The function of a new regulator of epidermal differentiation, HOP, and pathomechanisms underlying epidermolytic hyperkeratosis

The process of epidermal differentiation involves proliferation, differentiation, migration and maturation of keratinocytes to form an impermeable barrier against water loss and outside environment. It is controlled by highly balanced regulatory machinery, involving many molecules that are still under investigation.Homeobox proteins are involved in body patterning and morphogenesis of organs and are studied as potentially good candidates to regulate this process. In the first project we investigated the role of a protein named HOP which belongs to a group of homeobox proteins. Even if HOP is a small protein almost completely composed of the homeodomain and without DNA binding capacity, it is considered as transcriptional regulator in different tissues. HOP interacts with serum response factor (SRF) and histone deacetylase type 2 (HDAC2). By microarray analysis we found that HOP expression increases in cultured human primary keratinocytes (NHK) which undergo calcium-induced differentiation. HOP protein was localized in granular layer of the epidermis of healthy individuals. Lack of HOP was demonstrated in psoriatic lesions, whereas a strong expression was demonstrated in the lesional skin of patients affected with lichen planus (LP). Since LP is characterized by hypergranulosis while psoriatic lesions by progressive lack of the granular layer, the obtained data indicated that HOP might have a potential function in granular layer of epidermis. To investigate HOP function, we inhibited its expression by using HOP specific StealthRNAi and we overexpressed HOP using lentiviral vectors in differentiating NHK. The conclusion of both experiments indicated that HOP positively regulates the expression of late differentiation markers, such as profilaggrin, loricrin and transglutaminase 1. The in vitro data were next confirmed in vivo using HOP knockout mouse model.The second part of my study involved analysis of mechanisms underlying the pathogenesis of epidermolytic hyperkeratosis (EHK). EHK is a genetic disorder characterized by erythema, skin blistering, keratinocyte hyperproliferation and hyperkeratosis. EHK is caused by mutations in keratin 1 or 10 (K1, K10) which are major structural proteins of differentiated keratinocytes and participate in the cellular scaffold formation. To investigate how the structural proteins carrying mutations alter cellular signaling, we established an in vitro model for EHK by overexpression of one of the most common K10 mutations reported so far (K10R156H), in primary human keratinocytes. In order to mimic the in vivo situation, mutated keratinocytes growing on silicone membranes were subjected to mechanical stretch. We observed strong collapse of KIF in K10R156H keratinocytes when subjected to stretch for 30 minutes. Our data demonstrated stronger activation of p38, a member of MAPK stress signaling pathways, in K10R156H when compared to control cells. We demonstrated also that K10R156H keratinocytes showed an induction of TNF-α and RANTES release in response to stretch.Taken together these studies characterize a novel regulator of epidermal differentiation - HOP and demonstrate new aspects implicated in the pathogenesis of EHK

Homeodomain-only protein HOP is a novel modulator of late differentiation in keratinocytes.

The homeodomain-only protein (HOP) contains an atypical homeodomain which is unable to bind to DNA due to mutations in residues important for DNA binding. Recently, HOP was reported to regulate proliferation/differentiation homeostasis in different cell types. In the present study, we performed transcriptional profiling of cultured primary human keratinocytes and noted a robust induction of HOP upon calcium-induced cell differentiation. Immunohistochemistry of human skin localized HOP to the granular layer in the epidermis. Overexpression of HOP using a lentiviral vector up-regulated FLG and LOR expression during keratinocyte differentiation. Conversely, decreasing HOP expression using small interfering RNA markedly reduced the calcium-induced expression of late markers of differentiation in vitro, with the most prominent effect on profilaggrin (FLG) mRNA. Moreover, mRNA levels of profilaggrin and loricrin were downregulated in the epidermis of HOP knockout mice. Analysis of skin disorders revealed altered HOP expression in lichen planus, psoriasis and squamous cell carcinoma (SCC). Our data indicate that HOP is a novel modulator of late terminal differentiation in keratinocytes

Tailored degradation of biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/calcium silicate/poly(lactide-co-glycolide) ternary composites: an in vitro study

Biodegradable materials, which are currently available for bone tissue regeneration, still have limitations regarding their degradation rate, mechanical stability and/or biological response. Thus, a novel generation of materials for bioactive bone scaffolds is needed that triggers hydroxyapatite formation and can be tailored to suit application-specific requirements. In this study we developed ternary bioactive composite materials composed of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), calcium silicate and poly(lactide-co-glycolide) (PHBV/CS/PLGA), which merged the good bioactivity of CS/PHBV composite and the improved degradation velocity of PHBV/PLGA blend. Bioactive character of all composites was proven by formation of hydroxyapatite-like crystals after already one week of incubation in simulated body fluid. Addition of PLGA significantly increased initial ultimate tensile strength (UTS0) and Young's modulus of the ternary composites from 14.3 ± 1.1 MPa (binary composite) to 22.3 ± 2.6 MPa and 1.23 ± 0.05 GPa up to 1.64 ± 0.14 GPa, respectively. Furthermore the degradation rate (measured as a decrease of UTS during degradation) could be successfully tailored and was in range of − 0.033 UTS0 to − 0.118 UTS0 MPa/week. The bioacceptance of the materials was proven in vitro using 2-D (conventional setup) and 3-D (multicellular spheroids) human bone marrow stromal cell cultures

Pyrene-end-functionalized poly(L-lactide) as an efficient carbon nanotube dispersing agent in poly(L-lactide): mechanical performance and biocompatibility study

In order to improve the mechanical properties of poly(L-lactide) (PLLA) based implants, a study was made of how far well dispersed multi-walled carbon nanotubes (MWCNTs) within a PLLA matrix were able to positively affect these properties. To this end, pyrene-end-functionalized poly(L-lactide) (py-end-PLLA) was evaluated as a dispersing agent. Transmission electron microscopy (TEM) analyses and mechanical tests of MWCNTs-based materials demonstrated an enhancement of MWCNT dispersion in the PLLA matrix and improved Youngâs modulus (E) when 4âwt% of py-end-PLLA was used as the dispersing agent. Subsequently, the bioacceptance of PLLA/py-end-PLLA/MWCNTs nanocomposites was evaluated using human bone marrow stromal cells (HBMC) in vitro .The inclusion of py-end-PLLA and MWCNTs supported HBMC adhesion and proliferation. The expression levels of the bone-specific markers indicated that the cells kept their potential to undergo osteogenic differentiation. The results of this study indicate that the addition of MWCNT combined with py-end-PLLA in PLLA/py-end-PLLA/MWCNTs nanocomposites may widen the range of applications of PLLA within the field of bone tissue engineering thanks to their mechanical strength and cytocompatibility.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Spontaneous Atopic Dermatitis-Like Symptoms in a/a ma ft/ma ft/J Flaky Tail Mice Appear Early after Birth.

Loss-of-function mutations in human profilaggrin gene have been identified as the cause of ichthyosis vulgaris (IV), and as a major predisposition factor for atopic dermatitis (AD). Similarly, flaky tail (a/a ma ft/ma ft/J) mice were described as a model for IV, and shown to be predisposed to eczema. The aim of this study was to correlate the flaky tail mouse phenotype with human IV and AD, in order to dissect early molecular events leading to atopic dermatitis in mice and men, suffering from filaggrin deficiency. Thus, 5-days old flaky tail pups were analyzed histologically, expression of cytokines was measured in skin and signaling pathways were investigated by protein analysis. Human biopsies of IV and AD patients were analyzed histologically and by real time PCR assays. Our data show acanthosis and hyperproliferation in flaky tail epidermis, associated with increased IL1β and thymic stromal lymphopoietin (TSLP) expression, and Th2-polarization. Consequently, NFκB and Stat pathways were activated, and IL6 mRNA levels were increased. Further, quantitative analysis of late epidermal differentiation markers revealed increased Small proline-rich protein 2A (Sprr2a) synthesis. Th2-polarization and Sprr2a increase may result from high TSLP expression, as shown after analysis of 5-days old K14-TSLP tg mouse skin biopsies. Our findings in the flaky tail mouse correlate with data obtained from patient biopsies of AD, but not IV. We propose that proinflammatory cytokines are responsible for acanthosis in flaky tail epidermis, and together with the Th2-derived cytokines lead to morphological changes. Accordingly, the a/a ma ft/ma ft/J mouse model can be used as an appropriate model to study early AD onset associated with profilaggrin deficiency