Motoshi Kinouchi

Similarly potent action of 1,25-dihydroxyvitamin D3 and its analogues, tacalcitol, calcipotriol, and maxacalcitol on normal human keratinocyte proliferation and differentiation

BACKGROUND The active vitamin D3 regulates proliferation and differentiation of epidermal keratinocytes. Recently topical vitamin D3, tacalcitol, calcipotriol, and maxacalcitol are widely used for psoriasis. OBJECTIVE To examine the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on cultured normal keratinocytes (NHK) and compared its effect with those of various vitamin D3 analogues. METHODS Cell proliferation of NHK cells was analyzed by MTS, BrdU and 3H-thymidine incorporation. The expression of involucrin, transglutaminase 1, keratin 5 and keratin 1 was investigated by western blot and PCR amplification and quantitative assay. Furthermore, we performed cornified cell envelope (CE) formation assay. RESULTS 1,25(OH)2D3, tacalcitol, calcipotriol, and maxacalcitol decreased NHK cell proliferation in a concentration-dependent manner and the maximal effect was observed at 10(-7) M. There was no significant difference in the anti-proliferative effect among the active vitamin D3 analogues. The expression of involucrin and transglutaminase 1 were induced by 1,25(OH)2D3 and its analogues in mRNA and protein levels. CE formation was also induced by 1,25(OH)2D3 and its analogues. There was no significant difference in the potency among these chemicals. Keratin 5 and 1 expression was not altered by these active vitamin D3 analogues. CONCLUSIONS The present study demonstrated that active vitamin D3 analogues, tacalcitol, calcipotriol, and maxacalcitol, suppress keratinocyte proliferation and induce differentiation with similar potency.

Copper, zinc–superoxide dismutase protects from ultraviolet B-induced apoptosis of SV40-transformed human keratinocytes: the protection is associated with the increased levels of antioxidant enzymes

It has been reported that cellular oxidative stress induces apoptosis. Ultraviolet radiation that generates reactive oxygen intermediates (ROIs) also induces apoptosis. Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Mammalian cells express two isozymes of SOD, copper, zinc-SOD (Cu, Zn-SOD) and manganese-SOD (Mn-SOD). Using SV40-transformed human keratinocytes (SVHK cells), we investigated the role of SODs in the ultraviolet B (UVB) irradiation-induced apoptosis. UVB irradiation decreased transiently Cu, Zn- and Mn-SOD activities and their protein levels, with subsequent recovery to the basal levels by 24 h. The UVB-induced decrease in SOD activity was dose-dependent and the maximal effect was obtained at 75 mJ/cm(2). The decrease in Cu, Zn-SOD was more marked than that in Mn-SOD. The cell death assay, annexin-V/propidium iodide flow cytometry, and DNA fragmentation analysis revealed that UVB irradiation induces apoptosis in SVHK cells. The UVB-induced apoptosis was suppressed by the treatment of antioxidants, catalase, glutathione, and alpha-tochopherol. The stable transfection of Cu, Zn-SOD expression vectors into SVHK cells was accompanied by the increased activities of antioxidant enzymes, catalase, and glutathione reductase, as well as glutathione and the cells were shown to be more resistant to UVB-induced apoptosis. In contrast, the transfection of Mn-SOD affected neither activities of antioxidant enzymes nor the UVB-induced apoptosis. The transfection of Cu, Zn-SOD antisense oligomers but not sense oligomers into SVHK or Cu, Zn-SOD cDNA-transfected SVHK (C2) cells significantly decreased the antioxidant enzyme activities and increased the UVB-induced apoptosis. On the other hand, the transfection of Mn-SOD antisense oligomers did not affect the UVB-induced apoptosis. These results suggest that the transfection of Cu, Zn-SOD expression vector, which is accompanied by the increased level of antioxidant enzymes, suppresses the UVB-induced apoptosis of SVHK cells.

Differential phosphorylation of mitogen-activated protein kinase families by epidermal growth factor and ultraviolet B irradiation in SV40-transformed human keratinocytes

SV-40 transformed human keratinocytes (SVHK cells) were stimulated with epidermal growth factor (EGF) and ultraviolet B (UVB) irradiation. Following the stimulation, cell growth, apoptosis, and the activities of mitogen-activated protein (MAP) kinase families were analyzed. EGF (100 ng/ml) increased SVHK cell number compared with control cells cultured in serum-free DMEM medium. The EGF-stimulated cells did not show DNA fragmentation. In contrast, UVB irradiation (40 mJ/cm(2)) markedly decreased viable cell number that was accompanied with DNA fragmentation. EGF stimulated extracellular signal-regulated kinase (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Following the EGF stimulation, phosphorylated ERK and JNK were detected by phospho-p42/44 MAP kinase antibody and phospho-SAPK/JNK antibody, respectively. On the other hand, UVB irradiation stimulated the phosphorylation of p38 and JNK but not of ERK. The stimulation of ERK and JNK induced by EGF was observed earlier than the stimulation of p38 and JNK induced by UVB. PD98059, a specific MAP kinase kinase (MAPKK) 1 (also referred to as MEK1) inhibitor, inhibited EGF-dependent cell proliferation, that was associated with the inhibition of ERK and JNK phosphorylation. In contrast, UVB-induced overall cell death was not significantly affected by PD98059, that inhibited phosphorylation of JNK but not of p38. PD98059, however, significantly augmented UVB-induced cell death earlier time points (30 min--2 h). These results indicate that ERK and JNK are activated following EGF stimulation that might be associated with cell proliferation. On the other hand, UVB-induced apoptosis seems to be mostly associated with the activation of p38. JNK stimulation might provide an anti-apoptotic tonus during the UVB-induced, p38-associated SVHK cell death.

Structure and Transcriptional Regulation of the Human Cystatin A Gene THE 12-O-TETRADECANOYLPHORBOL-13-ACETATE (TPA) RESPONSIVE ELEMENT-2 SITE (−272 TO −278) ON CYSTATIN A GENE IS CRITICAL FOR TPA-DEPENDENT REGULATION

Cystatin A, a cysteine proteinase inhibitor, is one of the precursor proteins of cornified cell envelope of keratinocytes and is expressed during the late stage of keratinocyte differentiation. We have isolated and characterized the human cystatin A gene. The cystatin A gene consists of three exons and two introns. The first, the second, and the third exons consist of coding sequences that are 66, 102, and 126 base pairs in length, respectively. The first and the second introns consist of 14 and 3.6 kilobase pairs, respectively. The transcription initiation site was located 55 base pairs upstream from the first translation site. The fragment, +77 to −2595 in the 5′-flanking region of the human cystatin A gene, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector, p2672CAT, produced a significant CAT activity in transiently transfected SV40-transformed human keratinocytes (SVHK cells), that were further stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator. Sequence analysis of the gene detected three TPA responsive elements (TRE-1, TRE-2, and TRE-3) and one AP-2 site on the 5′ upstream promoter region. Deletion analyses of the p2672CAT vector demonstrated that TRE-2, which was located between −272 and −278, was critical for the regulation by TPA. Gel shift analyses revealed that c-Jun, JunD, and c-Fos bound to the TRE-2 region and that the p2672CAT activity level was elevated by co-transfection with c-Jun and c-Fos or with JunD and c-Fos expression vectors. Furthermore, co-transfection of SVHK cells with the protein kinase C-α expression vector and the p2672CAT expression vector also resulted in an increased CAT activity. These results indicate that the 5′-flanking region of the human cystatin A gene confers promoter activity and contains a TRE (TRE-2) that mediates, at least in part, the enhanced expression of this gene by TPA.

Decreased β2‐adrenergic receptor‐mRNA and Ioricrin‐mRNA, and increased involucrin‐mRNA transcripts in psoriatic epidermis: analysis by reverse transcription–polymerase chain reaction

Psoriatic hyperproliferative epidermis is characterized by a decreased β2‐adrenergic adenylate cyclase response as well us by altered differentiation markers that include decreased loricrin and increased involucrin, Using a quantitative reverse transcription‐polymerase chain reaction, we analysed the expression of β2‐adrenergic receptor‐mRNA. loricrin‐mRNA, and involucrin‐mRNA in the epidermis of five patients with psoriasis vulgaris. The mRNAs of the β2‐adrenergic receptor and loricrin in the involved epidermis were significantly decreased, by 0.35‐fold (P < 0.01) and 0.55‐fold (P < 0.05) respectively, compared with uninvolved epidermis. In contrast, the involucrin mRNA expression of the involved epidermis was significantly increased, by 3.77‐fold (P < 0.01). No significant difference in β‐actin mRNA transcripts was detected between the involved and the uninvolved epidermis. These results indicate that the altered expression of the β2‐adrenergic receptor, loricrin. and involucrin. in the psoriatic involved epidermis, is associated with different amounts of each mRNA transcripts.

Interferon-γ-dependent induction of manganese superoxide dismutase activity of SV40-transformed human keratinocytes by anti-Fas antibody and by TNF-α

Abstract It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF- α on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF- α in the presence of interferon- γ (IFN- γ ) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN- γ , no increase in Mn-SOD activity was detected. The induction of IFN- γ -dependent Mn-SOD activity by anti-Fas antibody or TNF- α was concentration-dependent; the maximal effect was observed at 1–10 μ g/ml and 5–10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF- α in the presence of IFN- γ resulted in an additive increase in Mn-SOD activity. Although the addition of 12- o -tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN- γ -dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF- α . The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN- γ and that TPA augments the process through the activation of protein kinase C.

Analysis of psoriatic patients registered in Asahikawa Medical College Hospital from 1983 to 2007

Psoriasis is a chronic inflammatory skin disease, which has been increasing during the last 50 years in Japan. The aim of the present study is to analyze psoriatic patients registered from 1983–2007 in Asahikawa Medical College Hospital, which is located in the northern part of Japan. A total of 607 cases were registered at the first inspection in the Department of Dermatology, Asahikawa Medical College. Men (403 cases, 66.4%) were predominant over women (204 cases, 33.6%). The clinical types of psoriasis were psoriasis vulgaris (91.5%), guttate psoriasis (4.2%), psoriasis arthropathica (2.8%), psoriatic erythroderma (0.6%), generalized pustular psoriasis (0.6%), localized pustular psoriasis (0.15%) and infantile psoriasis (0.15%). Topical corticosteroids (78.1%) and vitamin D3 (18.1%) products were the main previous topical agents. Previous systemic treatments included etretinate (7.7%), cyclosporine (1.5%) and methotrexate (0.3%). Use of topical vitamin D3 and cyclosporine therapies have been gradually increasing during the past 25 years. Regarding the previous phototherapy, topical psoralen and ultraviolet A therapy (PUVA) (4.9%) was predominant over ultraviolet B (0.9%), and systemic PUVA (0.7%). Use of ultraviolet B phototherapy has been increasing during the past 5 years. The results are essentially similar to those of a survey of psoriasis in Japan from 1982–2001. Although the incidence of psoriasis might be higher in Hokkaido Prefecture, there is essentially no variation in the disease profile of psoriatic patients.

Adenylate cyclases in keratinocytes: FRSK cells express types I, II, III, IV, VI and VIII, and 1,25(OH)2D3, retinoic acid and TPA augment forskolin-induced cyclic AMP accumulation in the absence of altered isozyme expression.

Abstract Molecular cloning analysis has detected at least nine adenylate cyclase isozymes in mammalian tissues. Using fetal rat skin keratinocytes (FRSK), we investigated adenylate cyclase expression and its modulation by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), a retinoid (Ro10-1670), and 12-O-tetradecanoylphorbol-13-acetate (TPA). Reverse transcription polymerase chain reaction (RT-PCR) indicated that FRSK contain adenylate cyclases I, II, III, IV, VI and VIII. Treatment with 1,25(OH) 2 D 3 (1 × 10 –7 M ), Ro10-1670 (1 × 10 –6 M ), and TPA (100 ng/ml) resulted in increased forskolin-induced cyclic AMP accumulation by FRSK cells and normal human keratinocytes (NHK). Quantitative RT-PCR and Western blot analysis, however, detected no alteration in mRNA and protein levels of each adenylate cyclase isozyme for at least 48 h. These results indicate that FRSK contain at least six (I, II, III, IV, VI and VIII) adenylate cyclase isozyme mRNAs, suggesting a complex regulatory mechanism of cyclic AMP generation in keratinocytes. Although 1,25(OH) 2 D 3 , Ro10-1670, and TPA augmented forskolin-induced cyclic AMP accumulation, they do not seem to affect the expression of specific adenylate cyclase isozymes by FRSK cells.

Drug-induced hypersensitivity syndrome followed by chronic inflammatory demyelinating polyneuropathy

Dear Editor, Drug-induced hypersensitivity syndrome (DIHS)/drug reaction with eosinophilia and systemic symptoms (DRESS), one of the most severe drug-related adverse reactions, is characterized by febrile rash, hematological abnormalities, lymphadenopathy and internal organ involvement. Sequential reactivation of human herpes virus (HHV) families, such as HHV-6/7, cytomegalovirus (CMV) and Epstein–Barr virus (EBV), is crucially involved in the pathomechanism. Furthermore, various autoimmune disorders, such as autoimmune thyroiditis and type 1 diabetes mellitus, can subsequently occur. Here, we report a case of trimethoprim/sulfamethoxazole (TMP/SMX)-induced DIHS/DRESS followed by chronic inflammatory demyelinating polyneuropathy (CIDP). A 59-year-old man presented with febrile diffuse maculopapular eruptions with a swollen face (Fig. 1a,b). He had been treated for immunoglobulin (Ig)A nephropathy with 20 mg/day of prednisolone and administrated TMP/SMX for 5 weeks to prevent Pneumocystis pneumonia. Histopathology showed vacuolar degeneration at the dermoepidermal junction and perivascular lymphocytic infiltration (Fig. 1c). Laboratory test results showed atypical lymphocytes and elevated liver enzyme levels. The patient’s titer of HHV-6-specific IgG increased 16 times from the onset of symptoms (from 1:40 to 1:640), followed by CMV antigenemia (C7-HRP; 330/50000 cells). These clinical and laboratory findings led to the diagnosis of DIHS. TMP/SMX administration was discontinued, and prednisolone dose was increased to 60 mg/day under ganciclovir treatment. These steps succeeded in controlling the febrile skin eruptions and the abnormal liver function. Then, prednisolone was tapered and discontinued over 5 months. Two years after the episode of DIHS, the patient experienced progressive gait difficulty. Gradually, dysesthesias on the extremities emerged. Magnetic resonance imaging showed brachial plexus hypertrophy (Fig. 1d). In the cerebrospinal fluid, increased protein level (236.2 mg/dL) without pleocytosis was detected, and a nerve conduction study suggested demyelinating neuropathy. From these findings, the patient was diagnosed with CIDP. i.v. immunoglobulin and plasmapheresis were initiated and the patient responded favorably. Chronic inflammatory demyelinating polyneuropathy, an acquired neuropathy with progressive muscle weakness and sensory dysfunction, is induced by an autoimmune reaction against the myelin sheath of peripheral nerves. Evidence of peripheral nerve demyelination, clarified by either nerve conduction studies or nerve biopsy, is important in the diagnosis. To the best of our knowledge, this is the first description of CIDP following DIHS. Drug-induced hypersensitivity syndrome-related autoimmune disorders can occur several months or even years after resolution of DIHS. Recent evidence suggested that viral reactivation and fatigue of regulatory T cells can be involved in the pathogenesis. CMV or EBV reactivation has also been reported in patients with CIDP. Although the detailed mechanism is unclear, CMV reactivation might have been associated with the development of subsequent CIDP in our case. Although the clinical presentation and course are variable, CIDP generally responds to systemic corticosteroids, i.v. immunoglobulin and plasmapheresis. In contrast, delayed