Tatsuya Tsuda

Identification of preferred substrate sequences for transglutaminase 1 – development of a novel peptide that can efficiently detect cross‐linking enzyme activity in the skin

Transglutaminase 1 (TGase 1) is an essential enzyme for cornified envelope formation in stratified squamous epithelia. This enzyme catalyzes the cross‐linking of glutamine and lysine residues in structural proteins in differentiating keratinocytes. To gain insight into the preferred substrate structure of TGase 1, we used a phage‐displayed random peptide library to screen primary amino acid sequences that are preferentially selected by human TGase 1. The peptides selected as glutamine donor substrate exhibited a marked tendency in primary structure, conforming to the sequence: QxK/RψxxxWP (where x and ψ represent non‐conserved and hydrophobic amino acids, respectively). Using glutathione S‐transferase (GST) fusion proteins of the selected peptides, we identified several sequences as preferred substrates and confirmed that they were isozyme‐specific. We generated GST‐fused alanine mutants of the most reactive sequence (K5) to determine the residues that were critical for reactivity. Even in peptide form, K5 appeared to have high and specific reactivity as substrate. In situ analysis of mouse skin sections using fluorescence‐conjugated K5 peptide resulted in detection of TGase 1 activity with high sensitivity, but no signal was detected in a TGase 1‐null mouse. In conclusion, we were successful in generating a novel substrate peptide for sensitive detection of endogenous TGase 1 activity in the skin.

Markedly elevated serum levels of calcium-binding S100A8/A9 proteins in psoriatic arthritis are due to activated monocytes/macrophages

BACKGROUND Serum levels of S100A8/A9 may correlate with disease activity in psoriasis. OBJECTIVE We sought to elucidate the association of serum levels of S100A8/A9 heterodimers with the clinical subtypes of psoriasis and the major cell source. METHODS Serum samples were collected from patients with psoriasis vulgaris (n = 30), psoriatic arthritis (PA) (n = 16), pustular psoriasis (n = 24), and atopic dermatitis (n = 14) and from healthy control subjects (n = 21). Serum concentrations of S100A8/A9 were measured, and the expression levels were examined in psoriatic lesions. The messenger RNA levels were quantified in circulating monocytes and neutrophils. RESULTS Serum levels of S100A8/A9 were significantly increased in all subtypes of psoriasis as compared with healthy controls and atopic dermatitis. Among the psoriatic subtypes, PA and pustular psoriasis showed remarkably high concentrations of S100A8/A9 heterodimers. The higher serum levels were associated with the presence of articular symptoms, but not significantly correlated with body surface areas of psoriatic lesions. S100A8 was expressed by both keratinocytes and infiltrating mononuclear cells, whereas S100A9 was predominantly expressed by neutrophils. The expression levels of S100A8 and S100A9 messenger RNA in monocytes were increased by approximately 2.25- and 1.91-fold in PA, respectively, whereas no significant increase was observed in psoriasis vulgaris and pustular psoriasis. LIMITATIONS Difficulty in acquisition of clinical and laboratory samples in untreated patients, and of a sufficient number of subjects, were limitations. CONCLUSIONS Although serum levels of S100A8/A9 were increased in all types of psoriasis examined, patients with PA had higher levels of S100A8/A9, probably because of an activated monocyte/macrophage system.

Tetracyclines Modulate Protease-Activated Receptor 2-Mediated Proinflammatory Reactions in Epidermal Keratinocytes

ABSTRACT In addition to their antibiotic effects, tetracyclines have anti-inflammatory action that is often beneficial in the control of inflammatory skin disorders. In this study, we examined the effects of tetracycline (TET) and two of its derivatives, doxycycline (DOX) and minocycline (MIN), on the production of interleukin-8 (IL-8) elicited by the activation of protease-activated receptor 2 (PAR2) in normal human epidermal keratinocytes (NHEK). In NHEK, the production of IL-8 stimulated by an agonist peptide of PAR2, SLIGKIV-NH2, at 100 μM was significantly reduced by TET, DOX, or MIN at 5 and 10 μM, concentrations that are noncytotoxic. The tumor necrosis factor alpha (TNF-α)-induced production of IL-8 was synergistically augmented by SLIGKIV-NH2, and that synergistic increase in the production of IL-8 was suppressed by 100 nM PAR2-specific small interfering RNA. It was also suppressed by TET, DOX, or MIN but not by the 14-membered-ring macrolide antibiotics erythromycin, roxithromycin, and clarithromycin, which also have anti-inflammatory activities, at 10 μM. These results suggest that tetracyclines attenuate the PAR2-IL-8 axis in keratinocytes and thereby effectively modulate proinflammatory responses in the skin.

Interleukin-17- and protease-activated receptor 2-mediated production of CXCL1 and CXCL8 modulated by cyclosporine A, vitamin D3 and glucocorticoids in human keratinocytes

Protease‐activated receptor 2 (PAR2) is a G protein‐coupled receptor which mediates a variety of functions in the skin including cutaneous inflammation. SLIGKV‐NH2, an agonist peptide for PAR2, enhanced the interleukin (IL)‐17‐induced production of two CXC chemokines, CXCL1 (GRO‐α) and CXCL8 (IL‐8), in normal human epidermal keratinocytes (NHEK) in a concentration‐dependent manner. The enhanced production of those chemokines was suppressed by a PAR2‐specific siRNA. The SLIGKV‐NH2‐induced production of both CXCL1 and CXCL8 was markedly reduced by cyclosporine A. The enhanced production of CXCL1 was suppressed by 1α, 24R‐dihydroxyvitamin D3, an active form of vitamin D3, and weakly by glucocorticoids, dexamethasone and clobetasol propionate, whereas production of CXCL8 was not altered by any of those receptor agonists. In psoriatic skin, the thickened upper spinous layer of the epidermis was positive for PAR2 protein and the expression of the IL17A mRNA was increased. These results suggest that the IL‐17‐induced pro‐inflammatory reaction is enhanced by the activation of PAR2 in keratinocytes, and that the effect of PAR2 is differentially modulated by cyclosporine A, the active form of vitamin D3 and glucocorticoids.

Knocking-in the R142C mutation in transglutaminase 1 disrupts the stratum corneum barrier and postnatal survival of mice.

BACKGROUND Mutations in the gene encoding transglutaminase 1 (TG1) are responsible for various types of autosomal recessive congenital ichthyosis (ARCI), such as lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) and some minor variants of ARCI. A point mutation of R143C in the β-sandwich domain of TG1 has been often identified in patients with LI or CIE. OBJECTIVE To elucidate the effect of that point mutation on skin barrier structures and functions, we generated mice with a point mutation of R142C, which corresponds to the R143C mutation in human TG1. METHODS A mouse line with the R142C point mutation in TG1 was established using a gene targeting technique and the Cre-loxP system. The skin phenotypes were analyzed in homozygous mutant Tgm1(R142C/R142C) mice. RESULTS In the skin of Tgm1(R142C/R142C) mice, expression of the mutant transcripts was comparable with wild-type or Tgm1(+/R142C) mice. However, the amount of mutated protein in the skin was markedly decreased in Tgm1(R142C/R142C) mice, and the TG1 activity of Tgm1(R142C/R142C) keratinocytes was almost lost. Tgm1(R142C/R142C) mice exhibited morphological and functional skin barrier defects and neonatal lethality. The stratum corneum of those mice lacked cornified envelopes, and loricrin, the major structural component, failed to assemble at the corneocyte cell periphery. Tgm1(R142C/R142C) mice showed a marked increase in transepidermal water loss and their skin was easily permeable to toluidine blue dye. The intercellular lipid lamellar structures of the stratum corneum were irregular and the 13-nm periodic X-ray diffractions from the stratum corneum lipid molecules were lost in vivo. CONCLUSION From these results, we suggest that the R142C mutation of TG1 reduces the enzyme stability which is indispensable for development of the stratum corneum and skin barrier function and for postnatal survival of mice.

Effect of 14-membered-ring macrolides on production of interleukin-8 mediated by protease-activated receptor 2 in human keratinocytes.

ABSTRACT The production of interleukin-8 induced by the activation of protease-activated receptor 2 and its synergism with interleukin-1β were modulated by 14-membered-ring macrolides, namely, roxithromycin, erythromycin, and clarithromycin, in cultured normal human epidermal keratinocytes. Those macrolides may attenuate the protease-activated receptor 2-interleukin-8 axis and thereby modulate proinflammatory responses in the skin.

Adult cutaneous alveolar rhabdomyosarcoma on the face diagnosed by the expression of PAX3‐FKHR gene fusion transcripts

A 26‐year‐old woman presented with an indurated subcutaneous tumor on her left cheek. The histology was compatible with alveolar rhabdomyosarcoma, but immunohistochemistry showed that the tumor cells were negative for desmin, α‐smooth muscle actin and α‐Sr‐1, but were positive for CD56, vimentin and myogenin. The diagnosis of alveolar rhabdomyosarcoma was confirmed by the detection of PAX3‐FKHR fusion gene transcripts in the paraffin‐embedded tumor tissue. The tumor was unresponsive to chemotherapy with pirarubicin, carboplatin and ifosfamide, and the patient died 9 months after the diagnosis. This adult case of an alveolar rhabdomyosarcoma primarily occurring on the face is very rare, and the detection of PAX3‐FKHR fusion gene transcripts was useful for diagnosis of the disease.

Lamellar ichthyosis with pseudoexon activation in the transglutaminase 1 gene

We report a case of a 12‐year‐old boy who was born as a collodion baby after which thick scales developed on his entire body surface. His younger brother showed a similar skin condition. Arcuate‐shaped, large, brownish scales covered his face with ectropion. His lower legs were also covered with larger thick, brownish, plate‐like scales, but other areas were covered with smaller scales. Next‐generation sequencing for exons and splice sites detected a stop‐gain TGM1 mutation leading to p.R71* in transglutaminase 1 (TG1). Another mutation identified was a cryptic mutation in intron 3 that activated a pseudoexon, which was detected by RNA‐based analysis of hair bulbs. The deep intronic mutation caused another truncation mutation, p.N171Tfs*45, in TG1. An in situ TG1 assay demonstrated that TG1 activity was totally lost in this case. Thus, we conclude that the severe phenotype of the patient developed due to those novel compound heterozygous null truncation mutations in TGM1.

Upregulation of interleukin‐33 in the epidermis of two Japanese patients with Netherton syndrome

Netherton syndrome (NS) is a rare autosomal recessive disorder which is caused by mutations in the SPINK5 gene encoding the serine‐protease inhibitor LEKTI. Characteristic symptoms of NS include erythroderma with diffuse desquamation, hair abnormalities and atopic manifestations. Here, we report two Japanese patients with NS, one of whom had a novel mutation in the SPINK5 gene which leads to p.C367Lfs*3. The upregulation of interleukin‐33 (IL‐33) was evident in basal and thickened lower spinous layers of the epidermis in those cases. This suggests that IL‐33 may be involved in the pathophysiology of NS as well as in atopic dermatitis.

Cutaneous malignant fibrous histiocytoma (undifferentiated pleomorphic sarcoma) arising in a chronic scalp ulcer of a patient with non‐bullous congenital ichthyosiform erythroderma

Editor An increased incidence of squamous cell carcinoma (SCC) has been pointed out in patients with lamellar ichthyosis or nonbullous congenital ichthyosiform erythroderma (NBCIE).1,2 However, those ichthyoses accompanied by cutaneous sarcomas have not previously been reported. We herein present a case of a cutaneous malignant fibrous histiocytoma (MFH) arising from a chronic scalp ulcer of a patient with NBCIE. A 38-year-old woman presented in July 2004. She had generalized erythemas with severe ivory-coloured thick scales after birth (Fig. 1a). Her toes and fingers were contracted with severe hyperkeratosis and all nails were thickened. A family history of the same disease was absent in her pedigree. At the age of 25, SCC developed on her left parietal region. The tumour was completely resected, and the defect was covered with a free skin graft from her left axilla. After the treatment, a localized ulcer appeared on the left temporal region of her scalp. The ulcer gradually enlarged with reddish granulations. In September 2005, an elevated nodule appeared on the temporal region of the ulcer and rapidly enlarged up to 4 cm in its major axis in 1 month (Fig. 1b). Her ichthyosiform skin showed acanthosis with hypergranulosis and severe hyperkeratosis. Involucrin was localized on the cell membrane of the middle to upper layers, and that transglutaminase 1 was on those of the upper layers of the epidermis. In situ transglutaminase assay revealed the incorporation of the substrate cadaverine into the cell periphery of the granular layers (Fig. 1c). The tumour of her scalp was composed of atypical fibroblastic spindle cells forming storiform or fascicular patterns (Fig. 1d) and numerous pleomorphic plump histiocytic cells with mitotic figures involving the dermis. The tumour cells were positive for vimentin, weakly positive for α1-antitrypsin, α1antichymotrypsin, and partly positive for α-smooth muscle actin, but negative for desmin, S-100, Factor VIII, CD68, CD31 and CD34. From those findings, an NBCIE accompanied by MFH was thus diagnosed. The tumour was completely resected followed by radiation therapy of a total of 66 Gy. No local recurrence or metastasis of the tumour has been detected for 2 years. The MFH of the present case originated from ulcerated skin and involves the dermis and only a part of the subcutaneous tissue. Dermatofibrosarcoma protuberans, pleomorphic leiomyoma, malignant melanoma and spindle cell carcinoma were excluded by histology and by immunohistochemistry, but the rapid growth of the tumour centred in the dermis might be suggestive of an atypical fibroxanthoma (AFX). However, our case is