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. 2023 Jul 14;24(14):11475.
doi: 10.3390/ijms241411475.

Transglutaminase 2 Facilitates Murine Wound Healing in a Strain-Dependent Manner

Ting W Yiu  1 , Sara R Holman  1 , Xenia Kaidonis  1 , Robert M Graham  1   2 , Siiri E Iismaa  1   2
Affiliations

Transglutaminase 2 Facilitates Murine Wound Healing in a Strain-Dependent Manner

Ting W Yiu et al. Int J Mol Sci. .

Abstract

Transglutaminase 2 (TG2) plays a role in cellular processes that are relevant to wound healing, but to date no studies of wound healing in TG2 knockout mice have been reported. Here, using 129T2/SvEmsJ (129)- or C57BL/6 (B6)-backcrossed TG2 knockout mice, we show that TG2 facilitates murine wound healing in a strain-dependent manner. Early healing of in vivo cutaneous wounds and closure of in vitro scratch wounds in murine embryonic fibroblast (MEF) monolayers were delayed in 129, but not B6, TG2 knockouts, relative to their wild-type counterparts, with wound closure in 129 being faster than in B6 wild-types. A single dose of exogenous recombinant wild-type TG2 to 129 TG2-/- mice or MEFs immediately post-wounding accelerated wound closure. Neutrophil and monocyte recruitment to 129 cutaneous wounds was not affected by Tgm2 deletion up to 5 days post-wounding. Tgm2 mRNA and TG2 protein abundance were higher in 129 than in B6 wild-types and increased in abundance following cutaneous and scratch wounding. Tgm1 and factor XIIA (F13A) mRNA abundance increased post-wounding, but there was no compensation by TG family members in TG2-/- relative to TG2+/+ mice in either strain before or after wounding. 129 TG2+/+ MEF adhesion was greater and spreading was faster than that of B6 TG2+/+ MEFs, and was dependent on syndecan binding in the presence, but not absence, of RGD inhibition of integrin binding. Adhesion and spreading of 129, but not B6, TG2-/- MEFs was impaired relative to their wild-type counterparts and was accelerated by exogenous addition or transfection of TG2 protein or cDNA, respectively, and was independent of the transamidase or GTP-binding activity of TG2. Rho-family GTPase activation, central to cytoskeletal organization, was altered in 129 TG2-/- MEFs, with delayed RhoA and earlier Rac1 activation than in TG2+/+ MEFs. These findings indicate that the rate of wound healing is different between 129 and B6 mouse strains, correlating with TG2 abundance, and although not essential for wound healing, TG2 facilitates integrin- and syndecan-mediated RhoA- and Rac1-activation in fibroblasts to promote efficient wound contraction.

Keywords: TG2; bupivacaine; cytoskeletal dynamics; integrin; mouse strain; murine embryonic fibroblasts; scratch wound; syndecan; wound healing; β-sandwich-core domain.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Early healing of cutaneous wounds was slower in 129 TG2−/− mice than in 129 TG2+/+, B6 TG2+/+ or B6 TG2−/− mice. (A,D) Representative photographs of cutaneous wounds in (A) 129 TG2+/+ or 129 TG2−/− or (D) B6 TG2+/+ or B6 TG2−/− mice at 0, 2, 4, 6, 8 and 10 days post-wounding. (B,E) Wound areas expressed as a fraction of the day 0 area of (B) 129 TG2+/+ (n = 16) or 129 TG2−/− (n = 16), respectively, or (E) B6 TG2+/+ (n = 10) or B6 TG2−/− (n = 10), respectively, until wound closure. (C,F) Integrated wound closure time: (C) calculated from total area under the curves from (B); (F) calculated from total area under the curves from (E). Scale bar = 5 mm. ***, p < 0.001 for individual post hoc Bonferroni test results between TG2−/− and TG2+/+ from repeated measure two-way ANOVA; ###, p < 0.001 for two tailed Student’s t test.
Figure 2
Figure 2
Bupivacaine injection prior to skin punch biopsy delayed wound healing in 129 TG2−/− and 129 TG2+/+mice. (A) Wound areas expressed as a fraction of the day 0 area of 129 TG2+/+ (n = 16) or 129 TG2−/− (n = 16), respectively, until wound closure. (B) Integrated wound closure time calculated from total area under the curves from (A).
Figure 3
Figure 3
Neutrophil and monocyte recruitment to cutaneous wounds was no different between 129 TG2−/− and 129 TG2+/+. (A) A typical random field (60× magnification) of a transverse cutaneous wound section showing an example of a multi-lobed nucleus of a neutrophil (black arrow) and a bean-shaped nucleus of a monocyte (white arrow); scale bar = 10 μm. (B) Number of neutrophils and monocytes per field (average of 10 random 20× magnification fields from n = 3 samples per genotype) in transverse sections of 129 TG2+/+ or 129 TG2−/− cutaneous wounds 1–5 days post-wounding. *, p < 0.05; **, p < 0.01, ***, p < 0.001 for individual post hoc Bonferroni test results from two-way ANOVA.
Figure 4
Figure 4
mRNA abundance of wound-related genes was increased to the same levels in 129 TG2−/− and 129 TG2+/+ mice on day 2 post-wounding. cDNA generated from total RNA isolated from unwounded skin samples (n = 3 per genotype) or skin samples on day 2 post-wounding (n = 3 per genotype) was examined in triplicate for the abundance of mRNAs for (A) TLR pathway genes Ticam1 (encoding TICAM1), Myd88 (encoding MyD88), Nfkb1 (encoding NF-κB subunit 1), Nfkbib (encoding I-κBβ), (B) early inflammatory cytokines Il6 (encoding IL6), Ifng (encoding IFN-γ) and Tnf (encoding TNF-α), and growth factors Egf (encoding EGF), Fgf2 (encoding basic fibroblast growth factor) and Tgfb2 (encoding TGFβ2), (C) ECM-related genes Fndc4 (encoding Fibronectin Type III Domain-Containing Protein 4), Fndc3a (encoding the wound-related fibronectin extra-domain A splice variant), Col1a1 (encoding collagen type I α1 chain), Col1a2 (encoding collagen type I α2 chain), Col3a1 (encoding collagen type III α1 chain), Sdc4 (encoding syndecan-4), (D) TG family members Tgm1 (encoding TG1), Tgm2 (encoding TG2), Tgm3 (encoding TG3), Tgm4 (encoding TG4), Tgm5 (encoding TG5), Tgm6 (encoding TG6), Tgm7 (encoding TG7), f13a (encoding F13A), normalized to Hprt (encoding hypoxanthine phosphoribosyltransferase 1) as the reference gene (expression level unaffected by the experimental treatment). *, p < 0.05; **, p < 0.01; ***, p < 0.001 for two tailed Student’s t test between respective different treatment groups of the same genotype. ###, p < 0.001 for two tailed Student’s t test between different genotypes in the same respective treatment group.
Figure 5
Figure 5
A single dose of TG2 protein to cutaneous wounds in 129 TG2−/− mice restored rate of wound healing to that in 129 TG2+/+ mice. (A,B) Wound areas expressed as a fraction of the day 0 area of (A) 129 TG2−/− wounds (n = 9) or (B) 129 TG2+/+ wounds (n = 9) treated with vehicle (PBS) or TG2 until wound closure. (C) Integrated wound closure time calculated from the total area under the curves from (A,B). *, p < 0.05; **, p < 0.01 for individual post hoc Bonferroni test results between TG2−/− + PBS and TG2−/− + TG2 from repeated measure two-way ANOVA with post hoc Bonferroni correction. ###, p < 0.001; ns, not significant for one-way ANOVA with Bonferroni’s multiple group comparisons.
Figure 6
Figure 6
The number of 129 TG2−/− MEFs adherent to Fn-coated plates was less compared to 129 TG2+/+ MEFs but was no different from B6 TG2+/+ or B6 TG2−/− MEFs. (A,B) Representative photographs of fixed crystal violet-stained 129 (A) or B6 (B) TG2+/+ or TG2−/− MEFs adhered to plates coated with Fn (10 μg/cm2) after 10, 20 and 120 min; 10× magnification, scale bar = 30 μm. (C) Quantitation of adherent 129 TG2+/+, 129 TG2−/−, B6 TG2+/+ and B6 TG2−/− MEFs after 10, 20, 30, 60, 90 and 120 min incubation on Fn-coated plates, expressed as a percentage of the total number of cells seeded (n = 4 experiments performed in triplicate). ***, p < 0.001 for individual post hoc Bonferroni test results from two-way ANOVA comparing 129 TG2+/+ with 129 TG2−/−, B6 TG2+/+ or B6 TG2−/− MEFs.
Figure 7
Figure 7
The addition of recombinant wild-type, transamidase-deficient or GTP-binding-deficient TG2 to Fn-coated plates increased the number of adherent MEFs. (A–C) Quantitation of adherent 129 TG2+/+, 129 TG2−/− and (A) B6 TG2+/+ and B6 TG2−/− MEFs, after 30 min incubation on plates coated with Fn (10 μg/cm2) plus (A) wild-type (0, 10, 20, 40, 60, 80 μg/cm2), (B) W241A transamidase-deficient (0, 10, 20, 40 μg/cm2) or (C) R579A GTP-binding-deficient (0, 10, 20, 40 μg/cm2) TG2, expressed as a percentage of the total number of cells (5 × 104) seeded (n = 5–6 experiments performed in triplicate). *, p < 0.05; **, p < 0.01, ***, p < 0.001 for individual post hoc Bonferroni test results from two-way ANOVA comparing 129 TG2+/+ with 129 TG2−/−, B6 TG2+/+ or B6 TG2−/− MEFs.
Figure 8
Figure 8
Adhesion of 129 TG2+/+ or 129 TG2−/− MEFs to a TG2-Fn matrix was dependent on syndecan binding in the presence, but not absence, of RGD peptides, and the exogenous addition of recombinant wild-type TG2 to an Fn matrix rescued RGD-impaired adhesion of 129 TG2+/+ and 129 TG2−/− MEFs. (A,B) Quantitation of adherent 129 TG2+/+ or 129 TG2−/− after 30 min incubation on plates coated with Fn (10 μg/cm2) and wild-type TG2 (0, 10, 20, 40, 60, 80 μg/cm2) in the absence or presence of (A) RGD or RAD peptides (84 μg/cm2) or (B) heparin (Hep, 42 μg/cm2) or heparin and RGD peptide (84 μg/cm2), expressed as a percentage of the total number of cells (5 × 104) seeded (n = 4 experiments performed in triplicate). ns, p > 0.05, *, p < 0.05; ***, p < 0.001 for two-way ANOVA.
Figure 9
Figure 9
Spreading of 129 TG2−/−, B6 TG2+/+ and B6 TG2−/− MEFs was equivalent but delayed relative to 129 TG2+/+ MEFs and the exogenous addition of recombinant TG2, independent of transamidase or GTP-binding activity, accelerated spreading in all genotypes. (A,C) Representative photographs of phalloidin-stained actin stress fibers in (A) 129 or (C) B6 TG2+/+ or TG2−/− MEFs after 30, 60 or 90 min incubation on plates coated with Fn (10 μg/cm2) without or with recombinant wild-type TG2 (+TG2, 20 μg/cm2) added; scale bar = 10 μm. (B,D,E) Quantitation of adherent (B,E) 129 TG2+/+ or 129 TG2−/− and (D) B6 TG2+/+ or B6 TG2−/− MEF cell areas after 30, 60 or 90 min incubation on plates coated with Fn in the absence or presence of (B,D,E) added recombinant wild-type TG2 (+TG2) or (E) added recombinant W241A transamidase-deficient or R579A GTP-binding-deficient TG2, normalized to mean 129 TG2+/+ cell area at 30 min post-seeding (n = 4–5 experiments performed in triplicate, 10 random cells quantitated per treatment per experiment). (F) Quantitation after 30, 60 or 90 min incubation on Fn-coated plates of cell areas of adherent 129 TG2−/− MEFs that were untransfected, or transfected with cDNAs encoding wild-type TG2 (TG2t), transamidase-deficient TG2 (C277St, W241At), GTP-binding-deficient TG2 (R579At, S171Et) or empty vector (Mock), normalized to mean 129 TG2+/+ cell area at 30 min post-seeding (n = 5 experiments performed in triplicate, 10 random cells quantitated per treatment per experiment). *, p < 0.05; **, p < 0.01, ***, p < 0.001 for individual post hoc Bonferroni test results from two-way ANOVA.
Figure 10
Figure 10
Activation dynamics of the GTPases RhoA and Rac1 that control cell adhesion and spreading on Fn were altered in 129 TG2−/− relative to TG2+/+ MEFs. (A,C) Representative western blots and (B,D) quantitation of activated GTP-bound RhoA (GTP-RhoA) relative to total RhoA (B) or activated GTP-bound Rac1 (GTP-Rac1) relative to total Rac1 (D) in lysates from Fn-adherent 129 TG2+/+ MEFs, TG2−/− MEFs, TG2−/− MEFs transfected with TG2 cDNA (TG2t) or TG2−/− MEFs with recombinant wild-type TG2 added to the Fn matrix (+TG2, 20 μg/cm2) at 10, 30, 60 or 90 min post-seeding, with GTPγS-treated TG2+/+ lysates as a positive control (+ve) (n = 3 experiments). *, p < 0.05; **, p < 0.01, ***, p < 0.001 for individual post hoc Bonferroni test results from two-way ANOVA. #, p < 0.05; ##, p < 0.01, ###, p < 0.001 for individual post hoc Bonferroni test results two-way ANOVA comparing TG2−/− with TG2+/+ MEFs at the indicated time points.
Figure 11
Figure 11
Closure of an in vitro scratch wound in confluent cell monolayers was delayed in 129 but not B6 TG2−/− MEFs, relative to their TG2+/+ counterparts, and the exogenous addition of recombinant wild-type TG2 immediately post-wounding accelerated wound closure in all genotypes. Confluent monolayers of (A,B) 129 TG2+/+ or TG2−/− MEFs or (C,D) B6 TG2+/+ or TG2−/− MEFs were scratch wounded and the scratch wound area was quantitated over time in the absence or presence (+TG2) of exogenous addition of recombinant wild-type TG2 immediately post-wounding; representative images (A,C) and quantitation of scratch wound area as a fraction of day 0 area (B,D) (n = 3 experiments); scale bar = 100 μm. ***, p < 0.001 for individual post hoc Bonferroni test results from two-way ANOVA. #, p < 0.05; ##, p < 0.01, ###, p < 0.001 for individual post hoc Bonferroni test results from two-way ANOVA comparing MEFs + TG2 with their respective MEFs at the indicated time points.
Figure 12
Figure 12
TG2 expression levels are higher in 129 than in B6 mice. (A,B) cDNA generated from total RNA isolated from 129 or B6 TG2+/+ and TG2−/− unwounded skin samples (n = 3 per genotype) (A) or skin samples on day 2 post-wounding (n = 3 per genotype) (B) was examined in triplicate for the abundance of mRNAs for TG family members: Tgm1 (encoding TG1), Tgm2 (encoding TG2), Tgm3 (encoding TG3), Tgm4 (encoding TG4), Tgm5 (encoding TG5), Tgm6 (encoding TG6), Tgm7 (encoding TG7), f13a (encoding F13A), normalized to Hprt (encoding hypoxanthine phosphoribosyltransferase 1) as the reference gene (expression level unaffected by the experimental treatment). *, p < 0.05; ***, p < 0.001 for individual post hoc Bonferroni test results from two-way ANOVA comparing 129 TG2+/+ with B6 TG2+/+ samples. (C–F) Representative western blots (C,E) and quantitation (D,F) of TG2 abundance relative to the reference proteins (expression level unaffected by genotype) pan-Cadherin (C,D) or GAPDH (E,F) in membrane or cytosolic fractions, respectively, of 129 or B6 TG2+/+ and TG2−/− MEF lysates (n = 4 experiments).
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