Noboru Nakagawa

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.

Penile pseudomyogenic hemangioendothelioma/epithelioid sarcoma-like hemangioendothelioma with a novel pattern of SERPINE1-FOSB fusion detected by RT-PCR – Report of a case

We experienced a rare case of penile mesenchymal tumor in a 43-year-old Japanese man. At least three nodules were observed around the penis. The tumors were composed of spindle- to oval-shaped atypical cells with and without prominent nucleoli. These cells were like myogenic cells, but negative for myogenic markers. They were positive for endothelial markers, such as ERG, Fli1 and CD31. They were also positive for nuclear and cytoplasmic FOSB which are not expressed in epithelioid hemangioendothelioma or epithelioid sarcoma. These pathological and immunohistochemical findings strongly suggested pseudomyogenic hemangioendothelioma/epithelioid sarcoma-like hemangioendothelioma (PHE/ES-HE). Since a recent report directly proved that two cases of PHE/ES-HE have SERPINE1-FOSB fusion gene by reverse transcription-polymerase chain reaction (RT-PCR), we examined whether the fusion gene is present or not in the present case by RT-PCR using fresh frozen surgical material. Sequencing of the PCR product revealed that this case has SERPINE1-FOSB fusion. The fusion pattern of our case was different from those of two previously reported cases. In our case, 86 nucleotides of SERPINE1 intron 1 were inserted between SERPINE1 exon 1 and the middle portion of FOSB exon 1, and a putative translation start codon was identified in SERPINE1 intron 1. Thus, this is the third case of PHE/ES-HE with SERPINE1-FOSB fusion proved by RT-PCR.

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.

Bathing suit ichthyosis with summer exacerbation: a temperature-sensitive case.

MADAM, A 27-year-old man complaining of generalized scales came to our clinic. He was born as a collodion baby and had developed ichthyosis after birth. No members of his family or pedigree had the same symptoms. He had no past history of serious diseases other than ichthyosis. On examination, his neck, chest and both sides of his trunk and vertebral regions were covered with slightly brownish, lamellar scales (Fig. 1a,e,g). More severe scales with erythema were on his postauricular and occipital regions, axillary regions, upper back, buttocks, lower abdomen and crural regions covered by his undershorts, and cubital and popliteal regions (Fig. 1b,c,e,i). The distribution of his areas of severe ichthyosis was almost compatible with thermographic images of areas with warmer body temperatures (Fig. 1g–j). Other regions of his face, trunk and extremities revealed only mild scaling. His palms and soles had thin lamellar scales and his fingernails and toenails were thickened (Fig. 1d). Teeth abnormalities, eclabium and ectropion were absent. The patient complained of seasonal changes in his ichthyosis. Indeed, his ichthyosis markedly worsened between 6 May 2010 (Fig. 1e) and 5 August 2010 (Fig. 1f). That summer, an extreme heat wave, over 37 C, occurred during July and August in Japan and the average monthly outside air temperature in the patient’s district increased by more than 10 C between his visits. Haematoxylin and eosin staining of biopsies from his left lumbar lesions showed that the cornified layers were thickened, and epidermal acanthosis and dermal perivascular inflammatory infiltrates were found in severe lesions with erythema and thick scales (Fig. 2a, lower left). In contrast, those characteristics were not evident in mild lesions with only slight scales (Fig. 2a, upper left). Ultrastructure of the lesions revealed that the stratum corneum had piled up to form 36–40 layers in the severe lesions, but was only 10–12 layers thick in the mild lesions. The cornified envelope (marginal band) was hypoplastic in both mild and severe lesions (Fig. 2a, right) and the degradation of corneodesmosomes was delayed in severe lesions (data not shown). The characteristics of the severe lesions represented the skin symptoms of lamellar ichthyosis (LI) and therefore, following approval of the Ethical Committee of the Hyogo College of Medicine and with informed consent of the patient, possible TGM1 mutations were characterized. Mutation analysis of cDNA derived from his hair bulbs revealed compound heterozygous mutations of TGM1, c.430G>A and c.919C>T, which result in p.G144R and p.R307W of transglutaminase 1 (TG1), respectively (Fig. 2b). The same mutations were identified in (a) (b)

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.

A novel point mutation of keratin 17 (KRT17) in a Japanese family with pachyonychia congenita type 2: an RNA-based genetic analysis using a single hair bulb

Pachyonychia congenita type 2 (PC-2) (MIM 167210; Jackson–Lawler syndrome) is an autosomal dominant keratin disorder characterized by hypertrophic nail dystrophy with multiple pilosebaceous cysts. Genetic defects in PC-2 have been correlated with two different keratin genes: keratin 17 (KRT17) and keratin 6b (KRT6B). We report here a novel missense mutation, methionine at codon 88 to lysine (p.M88K), of KRT17, which was detected using RNA from only an individual hair bulb.

Reply: Letters to the Editor

Dear Editor, We appreciate Dr Utani’s comments about our manuscript. Dr Utani mentions that additional or numerous names should not be used in closely related entities and our case should be simply diagnosed as ‘‘linear lichen planus, blaschkitis or adult-onset lichen striatus’’. If the histopathological features of our case with minor interface alterations and no spongiotic changes are considered as a phenotypic variation, it might be included in linear lichen planus or in adult-onset lichen striatus. Concerning the nomenclature of dermatoses occurring along the Blaschko lines of the skin, Müller et al. recently published an interesting report. They examined the clinical and histopathological features of six cases with such characteristics and proposed the concept of a wide spectrum of blaschkolinear dermatoses including lichen striatus and blaschkitis. According to their proposal, our case is located somewhere within the blaschkolinear dermatoses. However, if genetic abnormalities in keratinocytes displaying mosaicism are identified in the future, these rare disorders including variations might need to be re-classified.

Statistical Analysis of Basal Cell Carcinoma

1985年から2002年までの18年間に, 当科において病理組織学的に基底細胞癌と診断した207症例, 247病変について検討した。男性123例, 女性84例で男性にやや多いが, 18年間で増加, 減少傾向は認めなかった。部位別では, 顔面が70.4%と最も多く, 鼻部, 眼瞼部, 頬部で75%を占めていた。WHO分類に準じて病理組織学的分類の再検討を行ったが, nodular typeが37.7%と最も多く, 顔面だけでみるとその比率は44.9%に達した。WHO分類に含まれていないmixed typeが28.3%存在し, これらの取り扱いを含め, 病理組織学的分類の確立が重要であると思われる。