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Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

Akimov SS, Krylov D, Fleischman LF, Belkin AM - J. Cell Biol. (2000)

Bottom Line: These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII.Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins.Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

ABSTRACT
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

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TPA-induced adhesion and spreading of HEL cells on 42-kD Fn fragment is mediated by surface tTG. (A) Expression levels of tTG on the surface of live untreated and TPA-treated cells were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Transglutaminase activity on the surface of live untreated and TPA-treated cells was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars show the means of triplicate measurements. (C) Quantitative adhesion assays with untreated and TPA-treated cells plated for 1 h on Fn (open bars), 110-kD (crossed bars), and 42-kD (filled bars) fragments either without or in the presence of 10 μg/ml polyclonal anti-tTG antibody. (D) Spreading assays with untreated and TPA-treated cells. Cells were plated for 4 h on Fn, 110-kD, or 42-kD Fn fragments without any treatment or after TPA treatment either in the absence or with 10 μg/ml polyclonal anti-tTG antibody. Bar, 20 μM. (C and D) For adhesion and spreading assays HEL cells were plated in serum-free medium in the presence of cycloheximide.
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Figure 2: TPA-induced adhesion and spreading of HEL cells on 42-kD Fn fragment is mediated by surface tTG. (A) Expression levels of tTG on the surface of live untreated and TPA-treated cells were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Transglutaminase activity on the surface of live untreated and TPA-treated cells was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars show the means of triplicate measurements. (C) Quantitative adhesion assays with untreated and TPA-treated cells plated for 1 h on Fn (open bars), 110-kD (crossed bars), and 42-kD (filled bars) fragments either without or in the presence of 10 μg/ml polyclonal anti-tTG antibody. (D) Spreading assays with untreated and TPA-treated cells. Cells were plated for 4 h on Fn, 110-kD, or 42-kD Fn fragments without any treatment or after TPA treatment either in the absence or with 10 μg/ml polyclonal anti-tTG antibody. Bar, 20 μM. (C and D) For adhesion and spreading assays HEL cells were plated in serum-free medium in the presence of cycloheximide.

Mentions: It was reported earlier that HEL cells that normally grow in suspension adhere to Fn after treatment with TPA (Jarvinen et al. 1987; Ylanne et al. 1990). Using flow cytometry with live HEL cells, we found that TPA treatment increased the level of surface tTG (Fig. 2 A). In parallel, transglutaminase activity on the surface of live TPA-treated HEL cells rose ∼10-fold compared with untreated cells (Fig. 2 B). Quantitative adhesion experiments demonstrated that TPA treatment increased adhesion to Fn, and even more so to the 42-kD Fn fragment, without affecting adhesion to the 110-kD fragment of Fn (Fig. 2 C). Importantly, the antibody against tTG negated the effects of TPA on cell adhesion to Fn and to the 42-kD Fn fragment. Similarly, spreading of HEL cells on Fn and its 42-kD fragment was enhanced by TPA, and this effect was abolished by anti-tTG antibody (Fig. 2 D). These observations indicate that the induction of cell surface tTG can play a major role in stimulation of cell adhesion and spreading due to its interaction with the 42-kD gelatin-binding domain of Fn.


Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

Akimov SS, Krylov D, Fleischman LF, Belkin AM - J. Cell Biol. (2000)

TPA-induced adhesion and spreading of HEL cells on 42-kD Fn fragment is mediated by surface tTG. (A) Expression levels of tTG on the surface of live untreated and TPA-treated cells were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Transglutaminase activity on the surface of live untreated and TPA-treated cells was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars show the means of triplicate measurements. (C) Quantitative adhesion assays with untreated and TPA-treated cells plated for 1 h on Fn (open bars), 110-kD (crossed bars), and 42-kD (filled bars) fragments either without or in the presence of 10 μg/ml polyclonal anti-tTG antibody. (D) Spreading assays with untreated and TPA-treated cells. Cells were plated for 4 h on Fn, 110-kD, or 42-kD Fn fragments without any treatment or after TPA treatment either in the absence or with 10 μg/ml polyclonal anti-tTG antibody. Bar, 20 μM. (C and D) For adhesion and spreading assays HEL cells were plated in serum-free medium in the presence of cycloheximide.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169362&req=5

Figure 2: TPA-induced adhesion and spreading of HEL cells on 42-kD Fn fragment is mediated by surface tTG. (A) Expression levels of tTG on the surface of live untreated and TPA-treated cells were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Transglutaminase activity on the surface of live untreated and TPA-treated cells was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars show the means of triplicate measurements. (C) Quantitative adhesion assays with untreated and TPA-treated cells plated for 1 h on Fn (open bars), 110-kD (crossed bars), and 42-kD (filled bars) fragments either without or in the presence of 10 μg/ml polyclonal anti-tTG antibody. (D) Spreading assays with untreated and TPA-treated cells. Cells were plated for 4 h on Fn, 110-kD, or 42-kD Fn fragments without any treatment or after TPA treatment either in the absence or with 10 μg/ml polyclonal anti-tTG antibody. Bar, 20 μM. (C and D) For adhesion and spreading assays HEL cells were plated in serum-free medium in the presence of cycloheximide.
Mentions: It was reported earlier that HEL cells that normally grow in suspension adhere to Fn after treatment with TPA (Jarvinen et al. 1987; Ylanne et al. 1990). Using flow cytometry with live HEL cells, we found that TPA treatment increased the level of surface tTG (Fig. 2 A). In parallel, transglutaminase activity on the surface of live TPA-treated HEL cells rose ∼10-fold compared with untreated cells (Fig. 2 B). Quantitative adhesion experiments demonstrated that TPA treatment increased adhesion to Fn, and even more so to the 42-kD Fn fragment, without affecting adhesion to the 110-kD fragment of Fn (Fig. 2 C). Importantly, the antibody against tTG negated the effects of TPA on cell adhesion to Fn and to the 42-kD Fn fragment. Similarly, spreading of HEL cells on Fn and its 42-kD fragment was enhanced by TPA, and this effect was abolished by anti-tTG antibody (Fig. 2 D). These observations indicate that the induction of cell surface tTG can play a major role in stimulation of cell adhesion and spreading due to its interaction with the 42-kD gelatin-binding domain of Fn.

Bottom Line: These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII.Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins.Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

ABSTRACT
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

Show MeSH
Related in: MedlinePlus