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

Abstract

The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies.

Interleukin-17A pathway target genes are upregulated in Equus caballus supporting limb laminitis

“…The hoof is a modified epidermal appendage integrated into the musculoskeletal system by the epidermal and dermal lamellae, homologous to the nail bed, which in horses are extensively folded to form primary and secondary lamellae to increase the surface area of attachment ( Fig 1 ; [ 3 , 4 ]). Similar to skin, keratin 14 is expressed within lamellar epidermal basal cells [ 5 – 7 ]; however equine epidermal lamellae have several architectural and molecular modifications that differ from skin, including limited stratification into basal, suprabasal and cornified layers, and expression of unique keratins not found in equine or human skin [ 5 – 7 ]. These modifications are thought to impart critical functional differences that allow equine lamellar tissue to bear significantly greater mechanical tensile and compressive forces compared to skin, consistent with the functional integration of the hoof capsule and lamellae into the musculoskeletal system [ 8 ].…”

Interleukin-17 pathway activation inEquus caballussupporting limb laminitis

“…All control horses and one SLL case were donated for research and teaching; the remaining SLL cases were client-owned animals submitted for necropsy following euthanasia due to SLL. Lamellar tissue, including epidermal and dermal lamellae, was collected immediately after euthanasia as previously described [7; 11; 56; 57]. Tissue samples were immediately either snap frozen in liquid nitrogen and stored in liquid nitrogen until processed for RNA extraction, or formalin-fixed/paraffin-embedded (FFPE) and sectioned for microscopy experiments [7; 57; 58].…”

“…Lamellar tissue, including epidermal and dermal lamellae, was collected immediately after euthanasia as previously described [7; 11; 56; 57]. Tissue samples were immediately either snap frozen in liquid nitrogen and stored in liquid nitrogen until processed for RNA extraction, or formalin-fixed/paraffin-embedded (FFPE) and sectioned for microscopy experiments [7; 57; 58]. Tissue sections were oriented transverse to the axis of the limb and include the entire abaxial, middle, and axial regions (relative to the DP) of the primary epidermal lamellae as well as the sub-lamellar dermal tissue located between the DP and epidermal lamellae (see Fig 1).…”

“…In situ hybridization was performed using standard methods [62] as described previously [7], modified to include antibody incubation at room temperature. Briefly, tissue sections were deparaffinized in xylene, followed by rehydration in a graded ethanol series (100%, 75%, 50% and 25%) and digested for 5 min with proteinase K (10μg/mL; Ambion).…”

“…The hoof is a modified epidermal appendage integrated into the musculoskeletal system by the epidermal and dermal lamellae, homologous to the nail bed, which are extensively folded to form primary and secondary lamellae to increase the surface area of attachment (Fig 1; [3,4]). Similar to skin, keratin 14 is expressed within lamellar epidermal basal cells [5-7]; however equine epidermal lamellae have several architectural and molecular modifications that differ from skin, including limited stratification into basal, suprabasal and cornified layers, and expression of unique keratins not found in equine or human skin [5; 6]. These modifications are thought to impart critical functional differences that allow equine lamellar tissue to bear significantly greater mechanical tensile and compressive forces compared to skin, consistent with the functional integration of the hoof capsule and lamellae into the musculoskeletal system [8].…”