With establishment of pig cloning by somatic cell nuclear transfer it is now possible to produce transgenic animals from genetically engineered somatic cells. Recently, we established the use of SB DNA transposon-based vectors for development of transgenic pigs by DNA transposition in cultured cells serving as nuclear donors in handmade cloning 
. In the present report, we explore this approach for generating pig models of skin inflammation. We established Göttingen minipigs carrying human transgenes encoding either β1 or α2 integrin and demonstrated subrabasal expression of the exogenous integrins.
To ensure that transgene transcription was confined to the subrabasal layers, the integrin expression cassette was driven by the human involucrin promoter. We successfully generated six unique hITGB1 pigs containing between one and six transposon insertions. In case of the hITGA2 pigs, however, six of seven pigs were found to be genetically identical, suggesting that they were derived from a single clone out of approximately 30 pooled G418-resistant donor cells. We have previously observed this phenomenon 
which may reflect a large selection pressure in effect during in vitro
growth of somatic cells or during blastocyst and embryo development. It is currently not known whether any toxicity related to expression of the transgene or the SB transposase, or to the transposition process itself, may cause certain clones to exhibit growth advantages and increased survival relative to other clones.
The aggregation and ligation of membrane-bound integrins play a crucial role for strong and persistent activation of members of the large group of receptor tyrosine kinases (RTKs). The group of RTKs, including the EGFR family, serves as receptors for extracellular ligands including cytokines and growth factors and, upon activation, activates cellular signaling potentially through the MAPK pathway. Although mitogen-activated RTKs mediate strong– and integrin ligation only weak– MAPK activation, they are both temporary. In fact, induction of the MAPK pathway is diminished within one hour of integrin ligation 
. However, when such pathways of activation act in synergy, the activation of MAPK signaling is sufficient to induce cell growth 
. Integrins and RTKs are known to co-localize in higher-order complexes which may support prolonged MAPK activation by potentiating phosphorylation, protecting from dephosphorylation, or preventing internalization of the RTKs 
. Interestingly, β1 integrin may induce EGFR phosphorylation and thereby activate the MAPK pathway even in the absence of EGFR ligands 
. Several integrins, including β1 integrin, have been shown to cluster with other membrane receptors during cell-to-cell adhesion 59
. Thus, it is conceivable that integrin ligation caused by cell-to-cell interactions, in conjunction with integrin-directed activation of RTKs, elicit a strong activation of the MAPK pathway.
Subrabasal overexpression of β1 integrin in mice has been shown to entail keratinocyte hyperproliferation and disturbed differentiation. As a direct response to this, variable degrees of perturbations of the epidermis were evident causing chronic inflammation and in some instances conditions reminiscent of adult plaque psoriasis 
. Such visible cutaneous abnormalities either arose spontaneously or where induced by physical irritation (e.g. tape stripping) or topically administered chemicals (e.g. phorbol esters like TPA). The mechanism by which subrabasal integrins induce hyperproliferation and altered differentiation is still obscure. However, subrabasal MAPK activation coincides with β1 integrin expression. Moreover, β1 integrin ligation on the surface of cultured keratinocytes leads to MAPK induction, suggesting that the capability of β1 integrins to induce signal transduction plays a role during the induction of skin inflammation in the mouse 
Confocal microscopy of keratinocytes derived from hITGB1 transgenic pigs demonstrated membrane localization of the transgenic protein and the accumulation of β1 integrin at cell-to-cell interaction points. To investigate the potential activation of the MAPK pathway in transgenic animals, we first studied the level of Erk1/2 phosphorylation in TPA-stimulated HaCaT keratinocytes stably expressing β1 or α2 integrin. Notably, only β1 integrin triggered an elevated level of pErk1/2 in HaCaT cells, and the effect of β1 integrin was therefore explored in keratinocytes from β1 integrin-transgenic pigs. At least for one of three analyzed pigs (#2408) we could monitor a marked increase in the percentage of cells positive for phosphorylated Erk1/2 (also referred to as mitogen-activated protein kinase 3 and 1), suggesting that the MAPK pathway was activated upon stimulation with TPA. Similarly, the Erk/MAPK pathway was activated upon TPA stimulation of keratinocytes derived from α2β1 integrin double-transgenic mice 
, suggesting that constitutive expression of integrins in subrabasal keratinocytes rendered the keratinocytes hypersensitive to external stimuli. The fact that the level of pErk1/2 was unaffected by expression of α2 integrin, as based on our findings in TPA-stimulated HaCaT cells, suggests that β1, but not α2, integrin plays a crucial role for the activation of the Erk/MAPK pathway in this context. Previous work has demonstrated that the cytoplasmic tail of α2 integrin mediates signals via p38/MAPK 
. This effect is probably stimulated through the small G-proteins Cdc42 and Rac1 
, a route of signaling which is not stimulated by TPA. Interestingly, several reports have shown that p38 has an inhibitory effect on Erk1/2 which can be mediated either through p38-directed stimulation of protein phosphatase 2 (PP2A) 
or via a direct interaction between p38 isoforms and Erk1/2 
To further define the inflammatory phenotype of keratinocytes from transgenic pigs, we analyzed transgenic keratinocytes for potential induction of IL-1α as a marker for an altered inflammatory profile. The expression of IL-1α was found to be elevated in all transgenic pigs, indicating that both β1 and α2 integrin gave rise to transcriptional induction of IL-1α. However, the enhancement was far more pronounced for α2 integrin pigs relative to β1-transgenic animals. This suggests that overexpression of α2 integrin induces cellular signaling conveyed by routes different from β1 integrin overexpression. IL-1α is a very potent proinflammatory cytokine which is constitutively produced by epithelial cells, especially keratinocytes, leading to activation of the NF-κB pathway and recruitment of activated mononuclear cells– among a range of effects. Following skin wounding IL-1α is discharged from intracellular storage vesicles 
. Secreted IL-1α contributes to keratinocyte hyperproliferation by EGFR-dependent activation of the Erk/MAPK pathway and has been shown to induce inflammatory conditions in mouse and human epidermis 
. Under normal conditions the activity of IL-1α is tightly regulated 
by mechanisms that do not depend on ECM-induced integrin ligation 
. However, there are numerous examples of signal co-operativity between integrins and IL receptors, suggesting that integrins may assist in intensifying cytokine signaling 
. Indeed, integrin-mediated transcriptional activation of IL-1α has previously shown to involve Erk1/2 or NF-κB activation 
although the exact mechanism by which the release of IL-1α is stimulated is still uncertain. The demonstration of increased IL-1α production in β1 and α2 integrin-transgenic pigs supports the notion that increased integrin signaling correlates with enhanced levels of porcine cytokine production and an altered inflammation profile.
To clarify if the ectopic β1-integrin expression induced a general alteration of the cytokine and chemokine profile in the transgenic animals, we analyzed the mRNA levels of a series of inflammation markers in biopsies of untreated skin from two hITGB1-transgenic animals. The inflammatory response instigated by subrabasal integrin expression has previously shown to comprise TH
1- and TH
17-related cytokines like IL-1β, IFN-γ, GM-CSF and TNF-α 
. Importantly, the TH
1 cytokine signature has been associated with the pathogenesis of psoriasis 
. Notably, for all the cytokines investigated, except TNF-α, the level of mRNA was upregulated in both transgenic animals compared to an age-matched control. The level of TNF-α mRNA, in contrast, was only enhanced in one of the two pigs. A general upregulation of relevant chemokines was also observed. In addition, an increased level of psoriasin was detected in one of the two transgenic pigs. Psoriasin has been identified as a marker for hyperproliferative and inflammatory skin disorders such as psoriasis and atopic dermatitis 
. Overexpression of CXCL10 has also been linked to psoriasis 
. It is predominately activated by IFN-γ, and recruits activated T-lymphocytes and NK cells. Notably, there was a significant difference in the expression level of psoriasin and CXCL10 in the two transgenic animals. Interestingly, it has previously been shown that psoriasin expression is transcriptionally suppressed by IFN-γ 
. Taken together, the expression profile of inflammatory markers suggests that a wide-spread dysregulation of the immune system was prompted by the ectopic expression of β1-integrin. Peculiarly, the expression pattern seemed to vary between the two hITGB1-transgenic pigs, suggesting that the exact expression profile of the integrin itself played a role on downstream effects.
The expression of c-Fos is induced through both the p38/MAPK and the Erk/MAPK signaling pathways 
, and this marker is therefore, like IL-1α, a more general indicator of altered signaling caused by phosphorylation of transcription factors that bind c-Fos enhancer elements 
. We therefore measured the levels of c-Fos mRNA in transgenic keratinocytes. Whereas the increase was only moderate in hITGB1-transgenic keratinocytes (a significant increase in four of six analyzed animals), we observed a solid enhancement of c-Fos mRNA in all three hITGA2-transgenic animals that were analyzed. Although the total number of animals was small, these data support the notion that different signaling patterns were activated in the two groups of transgenic animals.
To date we have not registered any visible epidermal abnormality, or psoriasis-like phenotype, in any of the hITGB1- or hITGA2-transgenic pigs, neither by direct examination of the skin nor by analysis of skin morphology by staining of skin section. However, it is widely accepted that environmental influences, in conjunction with genetic components, play an important role in the pathogenesis of inflammatory diseases such as psoriasis. In α2/β1 integrin double-transgenic mice, skin irritation led to a chronic condition resembling psoriasis with persistent hyperplasia, cutaneous influx of CD4+
T-lymphocytes and secretion of pro-inflammatory cytokines like TNF-α, IFN-γ, IL-1β and IL-6 
. Based on our molecular analysis of central indicators of inflammation findings, we believe that the transgenic pigs may be pre-disposed for development of an exacerbated and potentially chronic inflammation by provocation. Ongoing studies have been designed to address the potential development of chronic plaque lesions in the transgenic pigs as a response to mechanically or chemically induced stress.
In conclusion, we have created Göttingen minipigs transgenic for the human integrins β1 and α2 and have demonstrated efficacious transgene expression in skin as well as induced inflammatory signaling in transgenic keratinocytes. We believe that such porcine models of skin inflammation will contribute to a better understanding of the pathogenesis of cutaneous diseases and will be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis.