Abstract
Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis.
Highlights
Development and refinement of topically administered medications directed against cutaneous inflammatory and hyperproliferative conditions, like psoriasis, scleroderma, and cutaneous lupus erythematosus, demand suitable animal models with a skin architecture that mimics the human counterpart
Integrins constitute a superfamily of ab-heterodimer transmembrane receptors that recognize predominately extracellular matrix (ECM) ligands and convey outside-in signaling events such as cytoplasmic alkalization, potassium channel activation, and activation of various signaling molecules, including mitogenactivated protein kinases (MAPKs) and protein kinase C (PKC)
Medium conditioned for 14 hours showed a 2- to 5-fold increment in secreted IL-1a by cells expressing b1 integrin compared to the average value obtained with medium conditioned by keratinocytes from the non-transgenic controls, pigs #301703 and #301706 (Fig. 6C)
Summary
Development and refinement of topically administered medications directed against cutaneous inflammatory and hyperproliferative conditions, like psoriasis, scleroderma, and cutaneous lupus erythematosus, demand suitable animal models with a skin architecture that mimics the human counterpart. Integrins constitute a superfamily of ab-heterodimer transmembrane receptors that recognize predominately extracellular matrix (ECM) ligands and convey outside-in signaling events such as cytoplasmic alkalization, potassium channel activation, and activation of various signaling molecules, including mitogenactivated protein kinases (MAPKs) and protein kinase C (PKC) This entails great importance for many cellular aspects such as proliferation, differentiation, and migration [3,5]. Subrabasal overexpression of a2 and/or b1 integrin (encoded by the human ITGA2 and ITGB1 genes, respectively) has previously been shown to entail dysregulation of epidermal proliferation and differentiation in mice, leading to cutaneous influx of activated cells of the leukocyte lineage and development of a psoriasis-like phenotype [14]. These findings mark the creation of the first cloned pig models with molecular markers of skin inflammation
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