Pathogenesis of a variant in the 5' untranslated region of ADAR1 in dyschromatosis symmetrica hereditaria.

Dyschromatosis symmetrica hereditaria (DSH) is a pigmentary genodermatosis caused by mutations in ADAR1. In this study, we performed mutation analysis on a family that included typical DSH patients. No mutations were found in any coding regions or exon-intron boundary regions of ADAR1, but a previously unreported non-coding heterozygous variant, c.-60A>G, was found in the 5' untranslated region (5'UTR) of ADAR1 in the proband and her mother. The function of 5'UTR in mRNA is not well-understood. To understand the pathogenesis of the variant and the function of the 5'UTR of ADAR1, we constructed two reporter genes carrying the ADAR1 5'UTR sequence with/without the variant between the PGK promoter and a luciferase coding sequence, and performed luciferase assays, semi-quantitative PCR analyses, and polysomal assays. In human melanocytes, c.-60A>G induced a 16% reduction in transcription and a 51% reduction in translation. Our results indicate that the 5'UTR c.-60A>G variant adversely affects the post-transcriptional step in gene expression, leading to DSH. Detailed functional assays of the 5'UTR of ADAR1 in the present study revealed the gene expression to be not only downregulated, but also upregulated by defects in 5'UTR depending on the locations. The regulation of translation by 5'UTR is very complicated.

Cutaneous collagenous vasculopathy: Report of first Japanese case and review of the literature.

Cutaneous collagenous vasculopathy (CCV) is a rare acquired idiopathic microangiopathy characterised by the progressive development of diffuse asymptomatic telangiectasias over the skin. Histologically, the presence of a thick hyaline collagenous wall around the affected capillaries, comprising the accumulation of collagen type IV, is noted. We herein report the case of a 17-year-old Japanese boy with symmetrical patches of diffuse telangiectasias on the bilateral extremities that persisted for 10 months. A histological examination revealed dilated capillaries in the papillary dermis surrounded by thick perivascular deposition of hyaline-like materials, which stained positive for periodic acid-Schiff and collagen type IV. We additionally performed a review of 26 CCV patients previously reported in the English literature and summarised the clinical and histological features of generalised telangiectatic disorders, such as CCV, generalised essential telangiectasia and hereditary haemorrhagic telangiectasia. To establish an accurate diagnosis, it is important for dermatologists to recognise the clinical and histological characteristics of CCV and the importance of the histological analysis.

Clinical and laboratory features of systemic sclerosis complicated with localized scleroderma.

Localized scleroderma (LSc) primarily affects skin, whereas systemic sclerosis (SSc) affects skin and various internal organs. LSc and SSc are considered to be basically different diseases, and there is no transition between them. However, LSc and SSc have several common characteristics, including endothelial cell dysfunction, immune activation, and excess fibrosis of the skin, and there exist several SSc cases complicated with LSc during the course of SSc. Clinical and laboratory characteristics of SSc patients with LSc remain unclear. We investigated the clinical and laboratory features of 8 SSc patients with LSc among 220 SSc patients (3.6%). The types of LSc included plaque (5/8), guttate (2/8), and linear type (1/8). All cases were diagnosed as having SSc within 5 years before or after the appearance of LSc. In three cases of SSc with LSc (37.5%), LSc skin lesions preceded clinical symptoms of SSc. Young age, negative antinuclear antibody, and positive anti-RNA polymerase III antibody were significantly prevalent in SSc patients with LSc. The positivity of anticentromere antibody tended to be prevalent in SSc patients without LSc. No significant difference in the frequency of complications, such as interstitial lung disease, reflux esophagitis, and pulmonary artery hypertension, was observed. The awareness of these characteristic of SSc with LSc are essential to establish an early diagnosis and treatment.

New insights into the mechanism of abnormal calcification in nephrogenic systemic fibrosis - gadolinium promotes calcium deposition of mesenchymal stem cells and dermal fibroblasts.

BACKGROUND: Recent studies have suggested that there is a close association between the administration of gadolinium (Gd)-based contrast agents and the development of nephrogenic systemic fibrosis (NSF), an acquired disorder characterized by systemic fibrosis and abnormal calcification in patients with severe renal dysfunction. However, the causative roles of Gd remain unknown. OBJECTIVE: The aim of this in vitro study was to investigate the effect of Gd on the development of fibrosis and calcification in cultured cells. METHODS: MC3T3-E1 cells (pre-osteoblastic cells), human adipose tissue-derived mesenchymal stem cells (HAMSCs), human subcutaneous preadipocytes, and human dermal fibroblasts (HDFs) were each cultured in differentiation medium with or without gadolinium chloride. Calcium deposition of MC3T3-E1 cells, HAMSCs, and HDFs was determined by alzarin red S staining. Adipogenic differentiation of human subcutaneous preadipocytes and HAMSCs was determined by oil red O staining. Fibrogenesis of HDFs was determined by real-time PCR to measure the mRNA expression of type I collagen. Cell proliferation was determined by MTS assay. RESULTS: Gd induced calcium deposition in MC3T3-E1 cells, HAMSCs and HDFs in osteogenic differentiation media. Gd did not induce adipogenic differentiation in human subcutaneous preadipocytes and HAMSCs. Gd did not increase the mRNA expression of type I collagen in HDFs, but did promote cell proliferation. CONCLUSIONS: We have demonstrated a direct effect of Gd on calcium deposition in cultured cells. The result will help us to understand the mechanism of abnormal calcification in NSF.

Case of livedoid vasculopathy with extensive dermal capillary thrombi.

Livedoid vasculopathy (LV) is thought to be a thrombogenic disorder. Here, we report a case of LV presenting livedo reticularis with leg ulcers clinically and many thrombogenic cutaneous vessels histologically. These features strongly suggested the presence of thrombogenic factors involved with the development of the lesions. After examination of various possible thrombogenic factors including phosphatidylserine-dependent anti-prothrombin antibody, we failed to detect any responsible thrombogenic factors for this case of LV. Recently, diverse thrombogenic factors have been reported to be involved in LV. This case may suggest that unknown thrombogenic factors are additionally related to LV pathogenesis.

Cutaneous manifestations and histological features of microscopic polyangiitis.

Purpura and livedo are common cutaneous manifestations of microscopic polyangiitis (MPA); however, only a few clinical analyses focusing on the relationship between clinical symptoms and the affected vessels in the skin have been reported. We herein report the cutaneous manifestations and histological features of four patients with MPA. In two patients, a Henoch-Shönlein purpura-like eruption developed with necrotizing vasculitis localized in the upper dermis. The other two patients presented with livedo racemosa; with histological findings of necrotizing vasculitis of the small vessels around the muscle fibers or from the deep dermis to subcutaneous tissue. Two patients' cases were complicated by systemic sclerosis and had poor prognoses. MPA can present with various cutaneous manifestations. Additional research is required to ascertain the relationship between the prognosis and the affected vessels.

Coincidental association of mycosis fungoides and occupational systemic sclerosis?

We report a 58-year-old man with mycosis fungoides (MF) and occupational systemic sclerosis (SSc) induced by silica exposure. He was engaged in tunnel construction from the age of 18 to 33 years. He developed MF at the age of 30. Diagnosis of silicosis was made at the age of 52 and SSc at the age of 58. Physical examinations revealed sclerotic skin changes on his forearms and fingers and poikiloderma on the left popliteal fossa and inguinal region. Both antinuclear antibody and antitopoisomerase-I antibody were positive. We could find no apparent difference between his clinical features and those of idiopathic SSc except for the presence of silicosis and MF. Systemic therapy with interferon-gamma for MF did not improve the skin sclerosis. We discuss the relationship of silica exposure to both MF and SSc.

Autopsy case of systemic sclerosis with severe pulmonary hypertension.

We report an autopsy case of a 60-year-old Japanese woman who died 27 years after the onset of systemic sclerosis and 4 years after the diagnosis of pulmonary arterial hypertension. Oral administration of bosentan was effective in improving her dyspnea but had to be stopped because of drug eruption along with fever and eosinophilia. During hospitalization for the treatment of multiple skin ulcers and gangrene, she suddenly complained of severe respiratory difficulty, followed by bradycardia, unconsciousness and cardiopulmonary arrest. The autopsy revealed concentric intimal proliferation and marked luminal obstruction in many small- and medium-sized vessels of the lungs. In addition to right ventricular hypertrophy and dilatation, similar vascular changes were also present in the myocardial tissue and the atrioventricular node. In our patient, these marked vascular changes caused pulmonary hypertension followed by the severe right heart failure. The vascular changes in the atrioventricular node were suspected as the cause of a fatal arrhythmia leading to sudden death.

Drug eruption due to bosentan in a patient with systemic sclerosis.

We present the case of a 60-year-old female patient with systemic sclerosis complicated by pulmonary hypertension. Ten days after the initiation of treatment with bosentan, high fever and skin eruptions were noted. In the previous reports, the frequency of drug-induced skin eruptions has not been well documented. Since the use of bosentan is expected to increase, we should be aware of the previously unknown adverse effects as well as skin eruptions.

Dihydrosphingosine 1-phosphate stimulates MMP1 gene expression via activation of ERK1/2-Ets1 pathway in human fibroblasts.

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes formation of important regulators of inter- and intracellular signaling, sphingosine-1 phosphate (S1P), and dihydrosphingosine 1-phosphate (dhS1P). In this study, we investigated the role of SphK1 in the regulation of expression of matrix metalloproteinase 1 (MMP1) in dermal fibroblasts, a key event in regulation of extra cellular matrix. We show that overexpression of SphK1 up-regulated MMP1 protein, MMP1 mRNA, and MMP1 promoter activity, and this action of SphK1 required activation of the ERK1/2-Ets1 and NF-kappaB pathways. Furthermore, experiments using SphK1 specific siRNA demonstrated that SphK1 is required for the TNF-alpha stimulation of MMP1. Additional data revealed a specific role of dhS1P, and not S1P, as a mediator of SphK1-dependent activation of ERK1/2 and up-regulation of MMP1. The stimulatory effect of dhS1P was sensitive to pertussis toxin, suggesting a possible involvement of a G-protein-coupled receptor. In contrast, S1P, but not dhS1P, stimulated the induction of COX-2, which demonstrated selective actions of these two closely related bioactive lipids. In conclusion, this study describes a novel mode of SphK1 signaling through generation of dhS1P with a key role in mediating transcriptional responses to TNF-alpha. This is the first report of selective function of dhS1P as compared with the better studied S1P.

Sphingosine kinase 1 (SPHK1) is induced by transforming growth factor-beta and mediates TIMP-1 up-regulation.

Transforming growth factor-beta (TGF-beta) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of sphingosine kinase (SphK) in TGF-beta signaling. TGF-beta markedly up-regulated SphK1 mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF-beta reduced sphingosine-1-phosphate phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that sphingosine 1-phosphate (S1P) was increased (approximately 2-fold), whereas sphingosine and ceramide were reduced after 24 h of TGF-beta treatment. Given the relatively early induction of SphK gene expression in response to TGF-beta, we examined whether SphK1 may be involved in the regulation of TGF-beta-inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-1 (TIMP-1). We demonstrate that decreasing SphK1 expression by small interfering RNA (siRNA) blocked TGF-beta-mediated up-regulation of TIMP-1 protein suggesting that up-regulation of SphK1 contributes to the induction of TIMP-1 in response to TGF-beta. The role of SphK1 as a positive regulator of TIMP-1 gene expression was further corroborated by using ectopically expressed SphK1 in the absence of TGF-beta. Adenovirally expressed SphK1 led to a 2-fold increase of endogenous S1P and to increased TIMP-1 mRNA and protein production. In addition, ectopic SphK1 and TGF-beta cooperated in TIMP-1 up-regulation. Mechanistically, experiments utilizing TIMP-1 promoter constructs demonstrated that the action of SphK1 on the TIMP-1 promoter is through the AP1-response element, consistent with the SphK1-mediated up-regulation of phospho-c-Jun levels, a key component of AP1. Together, these experiments demonstrate that SphK/S1P are important components of the TGF-beta signaling pathway involved in up-regulation of the TIMP-1 gene.

An increased transforming growth factor beta receptor type I:type II ratio contributes to elevated collagen protein synthesis that is resistant to inhibition via a kinase-deficient transforming growth factor beta receptor type II in scleroderma.

OBJECTIVE: Aberrant transforming growth factor beta (TGFbeta) signaling has been implicated in the pathogenesis of scleroderma (systemic sclerosis [SSc]), but the contribution of specific components in this pathway to SSc fibroblast phenotype remains unclear. This study was undertaken to delineate the role of TGFbeta receptor type I (TGFbetaRI) and TGFbetaRII in collagen overexpression by SSc fibroblasts. METHODS: Primary dermal fibroblasts from SSc patients and healthy adults were studied (n = 10 matched pairs). Adenoviral vectors were generated for TGFbetaRI (AdTGFbetaRI), TGFbetaRII (AdTGFbetaRII), and kinase-deficient TGFbetaRII (AdDeltakRII). TGFbetaRI basal protein levels were analyzed by (35)S-methionine labeling/immunoprecipitation and immunohistochemistry. Type I collagen and TGFbetaRII basal protein levels were analyzed by Western blot and newly secreted collagen by (3)H-proline incorporation assay. RESULTS: Analysis of endogenous TGFbetaRI and TGFbetaRII protein levels revealed that SSc TGFbetaRI levels were increased 1.7-fold (P = 0.008; n = 7) compared with levels in healthy controls, while TGFbetaRII levels were decreased by 30% (P = 0.03; n = 7). This increased TGFbetaRI:TGFbetaRII ratio correlated with SSc collagen overexpression. To determine the consequences of altered TGFbetaRI:TGFbetaRII ratio on collagen expression, healthy fibroblasts were transduced with AdTGFbetaRI or AdTGFbetaRII. Forced expression of TGFbetaRI in the range corresponding to elevated SSc TGFbetaRI levels increased basal collagen expression in a dose-dependent manner, while similar TGFbetaRII overexpression had no effect, although transduction of fibroblasts at higher multiplicities of infection led to a marked reduction of basal collagen levels. Blockade of TGFbeta signaling via AdDeltakRII resulted in approximately 50% inhibition of basal collagen levels in healthy fibroblasts and in 5 of 9 SSc cell lines. A subset of SSc fibroblasts (4 of 9 cell lines) was resistant to this treatment. SSc fibroblasts with the highest levels of TGFbetaRI were the least responsive to collagen inhibition via DeltakRII. CONCLUSION: This study indicates that an increased TGFbetaRI:TGFbetaRII ratio may underlie aberrant TGFbeta signaling in SSc and contribute to elevated basal collagen production, which is insensitive to TGFbeta signaling blockade via DeltakRII.

Hypoxia-induced increase of matrix metalloproteinase-1 synthesis is not restored by reoxygenation in a three-dimensional culture of human dermal fibroblasts.

BACKGROUND: Delayed wound healing is multi-factorial. Although ischemic change is considered to be crucial, little is known about the effects of hypoxia or reoxygenation on the connective tissue metabolism by human dermal fibroblasts. OBJECTIVE: The aim of this study is to determine whether or not hypoxia (2% O(2)) or reoxygenation (20% O(2)) affects mRNA expression and production of matrix metalloproteinase-1 (MMP-1), type I collagen, tissue inhibitors of metalloproteinase-1 (TIMP-1), and transforming growth factor-beta1 (TGF-beta1) by human dermal fibroblasts in a three-dimensional culture. METHODS: We introduced the three-dimensional culture of human dermal fibroblasts with experimental wound. After wounding, cells were incubated under hypoxic (2%) or normoxic (20%) condition, and harvested at 24, 36, 48, and 72 h (n=8). In the reoxygenation study (n=4), cells were first exposed to a hypoxic condition for 72 h and further incubated under a normoxic condition for 72 h. RESULTS: The relative ratio (hypoxia/normoxia) of MMP-1 mRNA expressions were significantly elevated at 36 and 48 h compared with those at 12 h (P<0.05). The relative ratio of proMMP-1 was also significantly increased at 48 and 72 h compared with that at 12 h (P<0.001 and P<0.05, respectively). There were no significant changes in mRNA and protein levels of type I collagen, TGF-beta1, and TIMP-1. In a reoxygenic condition, 72 h reoxygenation after 72 h hypoxia, the hypoxia-induced alterations of MMP-1 and carboxyterminal propeptide of type I procollagen (PIP) were not restored. CONCLUSION: Our results indicate that hypoxia may be responsible for delayed wound healing by inducing an increase of MMP-1 synthesis.