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Tenascin-C signaling through induction of 14-3-3 tau.

Martin D, Brown-Luedi M, Chiquet-Ehrismann R - J. Cell Biol. (2003)

Bottom Line: We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C.Furthermore, the growth rate on tenascin-C was increased compared with the parental cells.Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute, Novartis Forschungsstiftung, CH-4002 Basel, Switzerland.

ABSTRACT
We searched by a cDNA subtraction screen for differentially expressed transcripts in MCF-7 mammary carcinoma cells grown on tenascin-C versus fibronectin. On tenascin-C, cells had irregular shapes with many processes, whereas on fibronectin they were flat with a cobble stone-like appearance. We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C. To investigate the consequences of an increased level of this phospho-serine/threonine-binding adaptor protein, we transfected MCF-7 cells with a construct encoding full-length 14-3-3 tau protein and selected clones with the highest expression levels. The morphology of these cells on tenascin-C was flat, resembling that of cells on fibronectin. This was reflected by a similar pattern of F-actin staining on either substratum. Furthermore, the growth rate on tenascin-C was increased compared with the parental cells. After transient transfection of HT1080 fibrosarcoma and T98G glioblastoma cells with 14-3-3 tau, only the 14-3-3 tau-expressing cells were able to adhere and survive on tenascin-C, whereas all cells adhered well on fibronectin. Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth.

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Induction of 14-3-3 tau in cells grown on tenascin-C. (A) Photographs of ethidium bromide-stained agarose gels shows RT-PCR reactions of mRNA isolated from MCF-7 cells grown for 24 h on a fibronectin (FN) or on a tenascin-C (TN) substratum. They reveal an increase in 14-3-3 transcripts in cells from the tenascin-C substratum, whereas GAPDH expression is equal in both situations. The same result was obtained in two independently performed experiments (exp. 1 and exp. 2). (B) Amidoblack staining of a 12% SDS gel blotted to a PVDF membrane showing equal protein content of cell extracts loaded from cells grown on fibronectin (FN) or tenascin-C (TN; left). The immunoblot of the same membrane obtained with anti–14-3-3 tau reveals a stronger signal in the extract from cells grown on tenascin-C than on fibronectin (right).
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fig2: Induction of 14-3-3 tau in cells grown on tenascin-C. (A) Photographs of ethidium bromide-stained agarose gels shows RT-PCR reactions of mRNA isolated from MCF-7 cells grown for 24 h on a fibronectin (FN) or on a tenascin-C (TN) substratum. They reveal an increase in 14-3-3 transcripts in cells from the tenascin-C substratum, whereas GAPDH expression is equal in both situations. The same result was obtained in two independently performed experiments (exp. 1 and exp. 2). (B) Amidoblack staining of a 12% SDS gel blotted to a PVDF membrane showing equal protein content of cell extracts loaded from cells grown on fibronectin (FN) or tenascin-C (TN; left). The immunoblot of the same membrane obtained with anti–14-3-3 tau reveals a stronger signal in the extract from cells grown on tenascin-C than on fibronectin (right).

Mentions: MCF-7 cells were grown in medium containing 10% FCS for 24 h on plates coated with fibronectin or tenascin-C, respectively. The cells show completely different morphologies on these two substrates. They grow in epithelial patches on fibronectin but lose their cell–cell contacts and adopt irregular shapes with long processes on a tenascin-C substratum (Fig. 1). This is reminiscent of the results obtained with MCF-7 cells grown on collagen gels in the presence and absence of tenascin-C where the cells in the presence of tenascin-C were found to loose their cell–cell contacts and detached from the substratum (Chiquet-Ehrismann et al., 1989). To identify molecular differences between the cells grown on fibronectin versus tenascin-C, we isolated mRNA from these cells and performed a screen for differentially expressed transcripts. This screen resulted in the identification of a cDNA clone encoding the adaptor protein 14-3-3 tau. 14-3-3 proteins are a family of phospho-serine/threonine–binding proteins with >70 known ligands as diverse as kinases, phosphatases, receptors, structural proteins, and transcription factors (for reviews see Fu et al., 2000; van Hemert et al., 2001; Tzivion and Avruch, 2002). We confirmed the higher level of 14-3-3 tau transcripts in two mRNA batches isolated from independently cultured MCF-7 cells on tenascin-C versus fibronectin substrates by semiquantitative PCR. As shown in Fig. 2 A, transcript levels of GAPDH were equal, whereas 14-3-3 tau was highly increased in the samples from the cells cultured on tenascin-C compared with fibronectin. We next tested whether the level of the 14-3-3 tau protein was also affected. We loaded equal amounts of cellular protein on an SDS-PAGE followed by detection of 14-3-3 tau by a specific antiserum on an immunoblot. As shown in Fig. 2 B, the level of the 14-3-3 protein was also much higher in cell extracts of cells grown on a tenascin-C substratum than on fibronectin.


Tenascin-C signaling through induction of 14-3-3 tau.

Martin D, Brown-Luedi M, Chiquet-Ehrismann R - J. Cell Biol. (2003)

Induction of 14-3-3 tau in cells grown on tenascin-C. (A) Photographs of ethidium bromide-stained agarose gels shows RT-PCR reactions of mRNA isolated from MCF-7 cells grown for 24 h on a fibronectin (FN) or on a tenascin-C (TN) substratum. They reveal an increase in 14-3-3 transcripts in cells from the tenascin-C substratum, whereas GAPDH expression is equal in both situations. The same result was obtained in two independently performed experiments (exp. 1 and exp. 2). (B) Amidoblack staining of a 12% SDS gel blotted to a PVDF membrane showing equal protein content of cell extracts loaded from cells grown on fibronectin (FN) or tenascin-C (TN; left). The immunoblot of the same membrane obtained with anti–14-3-3 tau reveals a stronger signal in the extract from cells grown on tenascin-C than on fibronectin (right).
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Related In: Results  -  Collection

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fig2: Induction of 14-3-3 tau in cells grown on tenascin-C. (A) Photographs of ethidium bromide-stained agarose gels shows RT-PCR reactions of mRNA isolated from MCF-7 cells grown for 24 h on a fibronectin (FN) or on a tenascin-C (TN) substratum. They reveal an increase in 14-3-3 transcripts in cells from the tenascin-C substratum, whereas GAPDH expression is equal in both situations. The same result was obtained in two independently performed experiments (exp. 1 and exp. 2). (B) Amidoblack staining of a 12% SDS gel blotted to a PVDF membrane showing equal protein content of cell extracts loaded from cells grown on fibronectin (FN) or tenascin-C (TN; left). The immunoblot of the same membrane obtained with anti–14-3-3 tau reveals a stronger signal in the extract from cells grown on tenascin-C than on fibronectin (right).
Mentions: MCF-7 cells were grown in medium containing 10% FCS for 24 h on plates coated with fibronectin or tenascin-C, respectively. The cells show completely different morphologies on these two substrates. They grow in epithelial patches on fibronectin but lose their cell–cell contacts and adopt irregular shapes with long processes on a tenascin-C substratum (Fig. 1). This is reminiscent of the results obtained with MCF-7 cells grown on collagen gels in the presence and absence of tenascin-C where the cells in the presence of tenascin-C were found to loose their cell–cell contacts and detached from the substratum (Chiquet-Ehrismann et al., 1989). To identify molecular differences between the cells grown on fibronectin versus tenascin-C, we isolated mRNA from these cells and performed a screen for differentially expressed transcripts. This screen resulted in the identification of a cDNA clone encoding the adaptor protein 14-3-3 tau. 14-3-3 proteins are a family of phospho-serine/threonine–binding proteins with >70 known ligands as diverse as kinases, phosphatases, receptors, structural proteins, and transcription factors (for reviews see Fu et al., 2000; van Hemert et al., 2001; Tzivion and Avruch, 2002). We confirmed the higher level of 14-3-3 tau transcripts in two mRNA batches isolated from independently cultured MCF-7 cells on tenascin-C versus fibronectin substrates by semiquantitative PCR. As shown in Fig. 2 A, transcript levels of GAPDH were equal, whereas 14-3-3 tau was highly increased in the samples from the cells cultured on tenascin-C compared with fibronectin. We next tested whether the level of the 14-3-3 tau protein was also affected. We loaded equal amounts of cellular protein on an SDS-PAGE followed by detection of 14-3-3 tau by a specific antiserum on an immunoblot. As shown in Fig. 2 B, the level of the 14-3-3 protein was also much higher in cell extracts of cells grown on a tenascin-C substratum than on fibronectin.

Bottom Line: We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C.Furthermore, the growth rate on tenascin-C was increased compared with the parental cells.Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute, Novartis Forschungsstiftung, CH-4002 Basel, Switzerland.

ABSTRACT
We searched by a cDNA subtraction screen for differentially expressed transcripts in MCF-7 mammary carcinoma cells grown on tenascin-C versus fibronectin. On tenascin-C, cells had irregular shapes with many processes, whereas on fibronectin they were flat with a cobble stone-like appearance. We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C. To investigate the consequences of an increased level of this phospho-serine/threonine-binding adaptor protein, we transfected MCF-7 cells with a construct encoding full-length 14-3-3 tau protein and selected clones with the highest expression levels. The morphology of these cells on tenascin-C was flat, resembling that of cells on fibronectin. This was reflected by a similar pattern of F-actin staining on either substratum. Furthermore, the growth rate on tenascin-C was increased compared with the parental cells. After transient transfection of HT1080 fibrosarcoma and T98G glioblastoma cells with 14-3-3 tau, only the 14-3-3 tau-expressing cells were able to adhere and survive on tenascin-C, whereas all cells adhered well on fibronectin. Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth.

Show MeSH
Related in: MedlinePlus