Overcoming Expression Obstacles in Producing Functional Components of the G-Protein Coupled Receptor Pathway.

Elucidation of the cellular signaling pathways that are attributed to the progression of lameness must first begin with the identification of molecules that are involved in the normal homeostasis of hoof horn tissue. Epithelial-mesenchymal cell interactions direct growth and differentiation of hoof horn keratinocytes, but little is recognized about the means by which these interactions contributes to hoof horn disease. A greater understanding of factors orchestrating keratinocyte growth, differentiation and maturation in hoof horn production are pivotal to understanding hoof horn properties in health and disease. The specific objectives were to establish an in vitro culture system of bovine coronette keratinocytes, estimate stem cell numbers in keratinocyte populations, and determine epithelial-mesenchymal cell interactions involving keratinocyte growth and corresponding cytokine and growth factor expression patterns. We applied real-time PCR analysis of 16 genes to identify genes related to fetal development, and inflammatory diseases of the bovine hoof. The samples of fetal, adult normal, and adult ulcerated epidermal and corium tissue of the claw was taken from 6 donor Holstein cows per tissue type collected from an a local slaughterhouse. The real-time data demonstrated an overexpression of 7 genes in adult ulcerated tissue, such as: IL-1-beta, IL1RTI, IL12p35, IL12p40, IL-10, TNF-alpha, and iNOS, which are known markers involved in the inflammatory process. Additionally, ulcerated tissues exhibited extensive immune cell infiltration, edema, and signs of hyperproliferation. Alternatively, ulcerated tissues showed lower level of 3 genes such as IL1RTII, GM-CSF, and KGF, compared to fetal and adult normal tissues. There was very little difference in regional distribution between the bulb, sole, and wall of the bovine claw, with the exception of fetal IL-18 within the bulb segment and Ulcerated IL12p40 expression within the wall. Collectively, there was a prominent proinflammatory response within the ulcerated tissue relative to the fetal and normal adult tissue, and the effect of age between fetal and adult showed little differences apart from the GM-CSF levels, which were magnitudes higher in the fetal compared to the adult tissue. From this study we revealed molecules connected to hoof horn development and regulation in fetal, normal adult, and ulcerated adult tissue of the bovine claw, which are involved in known regulatory and transcriptional pathways. To establish an in vitro culture system that mimicked in vivo epithelial-mesenchymal cell interactions, we first had to characterize the keratinocyte compartment of the bovine claw. Abaxial coronette from the lateral, hindlimb claw was incubated in 1.5% trypsin (2h, 37C), minced into fine pieces and propagated 14-21d in Dulbecco's Media (10% FBS). Fibroblasts were collected using (0.7muM EDTA, 2min, 25C) and residual keratinocytes placed in keratinocyte growth medium containing epidermal growth factor (EGF) and bovine pituitary extract (BPE) for 7d. To assess stem cell numbers, keratinocytes were seeded onto bovine dermal fibroblast monolayers at 5 x 103, 7.5 x 103, or 1.0 x 104 viable keratinocytes/cm 2, incubated 10d in DMEM (10% FBS). The expression kinetics of different cytokines, growth factors and cognate receptors were determined at the mRNA level in parallel with functional colony formation assays. Keratinocytes formed 154 +/- 84, 241 +/- 97, 255 +/- 66 colonies at plating densities of 5 x 103, 7.5 x 103, or 1.0 x 104 viable keratinocytes/cm2, respectively. Mesenchymal cell requirements in keratinocyte growth were assessed by plating 5 x 103 and 7.5 x 103 viable...