Mariko Kashiwagi

HLA-B locus in Japanese patients with anti-epileptics and allopurinol-related Stevens-Johnson syndrome and toxic epidermal necrolysis.

INTRODUCTION Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but life-threatening severe cutaneous adverse reactions. Recently, strong associations of HLA-B*1502 and HLA-B*5801 with carbamazepine- and allopurinol-induced severe cutaneous adverse reactions were found in Han Chinese patients, respectively, but ethnic differences in the associations have been reported. The objective of this study is to clarify the involvement of HLA-B*1502 and HLA-B*5801 in Japanese SJS/TEN patients. METHODS HLA-B genotyping was performed on 58 Japanese SJS/TEN patients between July 2006 and April 2008 from multicenters in Japan. RESULTS There were no HLA-B*1502 carriers among 58 SJS/TEN patients. This patient group included seven carbamazepine-related and 11 aromatic anti-epileptic agent-related SJS/TEN patients. In addition, there were five HLA-B*5801 carriers, which included four allopurinol-related SJS/TEN patients. CONCLUSION While HLA-B*1502 is unlikely to be associated with carbamazepine-related or aromatic anti-epileptic agent-related SJS/TEN, HLA-B*5801 was significantly associated with allopurinol-related SJS/TEN in Japanese.

Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C.

ABSTRACT Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the α, δ, η, and ζ isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the α, δ, and η isoforms. Overexpression of the η isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The η-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the η-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the η isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative η isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1α,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the δ isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the α and ζ isoforms did not. These findings indicate that the η and δ isoforms of PKC are involved crucially in squamous cell differentiation.

β-catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair

The activity of keratinocytes in the hair follicle is regulated by signals from a specialized mesenchymal niche, the dermal papilla (DP). Here, mice expressing cre recombinase in the DP were developed to probe the interaction between follicular keratinocytes and the DP in vivo. Inactivation of the beta-catenin gene within DP of fully developed hair follicles results in dramatically reduced proliferation of the progenitors and their progeny that generate the hair shaft, and, subsequently, premature induction of the destructive phase of the hair cycle. It also prevents regeneration of the cycling follicle from stem cells. Gene expression analysis reveals that beta-catenin activity in the DP regulates signaling pathways, including FGF and IGF, that can mediate the DPs inductive effects. This study reveals a signaling loop that employs Wnt/beta-catenin signaling in both epithelial progenitor cells and their mesenchymal niche to govern and coordinate the interactions between these compartments to guide hair morphogenesis.

HLA-B*1511 is a risk factor for carbamazepine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis in Japanese patients

Stevens‐Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but life‐threatening severe cutaneous adverse reactions. Recently, strong associations of HLA‐B*1502 with carbamazepine‐induced SJS/TEN have been found in Han Chinese patients. These associations have been confirmed in several Asian populations, excluding Japanese. SJS patients carrying HLA‐B*1508, HLA‐B*1511, or HLA‐B*1521, which are members of the HLA‐B75 type along with HLA‐B*1502, were detected in studies in India and Thailand. In the current study, we genotyped the HLA‐B locus from 14 Japanese typical and atypical SJS/TEN patients in whom carbamazepine was considered to be involved in the onset of adverse reactions. Although there were no HLA‐B*1502 carriers, four patients had HLA‐B*1511. Our data suggest that HLA‐B*1511, a member of HLA‐B75, is a risk factor for carbamazepine‐induced SJS/TEN in Japanese.

Harnessing of the nucleosome-remodeling-deacetylase complex controls lymphocyte development and prevents leukemogenesis

Cell fate depends on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2β nucleosome-remodeling and histone-deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid lineage–determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active genes encoding molecules involved in lymphoid differentiation. Loss of Ikaros DNA-binding activity caused a local increase in chromatin remodeling and histone deacetylation and suppression of lymphoid cell–specific gene expression. Without Ikaros, the NuRD complex also redistributed to transcriptionally poised genes that were not targets of Ikaros (encoding molecules involved in proliferation and metabolism), which induced their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks.

HLA Class I markers in Japanese patients with carbamazepine‐induced cutaneous adverse reactions

Carbamazepine (CBZ) is frequently used for treating epilepsy, but this drug causes cutaneous adverse drug reactions (cADRs) that may range from mild to severe. It is reported recently that the human leukocyte antigen HLA‐B*1502 is associated with Stevens‐Johnson syndrome (SJS) induced by CBZ in Han Chinese. We examined HLA class I in 15 Japanese patients who fulfilled the diagnostic criteria for CBZ‐induced cADRs (mild in 10 and severe = SJS in 5). HLA‐B*1518, HLA‐B*5901 and HLA‐C*0704 alleles showed higher relative risks (above 10.0) for severe cADRs. The haplotype (HLA‐A*2402‐B*5901‐C*0102) had high relative risk (16.09) for severe cADRs. In patients with severe cADRs, frequencies of HLA‐A*1101, HLA‐A*3303, HLA‐B*1501, HLA‐B*4403, HLA‐B*5101, HLA‐B*5201, HLA‐C*0702, and HLA‐C*1202 alleles are relatively lower than in the Japanese population. These data may suggest that HLA‐B*5901 is one of the candidate markers for CBZ‐induced SJS in Japanese.

PKCα mediates TGFβ-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825–835). This paper addresses a question whether transforming growth factor β (TGFβ) shares the pathway with high Ca2+. On exposure of the cells to TGFβ1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C α (PKCα) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFβ1-induced growth inhibition was almost completely mitigated when PKCα activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCα–S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFβ1.

THE CHROMATIN REMODELER MI-2BETA IS REQUIRED FOR ESTABLISHMENT OF THE BASAL EPIDERMIS AND NORMAL DIFFERENTIATION OF ITS PROGENY

Using conditional gene targeting in mice, we show that the chromatin remodeler Mi-2β is crucial for different aspects of skin development. Early (E10.5) depletion of Mi-2β in the developing ventral epidermis results in the delayed reduction of its suprabasal layers in late embryogenesis and to the ultimate depletion of its basal layer. Later (E13.5) loss of Mi-2β in the dorsal epidermis does not interfere with suprabasal layer differentiation or maintenance of the basal layer, but induction of hair follicles is blocked. After initiation of the follicle, some subsequent morphogenesis of the hair peg may proceed in the absence of Mi-2β, but production of the progenitors that give rise to the inner layers of the hair follicle and hair shaft is impaired. These results suggest that the extended self-renewal capacity of epidermal precursors arises early during embryogenesis by a process that is critically dependent on Mi-2β. Once this process is complete, Mi-2β is apparently dispensable for the maintenance of established repopulating epidermal stem cells and for the differentiation of their progeny into interfollicular epidermis for the remainder of gestation. Mi-2β is however essential for the reprogramming of basal cells to the follicular and, subsequently, hair matrix fates.

PKCη associates with cyclin E/cdk2/p21 complex, phosphorylates p21 and inhibits cdk2 kinase in keratinocytes

PKC is activated on the cell membrane by phospholipids, thereby transducing signals to intracellular pathways. We provide here another function of PKC, namely, regulating cell cycle by interaction with the cyclin E/cdk2/p21 complex. Among the 10 isoforms of PKC, PKCη is predominantly expressed in squamous cell epithelia and induces terminal differentiation of keratinocytes. PKCη that is endogenously expressed or overexpressed was found to associate with the cyclin E/cdk2/p21 complex in keratinocytes of mice and humans. Requirement of a possible adaptor protein to the binding was suggested by the reconstitution of PKCη and the cyclin E/cdk2/p21 complex which were prepared from human keratinocytes or Sf9 insect cells. Colocalization of PKCη with cdk2 and cyclin E was observed in the cytoplasm, particularly in the perinuclear region. p21 was phosphorylated in the complex in a PKC-activator dependent manner. Association of PKCη with cdk2 resulted in marked inhibition of cdk2-kinase activity when measured by phosphorylation of Rb. Dominant negative PKCη associated with the cyclin E/cdk2/p21 complex, but caused a little inhibition of cdk2 kinase activity. Among the known regulatory mechanisms of cdk2 activity, dephosphorylation of Thr160 was demonstrated.

Human leukocyte antigen genotypes in carbamazepine‐induced severe cutaneous adverse drug response in Japanese patients

Dear Editor, Carbamazepine is a widely used antiepileptic drug, which occasionally induces adverse drug responses, including hepatoand nephrotoxicities, movement and behavioral disorders, and skin lesions. Various kinds of cutaneous adverse drug response (cADR) are relatively frequent for this drug, and on rare occasions it induces drug-induced hypersensitivity syndrome (DIHS), Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), which are the severer manifestations. It would be advantageous if genotyping prior to prescription could predict the occurrence of these severe cADR. Recently, carbamazepine-induced SJS/TEN has been shown to closely associate with a human leukocyte antigen (HLA) genotype B*1502 in a HanChinese population. Following this study, Lonjou et al. reported preliminary results from a European study (RegiSCAR) of twelve carbamazepine-induced SJS/TEN cases (nine French and three German). Among these, only four possessed the HLA-B*1502 allele and all of them had Asian ancestry. This indicates that this HLA genotype may not be a universal marker for cADR and that ethnicity may matter. To this end, we studied HLA genotypes in Japanese patients who experienced carbamazepine-induced severe cADR. All patients were recruited from Yokohama City University Hospital and Shizuoka National Epilepsy Center between April 2005 and March 2007. The study was approved by the local internal review boards. The patients with severe cADR incited by carbamazepine who needed hospitalization were eligible. We did not limit to SJS/TEN but broadly collected various types of serious carbamazepineinduced cADR. Altogether, 22 cases were included in the study. The original diseases for the prescription of carbamazepine were epilepsy (n = 11), post-therapeutic neuralgia (n = 4) and others (n = 7, including depression, obsessive compulsive disorder and stroke). Types of cADR were erythematous maculopapular and multiform (n = 6), erythroderma (n = 3), DIHS (n = 4), SJS (n = 2) and other drug eruptions (n = 7). High-resolution HLA typing was performed by using the polymerase chain reaction sequencespecific primer (PCR-SSP) method (MitsubishiChemical BCL Laboratory, Tokyo, Japan). Table 1 shows the HLA-B alleles and their frequencies in the patients, together with those reported for a general Japanese population. None of the 22 cases including the two cases of SJS possessed the B*1502 genotype. When each allele frequency was compared on a 2 × 2 contingency table using Fisher’s exact test, only the B*3902 allele gave a weak sign of association (P = 0.04), which did not remain statistically significant after adjusting with multiple testing. Thus, we concluded that the HLA-B genotypes of the patients do not to largely deviate from that in the general Japanese population. Table 2 shows the HLA-A alleles and their frequencies in the patients together with those reported for a general Japanese population. When each allele frequency was compared on a 2 × 2 contingency table using Fisher’s exact test, the A*3101 allele gave a statistically significant association (P = 0.0004), which remained after adjusting with multiple testing. The odds ratio was calculated to be 4.33 (95% confidence interval, 2.07–9.06). However, we should interpret this result cautiously because the sample size was small and there is a possibility of spurious association. After B*1502 was postulated as a genetic marker, many studies have been attempted to