Psoriasis is a complex chronic immune-mediated inflammatory cutaneous disease associated with the development of inflammatory plaques on the skin. Studies proved that the disease results from a deregulated interplay between skin keratinocytes, immune cells and the environment leading to a persisting inflammatory process modulated by pro-inflammatory cytokines and activation of T cells. However, a major hindrance to study the pathogenesis of psoriasis more in depth and subsequent development of novel therapies is the lack of suitable pre-clinical models mimicking the complex phenotype of this skin disorder. Recent advances in and optimization of three-dimensional skin equivalent models have made them attractive and promising alternatives to the simplistic monolayer cultures, immunological different in vivo models and scarce ex vivo skin explants. Moreover, human skin equivalents are increasing in complexity level to match human biology as closely as possible. Here, we critically review the different types of three-dimensional skin models of psoriasis with relevance to their application potential and advantages over other models. This will guide researchers in choosing the most suitable psoriasis skin model for therapeutic drug testing (including gene therapy via siRNA molecules), or to examine biological features contributing to the pathology of psoriasis. However, the addition of T cells (as recently applied to a de-epidermized dermis-based psoriatic skin model) or other immune cells would make them even more attractive models and broaden their application potential. Eventually, the ultimate goal would be to substitute animal models by three-dimensional psoriatic skin models in the pre-clinical phases of anti-psoriasis candidate drugs. Impact statement The continuous development of novel in vitro models mimicking the psoriasis phenotype is important in the field of psoriasis research, as currently no model exists that completely matches the in vivo psoriasis skin or the disease pathology. This work provides a complete overview of the different available in vitro psoriasis models and suggests improvements for future models. Moreover, a focus was given to psoriatic skin equivalent models, as they offer several advantages over the other models, including commercial availability and validity. The potential and reported applicability of these models in psoriasis pre-clinical research is extensively discussed. As such, this work offers a guide to researchers in their choice of pre-clinical psoriasis model depending on their type of research question.

C3G (RapGEF1), essential for mammalian embryonic development, shows dynamic nucleocytoplasmic exchange. Nuclear localization is regulated by NLSs, NES, and phosphorylation. C3G translocates to the nucleus in response to physiological stimuli and regulates chromatin modifications and gene expression.

RapGEF1 (C3G) is a ubiquitously expressed protein that is essential for mammalian embryonic development. We have shown earlier that C3G regulates cytoskeletal dynamics and is required for neuronal differentiation. To determine if C3G plays a wider role in differentiation of multiple tissue types, we examined its role in skeletal muscle differentiation using the model system of C2C12 cells in culture. C3G protein is highly expressed in mouse skeletal muscle and its transcript and protein levels increase as C2C12 cells are induced to differentiate. Increase in C3G was predominantly seen in the nuclei of myotubes. Ectopic expression of C3G promoted myotube formation when cells were cultured in growth as well as differentiation medium and, enhanced MHC levels were associated with C3G expression. C3G induced differentiation required its catalytic and protein interaction domains and was dependent on the function of cellular R-Ras. Knockdown of cellular C3G using small hairpin RNA reduced expression of muscle specific markers and β-catenin, resulting in impaired differentiation. Disabling C3G function also resulted in enhanced cell death suggesting that cellular C3G is required for cell survival. In cells grown in growth medium, over-expressed C3G increased Akt activity, and C3G knockdown reduced it. C3G expression also suppressed cyclin D1 levels, and induced p27 expression, molecules involved in regulating cell proliferation. Endogenous C3G localizes to focal adhesions in myotubes and C3G expressing cells show distinct stress fibers, elongation and parallel alignment. Expression of a dominant negative construct of C3G, disrupts actin cytoskeleton and formation of focal adhesions resulting in detachment of cells from the substratum and inhibition of differentiation. Our results provide evidence that C3G plays an important role in myogenic differentiation by coordinating cell cycle exit, actin dynamics and survival signaling.

The guanine nucleotide exchange factor C3G (RAPGEF1) regulates proliferation, migration, and differentiation of cells and is essential for mammalian embryonic development. The molecular effectors of C3G dependent functions are poorly understood. Here we report that C3G functions as a negative regulator of β-catenin, a major player in pathways known to be deregulated in human cancers. In mammalian cells, C3G is present in a complex with cellular β-catenin. The proline rich Crk binding region of C3G and residues 90-525 of β-catenin are sufficient for the interaction. Knockdown of cellular C3G stimulated, and its overexpression repressed, β-catenin/TCF transcription activity. C3G acts by destabilizing β-catenin protein and inhibiting its nuclear accumulation. Nuclear extracts of C3G overexpressing cells showed reduced binding to TCF consensus oligos. C3G exerts its effects independent of its function as an exchange factor. It also inhibits stability and activity of an N-terminal deletion construct of β-catenin that is not subject to GSK3β dependent phosphorylation, suggesting that C3G exerts its effect independent of GSK3β. β-catenin repression by C3G was not significantly altered in the presence of proteasome inhibitors, MG132 or lactacystin, suggesting that alternate mechanisms are engaged by C3G to cause β-catenin turnover. C3G expression represses β-catenin target gene expression, and stable clones of MCF-7 breast cancer cells expressing C3G showed reduced migration. Activation of cellular β-catenin or expression of constitutively active β-catenin resulted in reduced C3G expression, indicating that C3G gene expression is negatively regulated by β-catenin. Our results identify a novel property of C3G in functioning as a negative regulator of β-catenin signaling by promoting its degradation. In addition, we show that β-catenin inhibits C3G expression, forming a feedback loop. KeywordsC3G, β-catenin, guanine nucleotide exchange factor, signaling, GSK3βOriginal Article C3G interacts with and inhibits β-catenin function / Dayma et al.

TCPTP is an ubiquitously expressed tyrosine phosphatase with a predominant nuclear isoform (TC45) that binds DNA and has a role in G1-S cell cycle progression. Its deregulation by overexpression induces p53-dependent apoptosis, but the physiological role of its DNA-binding function is not known. Using immunocytochemistry and subcellular fractionation, we investigated changes in its localization in response to DNA damage and replication arrest. Rat fibroblasts showed an increase in endogenous TCPTP bound to nuclear components 3 h after exposure to sublethal dose of UV irradiation. Fractionation of nuclei showed an increase in chromatin and nuclear matrix associated component of TC45. After UV treatment, cells showed a concentration of TCPTP in discrete foci and enhanced colocalization with PCNA and p53BP1. Cells arrested at G1-S transition by hydroxyurea showed a loss of the predominant nuclear staining of TCPTP and an increase in cytoplasmic staining. Upon release from replication block, there was a time-dependent increase in number of cells showing prominent nuclear localization. This change in localization coincides with that of PCNA and Cdk2, two other nuclear proteins having functions in DNA replication. These results provide evidence for the regulation of TCPTP in response to DNA damage and replication stress. Dynamic changes in its localization coincident with that of PCNA suggest involvement of TCPTP in DNA repair and replication.