Atopic dermatitis is a chronic inflammatory skin disorder characterized by defects in the epidermal barrier and keratinocyte differentiation. The expression of filaggrin, a protein thought to have a major role in the function of the epidermis, is downregulated. However, the impact of this deficiency on keratinocytes is not really known. This was investigated using lentivirus-mediated small-hairpin RNA interference in a three-dimensional reconstructed human epidermis (RHE) model, in the absence of other cell types than keratinocytes. Similar to what is known for atopic skin, the experimental filaggrin downregulation resulted in hypogranulosis, a disturbed corneocyte intracellular matrix, reduced amounts of natural moisturizing factor components, increased permeability and UV-B sensitivity of the RHE, and impaired keratinocyte differentiation at the messenger RNA and protein levels. In particular, the amounts of two filaggrin-related proteins and one protease involved in the degradation of filaggrin, bleomycin hydrolase, were lower. In addition, caspase-14 activation was reduced. These results demonstrate the importance of filaggrin for the stratum corneum properties/functions. They indicate that filaggrin downregulation in the epidermis of atopic patients, either acquired or innate, may be directly responsible for some of the disease-related alterations in the epidermal differentiation program and epidermal barrier function.

Filaggrin-2 (FLG2), a member of the S100-fused type protein family, shares numerous features with filaggrin (FLG), a key protein implicated in the epidermal barrier functions. Both display a related structural organization, an identical pattern of expression and localization in human epidermis, and proteolytic processing of a large precursor. Here, we tested whether FLG2 was a substrate of calpain 1, a calcium-dependent protease directly involved in FLG catabolism. In addition, deimination being critical for FLG degradation, we analyzed whether FLG2 deimination interfered with its proteolytic processing. With this aim, we first produced a recombinant form of FLG2 corresponding to subunits B7 to B10 fused to a COOH-terminal His tag. Incubation with calpain 1 in the presence of calcium induced a rapid degradation of the recombinant protein and the production of several peptides, as shown by Coomassie Blue-stained gels and Western blotting with anti-FLG2 or anti-His antibodies. MALDI-TOF mass spectrometry confirmed this result and further evidenced the production of non-immunoreactive smaller peptides. The degradation was not observed when a calpain 1-specific inhibitor was added. The calpain cleavage sites identified by Edman degradation were regularly present in the B-type repeats of FLG2. Moreover, immunohistochemical analysis of normal human skin revealed colocalization of FLG2 and calpain 1 in the upper epidermis. Finally, the FLG2 deiminated by human peptidylarginine deiminases was shown to be more susceptible to calpain 1 than the unmodified protein. Altogether, these data demonstrate that calpain 1 is essential for the proteolytic processing of FLG2 and that deimination accelerates this process.In the epidermis, the program of keratinocyte terminal differentiation is an oriented process during which cells of the basal layer undergo a series of metabolic and structural changes throughout their migration to the surface of the tissue. The stratum corneum, the outermost layer of the epidermis, is formed by the stacking of so-called corneocytes, the end products of the process. The stratum corneum functions as an effective barrier between the body and its outside environment, limiting skin dehydration and preventing the penetration of outside pathogens, UV radiation, and exogenous chemicals. It also contributes to mechanical protection of the body. So that this function can be achieved, peculiar structures are formed, such as the cornified cell envelope, a resistant and insoluble protein shell that replaces the plasma membrane, and the intracorneocyte fibrous matrix made by the aggregation of keratin intermediate filaments.

No abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the COL7A1 gene encoding type VII collagen. Variations in severity between the different clinical forms of RDEB likely depend on the nature and location of COL7A1 mutations, but observed intrafamilial phenotypic variations suggest additional genetic and/or environmental factors. Candidate modifier genes include MMP1, encoding matrix metalloproteinase 1, the first gene implicated in RDEB before its primary role in the disease was excluded. Type VII collagen is a substrate of MMP1 and an imbalance between its synthesis and degradation could conceivably worsen the RDEB phenotype. Here, we studied a previously described family with three affected siblings of identical COL7A1 genotype but displaying great sibling-to-sibling variations in disease severity. RDEB severity did not correlate with type VII collagen synthesis levels, but with protein levels at the dermal-epidermal junction, suggesting increased degradation by metalloproteinases. This was supported by the presence of increased transcript and active MMP1 levels in the most severely affected children, who carried a known SNP (1G/2G) in the MMP1 promoter. This SNP creates a functional Ets binding site resulting in transcriptional upregulation. We next studied a French cohort of 31 unrelated RDEB patients harboring at least one in-frame COL7A1 mutation, ranging from mild localized RDEB to the severe Hallopeau-Siemens form. We found a strong genetic association between the 2G variant and the Hallopeau-Siemens disease type (odds ratio: 73.6). This is the first example of a modifier gene in RDEB and has implications for its prognosis and possible new treatments.

Background Deimination (also known as citrullination), the conversion of arginine in a protein to citrulline, is catalyzed by a family of enzymes called peptidyl-arginine deiminases (PADs). Three PADs are expressed in the epidermis, one of their targets being filaggrin. Filaggrin plays a central role in atopic dermatitis and is a key protein for the epidermal barrier. It aggregates keratins and is cross-linked to cornified envelopes. Following its deimination, it is totally degraded to release free amino acids, contributing to the natural moisturizing factor (NMF). The mechanisms controlling this multistep catabolism in human are unknown. Objective To test whether external humidity plays a role, and investigate the molecular mechanisms involved. Methods Specimens of reconstructed human epidermis (RHEs) produced in humid or dry conditions (>95% or 30–50% relative humidity) were compared. Results RHEs produced in the dry condition presented structural changes, including a thicker stratum corneum and a larger amount of keratohyalin granules. The transepidermal water loss and the stratum corneum pH were decreased whereas the quantity of NMF was greater. This highly suggested that filaggrin proteolysis was up-regulated. The expression/activity of the proteases involved in filaggrin breakdown did not increase while PAD1 expression and the deimination rate of proteins, including filaggrin, were drastically enhanced. Partial inhibition of PADs with Cl-amidine reversed the effect of dryness on filaggrin breakdown. Conclusion These results demonstrate the importance of external humidity in the control of human filaggrin metabolism, and suggest that deimination plays a major role in this regulation.

Atopic dermatitis is a chronic inflammatory skin disease with defects in the epidermal barrier. In a cohort of African-American children, a FLG2 nonsense mutation has been associated with the disease. In the epidermis of European patients, the expression of filaggrin-2, the filaggrin-related protein encoded by FLG2, is decreased. To describe the function of filaggrin-2 and evaluate the impact of its deficiency, its expression was downregulated using lentivirus-mediated shRNA interference in a three-dimensional reconstructed human epidermis (RHE) model. This resulted in parakeratosis and a compact stratum corneum, presence of abnormal vesicles inside the corneocytes, increased pH and reduced amounts of free amino acids at the RHE surface, leading to increased sensitivity to UVB radiations. The expression of differentiation markers was slightly modified. However, we observed reduced proteolytic processing of corneodesmosin, hornerin and filaggrin in parallel with reduced amounts of caspase-14 and bleomycin hydrolase. Our data demonstrated that filaggrin-2 is important for a proper cornification and a functional stratum corneum. Its downregulation in atopic patients may be involved in the disease-associated epidermis impairment.

Transforming growth factor-b (TGF-b) and retinoic acid (RA) are important regulators of cell growth and differentiation. The TGF-b receptors utilize Smad proteins to transduce signals intracellularly and regulate transcription of target genes, either directly or in combination with other sequence-specific transcription factors. Two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoic X receptors, are involved in mediating transcriptional responses to RA. Given the known interactions between the TGF-b and RAR pathways, we have investigated the role played by RAR ligands in modulating functional interactions between Smad3 and RARs. Using transient cell transfection experiments with an artificial Smad3/Smad4-dependent reporter construct, we demonstrate that RAR overexpression enhances Smad-driven transactivation, an effect that requires both Smad3 and Smad4. We provide evidence that RAR effect on Smad3/Smad4-driven transcription is prevented by natural and synthetic RAR agonists, and potentiated by synthetic RAR antagonists. The activity of two TGF-b-responsive human gene promoter constructs was regulated in a parallel fashion. Using both mammalian two-hybrid and immunoprecipitation/Western methods, we demonstrate a direct interaction between the region DEF of RARc and the MH2 domain of Smad3, inhibited by RAR agonists and enhanced by their antagonists. We propose that RARs may function as coactivators of the Smad pathway in the absence of RAR agonists or in the presence of their antagonists, a phenomenon that contrasts with their known role as agonist-activated transcriptional regulators of RA-dependent genes.

Epidermolysis bullosa acquisita (EBA) is a rare autoimmune bullous disease (AIBD). However, higher EBA incidence and predisposing genetic factor(s) involving an HLA haplotype have been suspected in some populations. This retrospective study assessed the overrepresentation of black patients with EBA, its link with HLA-DRB1*15:03, and their clinical and immunological characteristics. Between 2005 and 2009, 7/13 (54%) EBA and 6/183 (3%) other-AIBD patients seen consecutively in our department were black (P=10(-6)); moreover 7/13 (54%) black patients and 6/183 (3%) white patients had EBA (P=10(-6)). In addition, between 1983 and 2005, 12 black patients had EBA. Finally, among the 19 black EBA patients, most of them had very atypical clinical presentations, 9 were natives of sub-Saharan Africa, 1 from Reunion Island, 7 from the West Indies, and 2 were of mixed ancestry. HLA-DRB1*15:03 allelic frequencies were 50% for African patients, significantly higher than the control population (P<10(-3)), and 21% for the West Indians (nonsignificant). High EBA frequencies have already been reported in American blacks significantly associated with the HLA-DR2. In conclusion, black-skinned patients developing EBA seem to have a genetic predisposition, and EBA should be suspected systematically for every AIBD seen in this population.