Prolyl 4‐hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4‐hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta‐subunit of prolyl 4‐hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non‐coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta‐subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4‐hydroxylase tetramer.

Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. Here we investigated the release of the ectodomain and identi®ed the enzymes involved. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP-3, but not by inhibitors of other protease classes or by TIMP-2. This pro®le implicated MMPs or ADAMs as candidate sheddases. MMP-2, MMP-9 and MT1-MMP were excluded, but TACE, ADAM-10 and ADAM-9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE-de®cient cells shedding was signi®cantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.

The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1β, IL-6, TGF-β, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-β) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.

Philpott MP, Kealey T. Metabolic studies on isolated hair follicles: Hair follicles engaged in aerobic glycolysis and do not demonstrate the glucose fatty acid cycle. J Invest Dermatol 1990;96:875e9. Ramot Y, Mastrofrancesco A, Camera E, Desreumaux P, Paus R, Picardo M. The role of PPARgamma-mediated signalling in skin biology and pathology: new targets and opportunities for clinical dermatology. Exp Dermatol 2015;24:245e51.

Collagen XVII/BP180, an epidermal adhesion molecule, exists as a full-length transmembrane protein and as a soluble 120-kd ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. Despite a number of studies on autoantibody targets in blistering skin diseases, it has remained unclear whether the physiologically shed ectodomain of collagen XVII plays a role as an autoantigen. Here we isolated the authentic, soluble form of human collagen XVII and showed that it is an autoantigen recognized by IgG and IgA autoantibodies in different blistering skin diseases and is, in some cases, the preferential target. The ectodomain was isolated from the epidermis, keratinocyte media, amniotic fluid, and pemphigoid blister fluid, and autoantibodies affinity-purified with this ectodomain bound to the proximal surface of the epidermis in normal skin but not in collagen XVII-deficient skin. The antibody reactivity was not dependent on the native conformation or the N-glycosylation of the soluble ectodomain, but was abolished by collagenase treatment. Sera of 81 patients with a clinically active blistering skin disease were reacted with full-length collagen XVII, the authentic soluble ectodomain, and recombinant fragments. In bullous and cicatricial pemphigoid, IgG reactive with full-length collagen XVII also recognized the soluble ectodomain. In linear IgA dermatosis and chronic bullous dermatosis of childhood, IgA targeted the soluble ectodomain more efficiently than the full-length protein. The use of recombinant fragments demonstrated that epitopes were present in several noncollagenous and collagenous subdomains of the molecule, and that a significant portion of the sera targeted Col15 domain, a hitherto unrecognized epitope region.

The efficient healing of skin wounds is crucial for securing the vital barrier function of the skin, but pathological wound healing and scar formation are major medical problems causing both physiological and psychological challenges for patients. A number of tightly coordinated regenerative responses, including haemostasis, the migration of various cell types into the wound, inflammation, angiogenesis, and the formation of the extracellular matrix, are involved in the healing process. In this article, we summarise the central mechanisms and processes in excessive scarring and acute wound healing, which can lead to the formation of keloids or hypertrophic scars, the two types of fibrotic scars caused by burns or other traumas resulting in significant functional or aesthetic disadvantages. In addition, we discuss recent developments related to the functions of activated fibroblasts, the extracellular matrix and mechanical forces in the wound environment as well as the mechanisms of scarless wound healing. Understanding the different mechanisms of wound healing is pivotal for developing new therapies to prevent the fibrotic scarring of large skin wounds.

The genetic basis of epidermolysis bullosa, a group of genetic disorders characterized by the mechanically induced formation of skin blisters, is largely known, but a number of cases still remain genetically unsolved. Here, we used whole-exome and targeted sequencing to identify monoallelic mutations, c.1A>G and c.2T>C, in the translation initiation codon of the gene encoding kelch-like protein 24 (KLHL24) in 14 individuals with a distinct skin-fragility phenotype and skin cleavage within basal keratinocytes. Remarkably, mutation c.1A>G occurred de novo and was recurrent in families originating from different countries. The striking similarities of the clinical features of the affected individuals point to a unique and very specific pathomechanism. We showed that mutations in the translation initiation codon of KLHL24 lead to the usage of a downstream translation initiation site with the same reading frame and formation of a truncated polypeptide. The pathobiology was examined in keratinocytes and fibroblasts of the affected individuals and via expression of mutant KLHL24, and we found mutant KLHL24 to be associated with abnormalities of intermediate filaments in keratinocytes and fibroblasts. In particular, KLHL24 mutations were associated with irregular and fragmented keratin 14. Recombinant overexpression of normal KLHL24 promoted keratin 14 degradation, whereas mutant KLHL24 showed less activity than the normal molecule. These findings identify KLHL24 mutations as a cause of skin fragility and identify a role for KLHL24 in maintaining the balance between intermediate filament stability and degradation required for skin integrity.

Acquired or inherited junctional epidermolysis bullosa are skin diseases characterized by a separation between the epidermis and the dermis. In inherited nonlethal junctional epidermolysis bullosa, genetic analysis has identified mutations in the COL17A1 gene coding for the transmembrane collagen XVII whereas patients with acquired diseases have autoantibodies against this protein. This suggests that collagen XVII participates in the adhesion of basal keratinocytes to the extracellular matrix. To test this hypothesis, we studied the behavior of keratinocytes with null mutations in the COL17A1 gene. Initial adhesion of mutant cells to laminin 5 was comparable to controls and similarly dependent on ␣3␤1 integrins. The spreading of mutant cells was, however, enhanced, suggesting a propensity to migrate, which was confirmed by migration assays. In addition, laminin 5 deposited by collagen XVII-deficient keratinocytes was scattered and poorly organized , suggesting that correct integration of laminin 5 within the matrix requires collagen XVII. Collagen XVII (or BPAG2, 180-kd bullous pemphigoid antigen) is expressed by epithelial cells forming hemidesmosomes (HDs), such as basal keratinocytes in skin or mucosa. HDs are multiprotein complexes connecting the intracellular keratin network to anchoring filaments and fibrils located in the basement membrane (BM) and the upper dermis, respectively.1 Collagen XVII is expressed as a transmembrane full-length entity that is in part converted to an extracellular protein by shedding.2-4 Because the full-length protein is anchored in a type II orientation in the plasma membrane, its N-terminus is intracellular and the C-terminus, or ectodomain, is extracellular. The intracellular domain of collagen XVII is located in the cytoplasmic plaque of HDs where it colocalizes and interacts with the intracellular tail of the integrin ␤4 subunit, BPAG1 (230-kd bullous pemphigoid antigen) and HD1/plectin, 5 which in turn acts as a molecular bridge to the keratin-based cytoskeleton.1 A further interaction occurs between extracellular stretches adjacent to the transmembrane domains of collagen XVII and integrin ␣6 subunit.