In vivo Role of TGase 3 Figure 6. Wound healing in TGM32/2 skin. The rate of wound closure in TGM32/2 mice was not different compared to control animals (A) (n = 9), despite an increase in TGase 3 expression in healing epidermis (B). At day 9 post wounding the thickness of the epidermal layer in the wounded region was greater in wild type skin (C, E) when compared to mutant skin (D, F). Staining of healing skin with keratinocyte differentiation markers in wild type (G, J, M) and TGM32/2 (H, I, K, L, N, O) animals demonstrated that while there was no change in the expression pattern of the keratinocyte markers loricrin (G–I) or keratin10 (J–L) between the mouse lines, keratin14 (M–O) already showed a more restricted staining pattern reminiscent of unwounded skin. (scale bars C, D 400 mm, E–G 100 mm). doi:10.1371/journal.pone.0034252.g006 residues (23%) [36]. Trichohyalin is co-expressed with TGase 3 in other stratified epithelia, such as the filiform ridges of the tongue and the keratinized stomach of the rodent [37]. Even so, we failed to find any defects in these structures in TGM32/2 mice. Trichohyalin is also abundant in the medulla and in the inner root sheath of developing hair where it is cross-linked to keratin filaments [25]. After terminal differentiation trichohyalin cannot usually be extracted by conventional means and its release from TGM32/2 hair agrees with previous results suggesting that trichohyalin cross-linking depends on TGase 3 rather than TGase 1, despite the latter enzyme being present in the inner root sheath [24]. Further, in vitro studies showed that TGase 3 has a high preference for trichohyalin and keratin filaments as substrates [25,38,39]. Human data suggests that TGase 3 is the predominant and possibly only transglutaminase present in the hair cuticle and cortex, which also develop a cornified envelope [40,41]. Electron microscopy showed that many hairs in TGM32/2 mice had a highly distorted cuticle, which was poorly retained upon the underlying cortex. Mild heating of hair in a reducing solvent led to loss of the cuticle. It is not known whether this is caused by breakage of bonds between the cuticle and the cortex or breakdown of the cuticle cells itself. Transmission electron microscopy showed that the subcuticular zone failed to form normally in the TGM32/2 animals suggesting that the hair cuticular cells are directly compromised. Further, the Huxley’s layer which has a high expression of transglutaminases, including TGase 3 [24] appears fragile. Keratin filaments are cross-linked into the cornified envelope by both disulfide and isopeptide bonds. Indeed, many type 2 keratins are TGase 3 substrates in vitro [39] where a conserved lysine is utilized. Keratin6hf is present in the medulla and matrix of the hair shaft [42], co-localizing with keratin17 [43], and it appears that neither the other keratin6 isoforms nor the closely related keratin5 are expressed here. Our results provide evidence that keratin6hf and keratin17 form filaments in vivo, in agreement with the co-localizing seen by confocal microscopy [42]. The high solubility of these keratins and of trichohyalin in TGM32/2 mice strongly suggests that they form a complex stabilized by isopeptide bonds. Mutations in either keratin6hf or keratin17 are related to hair defects. K172/2 mice show alopecia with defects in primary hair growth when kept on a C57/Bl6 background [44]. The hair in these mice is extremely fragile with increased apoptosis in the matrix of the hair and PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34252