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First published online 10 June 2008
doi: 10.1242/jcs.011262

Journal of Cell Science 121, 2256-2264 (2008)
Published by The Company of Biologists 2008
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Factor XIIIA mobilizes transglutaminase 2 to induce chondrocyte hypertrophic differentiation

Kristen A. Johnson, David M. Rose and Robert A. Terkeltaub*

Veterans Affairs Medical Center, UCSD, La Jolla, CA 92161, USA


Figure 1
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  		Fig. 1. Both exogenous FXIIIA and TG2 induce chondrocyte hypertrophic differentiation. We studied induction by TG2 and FXIIIA of MMP13, VEGF and type X collagen in cultured chondrocytes. Normal human knee articular chondrocytes plated in 12-well dishes (at 105 cells per well) were incubated for 4 or 8 hours with 100 ng/ml of recombinant WT TG2 or FXIIIA in the ascorbate-containing medium A described in Materials and Methods. (A) Quantitative PCR. Using quantitative RT-PCR, we determined chondrocyte GAPDH mRNA expression levels relative to those of MMP13 and VEGF, and the mRNA expression ratio of type X collagen to type II collagen, studying data collected from three separate human donors (n=9, *P<0.05). (B) Bovine articular cartilage explants. Assessment of the induction by TG2 and FXIIIA of type X collagen in cartilage organ culture. Normal bovine articular cartilage explants in organ culture were incubated with 100 ng/ml recombinant WT TG2 or FXIIIA for 5 days in medium A, and frozen 10-µm sections were stained for expression of type X collagen (representative of five donors).

 

Figure 2
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  		Fig. 2. FXIIIA-stimulated expression of type X collagen is dependent upon TG2. Assessment of TG2- and FXIIIA-knockout mouse cells. Primary knee chondrocytes were removed from F13a1+/+, F13a1 –/–, Tgm2+/+ and Tgm2–/– mice. After two weeks in culture, aliquots of 5x103 cells in Medium A were stimulated for 5 days with 10 nM ATRA, 10 ng/ml CXCL8 or 100 ng/ml of sTG2 or sFXIIIA, and then type X collagen was examined by SDS-PAGE and western blotting, as described in the Materials and Methods. Representative of three experiments.

 

Figure 3
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  		Fig. 3. (A) FXIIIA. Site-directed FXIIIA mutations generated for structural analysis examination. Depicted are features of the primary structure of WT FXIIIA and the site-directed FXIIIA mutations generated and studied, listed her as 1-3. (B) Type X collagen. Aliquots of human articular chondrocytes (105 cells) were incubated with 100 ng/ml of each recombinant protein in medium A for 72 hours, and type X collagen assessed in cell lysates by SDS-PAGE and western blotting. Representative of three donors in three separate experiments (n=9).

 

Figure 4
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  		Fig. 4. Rapid mobilization of TG2 by FXIIIA is essential for stimulation of p38 phosphorylation. (A) Plasma-membrane-bound TG2. Aliquots of 5x103 human articular chondrocytes were starved in serum-free high-glucose DMEM for 2 hours and then stimulated with WT or mutant sFXIIIA. TG2-specific antibodies were used to detect membrane-bound TG2, quantified through successive incubations with biotin anti-rabbit and streptavidin-AP antibodies as described in Materials and Methods (n=9). (B) p38 MAP kinase phosphorylation. Aliquots of 3x105 human articular chondrocytes were starved in serum-free high-glucose DMEM for 2 hours and then stimulated with WT or mutant sFXIIIA for the time indicated. Cell lysates were analyzed by western blotting for phosphorylated p38 (p-p38) and total p38. Representative of three donors in three separate experiments (n=9).

 

Figure 5
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  		Fig. 5. Recombinant FXIIIA engages the {alpha}1-integrin subunit in human chondrocytes. (A) Western blot. Aliquots of 105 human articular chondrocytes were starved for 2 hours in serum-free DMEM and then incubated for the indicated times with 100 ng/ml sTG2 or sFXIIIA. The cells were washed prior to lysis and then examined for the presence of the Xpress epitope on the recombinant proteins by SDS-PAGE and western blotting. (B) Integrin immunoprecipitation. Aliquots of 106 human articular chondrocytes were incubated for 3 days in medium A containing 100 ng/ml of sTG2 or sFXIIIA where indicated. Cell lysates (200 µg protein) were immunoprecipitated using 1 µg/ml of {alpha}1- (clone TS2/7), {alpha}2-, {alpha}5- or {alpha}6-integrin-subunit-specific antibody, and precipitated proteins were analyzed for the Xpress tag or each respective {alpha}-integrin subunit by western blotting. Representative of three separate experiments using three different donors.

 

Figure 6
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  		Fig. 6. FXIIIA induction of type X collagen is associated with FAK and p38 MAP kinase phosphorylation and dependent upon the {alpha}1-integrin subunit. (A) Type X collagen. Aliquots of 105 human articular chondrocytes were starved and pre-treated for 2 hours with 1 µg/ml of IgG control, β1-integrin subunit or {alpha}1-integrin-subunit-blocking (FB12) antibodies in serum-free DMEM and then incubated for the indicated times with 100 ng/ml sTG2 or sFXIIIA, with type X collagen assessed by western blotting of cell lysates as above. (B) FAK and p38 MAP kinase phosphorylation. Aliquots of 105 cells were starved for 2 hours in serum-free DMEM and then incubated for the indicated times with 100 ng/ml sTG2 or sFXIIIA and 1 µg/ml of {alpha}1-integrin-subunit-blocking antibody (FB12) where indicated, with cell lysates examined for FAK and p38 phosphorylation by western blotting. Representative of results from three experiments employing three separate donors.

 

Figure 7
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  		Fig. 7. Rapid mobilization of TG2 to the cell surface by either sFXIIIA or antibody crosslinking of {alpha}1-integrin subunit. To determine whether FXIIIA-induced movement of TG2 to the cell surface is {alpha}1-integrin-subunit dependent, aliquots of 5x103 human articular chondrocytes were starved in serum-free high-glucose DMEM for 2 hours and then stimulated with sFXIIIA, the {alpha}1-integrin-subunit antibody TS2/7 (with and without crosslinking by anti-mouse IgG) versus an IgG control antibody. Additionally, after starvation the chondrocytes were pre-treated with the blocking {alpha}1-integrin-subunit antibody, FB12 and then stimulated for 5 or 10 minutes with WT sFXIIIA. After fixation of the cells, TG2-specific antibodies were used to detect membrane-bound TG2, quantified through successive incubations with biotin anti-rabbit and streptavidin-AP antibodies as described in Materials and Methods.

 

Figure 8
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  		Fig. 8. Model for how extracellular FXIIIA mobilizes TG2 in promoting chondrocyte hypertrophic differentiation that is dependent on both {alpha}1β1 integrin and endogenous FXIIIA. Extracellular GTP-bound TG2 was previously demonstated to induce β1-integrin-dependent signaling to stimulate chondrocyte maturation to hypertrophy, and associated with p38 MAP kinase activation. In this model, which summarizes results of this study, TG2 is rapidly mobilized from the cytosol to the extracellular surface of the plasma membrane by FXIIIA in a manner dependent on {alpha}1β1-integrin-dependent signaling, and reproduced by anti-{alpha}1-antibody-induced {alpha}1β1-integrin crosslinking. To promote chondrocyte hypertrophy, FXIIIA requires endogenous TG2 to be mobilized, and FXIIIA structurally requires the Pro37 residue that mediates thrombin induction of TG catalytic activity in latent FXIIIA. By contrast, TG catalytic activity of exogenous FXIIIA is not required for induction of hypertrophy, nor is the Met513 residue that mediates thrombin inactivation of FXIIIa TG catalytic activity. Exogenous FXIIIA is shown to be a ligand of the {alpha}1-integrin subunit, but {alpha}1β1-integrin signaling in response to FXIIIA is possibly mediated by other {alpha}1β1-integrin ligand(s) or trough their complex-formation with FXIIIA and {alpha}1β1 integrin. Last, we observed that exogenous FXIIIA (as well as retinoic acid) fail to induce chondrocyte hypertrophy in the absence of endogenous FXIIIA. Hence, endogenous FXIIIA modulates chondrocyte differentiation, including responsiveness to exogenous FXIIIA.

 

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