prx1 (left). FAK–wild-type cells were transiently transfected with the c-myc/FRNK fusion protein (right), and then stained for FRNK using c-myc antibody (green) and for Prx1 protein (red). Cell nuclei were visualized with DAPI (blue). Bar, 15 μm.

Mentions: To determine whether FAK regulates TN-C by inducing expression of Prx1, we conducted RT-PCR assays using total RNA isolated from FAK–wild-type and - fibroblasts, which showed that the steady-state levels of Prx1 mRNA are suppressed in FAK- cells (Fig. 5 A). Consistent with this, Western immunoblotting showed that expression of Prx1a and Prx1b was suppressed in FAK- cells (Fig. 5 B). Next, we directly determined whether FAK is required for Prx1 expression. It has been reported previously that although the Prx1 antibody recognizes a cross-reactive band in Western immunoblots, this antibody specifically recognizes Prx1 when used for immunocytochemistry (Chesterman et al., 2001). Immunostaining experiments confirmed that FAK- cells do not express Prx1 protein (Fig. 5 C, left), whereas FAK- cells transiently transfected with the plasmid encoding the IL2R-FAK wild-type fusion protein expressed Prx1 protein in their nuclei (Fig. 5 C, right). RT-PCR and immunofluorescence experiments using the FAK-reconstituted DA2 clone also showed that FAK is required for Prx1 mRNA, and protein expression (Fig. 5, D and E). As with TN-C mRNA expression, basal levels of Prx1 expression were greater in control cells for DA2 (Fig. 5 A), when compared with FAK- cells.


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 expression depends on activated FAK. (A) Semi-quantitative RT-PCR assays to assess steady-state levels of Prx1 and GAPDH in FAK–wild-type (+) and - (−) fibroblasts. The number of PCR cycles used to amplify Prx1 mRNA is indicated. (B) Western immunoblots for Prx1a and Prx1b (top) and α-actin (bottom) in FAK–wild-type (+) and - (−) fibroblasts. An additional cross-reactive band was also detected in both samples. (C) Representative fluorescence micrographs assessing Prx1 immunoreactivity in FAK- (−) fibroblasts (left), or in FAK- fibroblasts transiently transfected with the IL2R-FAK wild-type fusion protein (right). FAK is shown in green, whereas Prx1 is shown in red. Nuclei are visualized by DAPI staining. Bar, 15 μm. (D) Semi-quantitative RT-PCR assays were used to assess the steady-state levels of Prx1 and GAPDH mRNA levels in DA2 cells (i.e., FAK- cells stably transfected with FAK) and its control line (i.e., FAK- cells stably transfected with pcDNA3.1). The number of PCR cycles used to amplify Prx1 mRNA is indicated. (E) Photomicrographs showing DA2 cells and its control line plated onto FN after immunostaining for Prx1 protein (red). Bar, 30 μm. (F) Photomicrographs showing FAK–wild-type (+) cells replated onto FN followed by immunostaining for Prx1 (left). FAK–wild-type cells were transiently transfected with the c-myc/FRNK fusion protein (right), and then stained for FRNK using c-myc antibody (green) and for Prx1 protein (red). Cell nuclei were visualized with DAPI (blue). Bar, 15 μm.
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fig5: Prx1 expression depends on activated FAK. (A) Semi-quantitative RT-PCR assays to assess steady-state levels of Prx1 and GAPDH in FAK–wild-type (+) and - (−) fibroblasts. The number of PCR cycles used to amplify Prx1 mRNA is indicated. (B) Western immunoblots for Prx1a and Prx1b (top) and α-actin (bottom) in FAK–wild-type (+) and - (−) fibroblasts. An additional cross-reactive band was also detected in both samples. (C) Representative fluorescence micrographs assessing Prx1 immunoreactivity in FAK- (−) fibroblasts (left), or in FAK- fibroblasts transiently transfected with the IL2R-FAK wild-type fusion protein (right). FAK is shown in green, whereas Prx1 is shown in red. Nuclei are visualized by DAPI staining. Bar, 15 μm. (D) Semi-quantitative RT-PCR assays were used to assess the steady-state levels of Prx1 and GAPDH mRNA levels in DA2 cells (i.e., FAK- cells stably transfected with FAK) and its control line (i.e., FAK- cells stably transfected with pcDNA3.1). The number of PCR cycles used to amplify Prx1 mRNA is indicated. (E) Photomicrographs showing DA2 cells and its control line plated onto FN after immunostaining for Prx1 protein (red). Bar, 30 μm. (F) Photomicrographs showing FAK–wild-type (+) cells replated onto FN followed by immunostaining for Prx1 (left). FAK–wild-type cells were transiently transfected with the c-myc/FRNK fusion protein (right), and then stained for FRNK using c-myc antibody (green) and for Prx1 protein (red). Cell nuclei were visualized with DAPI (blue). Bar, 15 μm.
Mentions: To determine whether FAK regulates TN-C by inducing expression of Prx1, we conducted RT-PCR assays using total RNA isolated from FAK–wild-type and - fibroblasts, which showed that the steady-state levels of Prx1 mRNA are suppressed in FAK- cells (Fig. 5 A). Consistent with this, Western immunoblotting showed that expression of Prx1a and Prx1b was suppressed in FAK- cells (Fig. 5 B). Next, we directly determined whether FAK is required for Prx1 expression. It has been reported previously that although the Prx1 antibody recognizes a cross-reactive band in Western immunoblots, this antibody specifically recognizes Prx1 when used for immunocytochemistry (Chesterman et al., 2001). Immunostaining experiments confirmed that FAK- cells do not express Prx1 protein (Fig. 5 C, left), whereas FAK- cells transiently transfected with the plasmid encoding the IL2R-FAK wild-type fusion protein expressed Prx1 protein in their nuclei (Fig. 5 C, right). RT-PCR and immunofluorescence experiments using the FAK-reconstituted DA2 clone also showed that FAK is required for Prx1 mRNA, and protein expression (Fig. 5, D and E). As with TN-C mRNA expression, basal levels of Prx1 expression were greater in control cells for DA2 (Fig. 5 A), when compared with FAK- cells.

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