The expression of Involucrin, PCNA, P63 and B-1 Integrin In keratinocytes derived from the coronette region of the bovine claw

Brody, Lisa
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University of Delaware
Modern production practices, housing facilities and nutritional programs in the dairy industry have created a precipitous decline in leg and hoof health such that 1 in 4 cows in the US experienced one or more episodes of lameness in 2008. The costs of lameness arise from decreased milk yields, lower reproductive efficiency, increased involuntary culling rates and higher cow mortality. A majority of the lesions associated with lameness are located in the lateral, hind limb claw capsule and have been proposed to arise secondary to “poor quality” horn tissues. Claw horn lesions were associated with changes in expression of pro-and anti-inflammatory cytokines, growth factors and receptors that may underlie disturbances in keratinocyte proliferation and terminal differentiation and production of weak horn tissues.. The purpose of this work was to quantify expression of structural and functional proteins associated with progenitor and proliferating cells (p63 and proliferating cell nuclear antigen (PCNA) and β-1 integrin) and terminally differentiating cells (involucrin) in 21 day old, co-culture keratinocyte derived from the coronette region of the bovine hind limb, lateral claw. We hypothesized expression of P63, β-1 integrin and PCNA (proliferating cell nuclear antigen), (markers of stem cells and committed progenitor keratinocytes) were low and expression of involucrin (marker of terminal differentiated keratinocytes) was high in keratinocytes driven toward terminal differentiation. Tissues were collected from the coronette of the lateral hind limb and cells were isolated, fixed, and stained for fluorescent activity cell sorting analysis (FACS). While expression of p63 was inconsistently demonstrated in these cell populations 50.04% + 2.98 of the cell population was involucrin + (p=0.00017 ),16.6% + 1.09 was β-1 integrin + (p=0.0007) and 1.90% + 0.325 (p=0.09 ) was PCNA +. These data indicated for the first time bovine keratinocytes in 21 day cocultures expressed proteins indicative of progenitor cell and terminal differentiating cell activity. Because prevalence of p63 expression was very small, further quantification of p63 expression will require re-evaluation of p63 protein in the β-1 integrin + keratinocyte pool. The amount of each protein expression was similar to amounts described in human and murine models of keratinocyte proliferation and differentiation. Expression of these markers in primary cells isolated directly from the bovine claw may valuable in site into keratinocyte growth and maturation in normal as well as diseased claw horn tissues.