Loss of Keratin K2 Expression Causes Aberrant Aggregation of K10, Hyperkeratosis, and Inflammation

ORIGINAL ARTICLE Loss of Keratin K2 Expression Causes Aberrant Aggregation of K10, Hyperkeratosis, and Inflammation Heinz Fischer1, Lutz Langbein2, Julia Reichelt3, Silke Praetzel-Wunder2, Maria Buchberger1, Minoo Ghannadan1, Erwin Tschachler1 and Leopold Eckhart1 Keratin K2 is one of the most abundant structural proteins of the epidermis; however, its biological significance has remained elusive. Here we show that suprabasal type II keratins, K1 and K2, are expressed in a mutually exclusive manner at different body sites of the mouse, with K2 being confined to the ear, sole, and tail skin. Deletion of K2 caused acanthosis and hyperkeratosis of the ear and the tail epidermis, corneocyte fragility, increased transepidermal water loss, and local inflammation in the ear skin. The loss of K2 was partially compensated by upregulation of K1 expression. However, a significant portion of K2-deficient suprabasal keratinocytes lacked a regular cytoskeleton and developed massive aggregates of the type I keratin, K10. Aggregate formation, but not hyperkeratosis, was suppressed by the deletion of both K2 and K10, whereas deletion of K10 alone caused clumping of K2 in ear skin. Taken together, this study demonstrates that K2 is a necessary and sufficient binding partner of K10 at distinct body sites of the mouse and that unbalanced expression of these keratins results in aggregate formation. Journal of Investigative Dermatology (2014) 134, 2579–2588; doi:10.1038/jid.2014.197; published online 29 May 2014 INTRODUCTION keratins lead to epidermal fragility (Omary et al., 2004). The Keratins are the main cytoskeletal proteins of epithelial cells deletion of K10 causes epidermal thickening and an including epidermal keratinocytes (Morley and Lane, 1994; enlargement of sebaceous glands (Reichelt and Magin, 2002; Candi et al., 2005; Moll et al., 2008). Distinct pairs of type I Reichelt et al., 2004). This mild phenotype was attributed to and type II keratins heterodimerize to form keratin interme- compensatory upregulation of the basal epidermal keratin K14 diate filaments that increase the mechanical resilience of (Reichelt et al., 2001). By contrast, the deletion of K1 results in epithelial cells besides having additional nonstructural roles a defect in skin barrier formation, inflammation, and perinatal (Morley and Lane, 1994; Magin et al., 2007; Pan et al., 2013; lethality (Roth et al., 2012). Ramms et al., 2013; Seltmann et al., 2013). Different muta- The expression of the type II keratin K2 (formerly K2e; Moll tions in keratin genes cause distinct epidermolytic bullous et al., 2008) is restricted to the uppermost suprabasal layers of diseases and certain forms of ichthyoses (Uitto et al., 2007; the epidermis (Rentrop et al., 1987; Collin et al.,1992;Herzog Arin, 2009; McLean and Moore, 2011). et al., 1994) where it colocalizes with the type I keratin K10 Epidermal keratinocytes express keratins in a strictly differ- (Smith et al., 1999). In humans, K2 is expressed throughout the entiation stage–dependent manner. K5 and K14 are expressed interfollicular epidermis but strongly increased in palmo- in the basal layer, whereas K1, K2, and K10 are confined to plantar skin and in the epidermis on other mechanically the suprabasal layers (Fuchs, 1990; Collin et al., 1992; Morley stressed body sites (Rothnagel et al., 1994; Swensson et al. and Lane, 1994; for a review, see Moll et al.,2008).The 1998). This expression pattern has led to the suggestion that K2 primary role of suprabasal keratins is to enhance the provides additional mechanical resilience to the epidermis. mechanical strength of the epidermis, and mutations in these KRT2 mutations that lead to the substitution of amino acid residues critical for filament formation cause ichthyosis bullosa 1Department of Dermatology, Medical University of Vienna, Vienna, Austria; of Siemens (Kremer et al., 1994; McLean et al.,1994; 2Department of Genetics of Skin Carcinogenesis, German Cancer Research Rothnagel et al., 1994). In this disease cell lysis occurs in 3 Center, Heidelberg, Germany and Institute of Cellular Medicine and North the spinous and granular epidermal layers upon mild stress, East England Stem Cell Institute, Newcastle University, Newcastle upon Tyne, UK which manifests particularly on soles, palms, knees, and Correspondence: Leopold Eckhart, Department of Dermatology, Medical elbows. Clinically, these lesions are characterized by acan- University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. thosis, vacuolization of the granular layer, and hyperkeratosis. E-mail: [email protected] Skin inflammation has been observed in this condition Abbreviations: DTT, dithiothreitol; IL, interleukin; PAGE, polyacrylamide gel (Basarab et al., 1999). As ichthyosis bullosa of Siemens is eletrophoresis; SDS, sodium dodecyl sulfate; TEWL, transepidermal water loss; caused by mutated K2 proteins that act in a dominant-negative TNF-a, tumor necrosis factor alpha; TSLP, thymic stromal lymphopoietin manner, the symptoms of ichthyosis bullosa of Siemens Received 27 November 2013; revised 24 March 2014; accepted 2 April 2014; accepted article preview online 21 April 2014; published online 29 are not informative about the physiological function of May 2014 normal K2. & 2014 The Society for Investigative Dermatology www.jidonline.org 2579 HFischeret al. Role of Keratin Pair K2/K10 in Skin Barrier K2 K2 K1 K1 * * K2 + K1 K2 + K1 ##* Nail K2 + K1 ** Nail # # K2 + K1 Figure 1. Keratins K1 and K2 are differentially expressed in the epidermis of the mouse. (a–d) Immunofluorescence labeling of K1 (red) and K2 (green) in the skin of the ears (a), back (b), tail (c), and paw (d) of C57BL/6 wild-type mice. Nuclei were counterstained (blue) with Hoechst 33258. Asterisks mark infundibula of hair follicles. **The image of the paw is a compilation of pictures. (e, f) X-gal staining (blue) of beta-galactosidase expressed under the control of the Krt2 promoter in the tail (e) and toe (f)ofKrt2 þ / À mice. Arrows and arrowheads mark corresponding positions of the toe pad in d and f, whereas # symbols mark scales in c and e. Bars ¼ 40 mm(a, b), 100 mm(c), 200 mm(d). Here we investigate the role of K2 in epidermal homeostasis (Figure 1a–c). By contrast and surprisingly different from the by defining the expression patterns of suprabasal keratins in situation in human skin, K1 was absent from the interfolli- the mouse and by generating and characterizing K2-deficient cular epidermis of the mouse ear, whereas it was present in mice. We show that suprabasal epidermal keratinocytes of the infundibular regions (Figure 1a). In the back skin, which several murine body sites lack expression of K1 while expre- is densely covered by pelage hair follicles, K2 was absent, ssing K2 together with K10. The corresponding sites of K2 whereas K1 was expressed throughout the interfollicular knockout mice contain prominent K10 aggregates and epidermis (Figure 1b). In the tail skin, K1 and K2 were develop epidermal acanthosis and hyperkeratosis as well as confined to the interscale regions within which the infundi- skin inflammation. Together with our finding that K2 forms bulum regions of the sparse hair follicles contained K1 and aggregates in the absence of K10, our study establishes that the region more distant from the hair follicles contained K2 K2/K10 intermediate filaments are essential for normal epi- (Figure 1c). On the soles and footpads, K1 and K2 were also dermal morphology at specific body sites. expressed in an alternating pattern (Figure 1d, arrows). All skin sites that contained either K1 or K2 expressed K10 RESULTS (Supplementary Figure S1 online). Essentially the same Keratins K1 and K2 are differentially expressed in the suprabasal results were obtained by immunofluorescence analysis of ear and plantar epidermis the mouse strains C57BL/6, mixed 129/C57BL/6, mixed The expression patterns of K2 and K1 were determined by C57BL/6/Balb/c, and SKH-1. Together, these expression immunofluorescence labeling of the skin from various body patterns suggested that the suprabasal epidermis of the sites of wild-type (WT) mice (Figure 1). K2 was expressed in mouse has two mutually exclusive schemes of keratiniza- the suprabasal layers of the interfollicular epidermis of the tion, one involving the formation of intermediate filaments ear with the exception of the infundibulum of hair follicles made of K1/K10 heterodimers and the other involving the 2580 Journal of Investigative Dermatology (2014), Volume 134 HFischeret al. Role of Keratin Pair K2/K10 in Skin Barrier formation of intermediate filaments made of K2/K10 a Krt2 +/+ Krt2 –/– heterodimers. In an independent experimental approach, the activity of the murine Krt2 promoter was assessed in mice carrying a beta-galactosidase reporter gene inserted into one of the two Krt2 alleles (Supplementary Figure S2 online). Visualization of beta-galactosidase activity in situ revealed reporter gene expression in the ear skin (Supplementary Figure S3 online), in the interscale regions of tail skin (Figure 1e), and in those regions of the sole and toe pads that lacked eccrine sweat glands (Figure 1f, for overview and negative control see Supplementary Figure S3 online). No expression of the reporter gene was detected in the back skin of heterozygous mice (not shown) and in any tissue of WT mice that were used as negative controls (Supplementary Figure S3 online). Thus, +/+ –/– b Krt2 Krt2 the beta-galactosidase activity staining confirmed the expression sites of K2 as detected by immunofluorescence analysis of tissue sections and revealed a highly patterned organization of Krt2 promoter activity on the tail as well as on the soles and toe pads. Further investigations were focused on the epidermis of the ears because of the uniform expression of K2 in the interfollicular epidermis at this body site. c d 3 Targeted deletion of K2 leads to hyperkeratosis, increased water 100 * * loss, and localized inflammation 80 ) To investigate the role of K2 in vivo, we generated mice in –1 h À / À 2 which both alleles of the Krt2 gene were disrupted (Krt2 ) 60 –2 (Supplementary Figure S2 online).

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