The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae ofEquus caballus

Abstract

HGH)2 29 Abstract 30 The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which 31 increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, 32 a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant 33 keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are 34 lamellar tissue-specific and could serve as differentiation-and disease-specific markers. Our 35 objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to 36 generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) 37 KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or 38 hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, 39 to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and 40 abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A 41 monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar 42 keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three 43 monoclonal antibodies generated against N-and C-terminal K124 peptides detected a band of the 44 appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal 45 limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 46 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate 47 the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-48 K124 monoclonal antibodies. 3 49 1. Introduction 50 The skin and its appendages are made of stratified epithelia composed of keratinocytes, 51 defined by expression of keratin intermediate filament proteins (abbreviated K for proteins and 52 KRT for genes) [1]. Keratin filaments resist stretching (strain) and provide tensile strength to 53 epithelia and skin appendages. Tissue-and differentiation-specific variation in specific keratin 54 isoform content determines the physical and mechanical properties of diverse epithelial tissues 55 and of their keratinocyte building blocks [2;3]. Understanding how keratins function to provide 56 mechanical stability is crucial to our understanding of human diseases associated with keratin 57 mutations and abnormal keratin expression [4-8]. Here we describe unique keratins of the equine 58 (Equus caballus) epidermal lamellae, a highly specialized tissue that withstands extreme force 59 and provides a model for understanding how keratins provide mechanical strength to flexible 60 tissues. 61 Each single-toed foot of a 500 kg horse (E. caballus) must withstand peak ground reaction 62 forces of 2-5,000 N while protecting the underlying limb from trauma [9;10]. The equine 63 adaptation to single-toed unguligrade locomotion requires the ...

Studies on the pathogenesis of insulin-induced laminitis in horses

“…While a number of keratin and actin filament proteins expressed in the lamellar tissue are also expressed in other epithelial tissues [63], it has recently been demonstrated that keratin protein, K124, is lamellar tissue-specific, highlighting the uniqueness of the lamellar epithelial tissue [64]. In addition to providing mechanical stability, the cytoskeleton also plays an important role intracellular signaling via mechanically-induced signal transduction [65].…”