Atsushi Kon

A hyaluronan synthase suppressor, 4‐methylumbelliferone, inhibits liver metastasis of melanoma cells

4‐Methylumbelliferone (MU) inhibits the cell surface hyaluronan (HA) formation, and that such inhibition results in suppression of adhesion and locomotion of cultured melanoma cells. Here, we examine the effect of MU on melanoma cell metastasis in vivo. MU‐treated melanoma cells showed both decreased cell surface HA formation and suppression of liver metastasis after injection into the mice. Oral administration of MU to mice decreased tissue HA content. These HA knock‐down mice displayed suppressed liver metastasis. Thus, both cell surface HA of melanoma cells and recipient liver HA can promote liver metastasis, indicating that MU has potential as an anti‐metastatic agent.

4-methylumbelliferone, a hyaluronan synthase suppressor, enhances the anticancer activity of gemcitabine in human pancreatic cancer cells

Hyaluronan (HA) is a ubiquitous, major component of the pericellular matrix and is necessary for various physiological processes. It plays a very important role in biological barriers. We previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and pericellular HA matrix formation in cultured human skin fibroblasts, Streptococcus equi FM100, and B16F10 melanoma cells. We hypothesized that MU-mediated inhibition of HA synthesis and pericellular HA matrix formation would increase the efficacy of anticancer drugs. We have already demonstrated in vitro, using a sandwich binding protein assay and a particle exclusion assay, that MU inhibits HA synthesis and formation of the pericellular HA matrix, respectively, in human KP1-NL pancreatic cancer cells. AlamarBlue assay revealed that the anticancer effect of gemcitabine in KP1-NL cells was increased by pretreatment with MU. In vivo simultaneous administration of MU and gemcitabine to tumor-bearing mice with severe combined immunodeficiency disease (SCID) decreased the size of the primary and metastatic tumors more than did gemcitabine alone. These data strongly suggest that a combination of MU and gemcitabine is effective against human pancreatic cancer cells. MU may have potential as a chemosensitizer and may provide us with a new anticancer strategy.

Cooperation between SMAD and NF-κB in growth factor regulated type VII collagen gene expression

We have previously demonstrated that transforming growth factor-β (TGF-β) and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) or interleukin-1β, synergistically enhance the expression of type VII collagen gene (COL7A1) in human dermal fibroblasts in culture (Mauviel et al., 1994). Recently, we identified a SMAD-containing complex, rapidly induced by TGF-β and binding the region [−496/−444] of the COL7A1 promoter, responsible for COL7A1 gene transactivation (Vindevoghel et al., 1998a). In this report, we demonstrate that TGF-β and TNF-α response elements are distinct entities within the COL7A1 promoter. In particular, we demonstrate that the TNF-α effect is mediated by NF-κB1/RelA (p50/p65) and RelA/RelA (p65/p65) NF-κB complexes binding the TNF-α response element (TaRE) located in the region [−252/−230], with RelA acting as the transcriptional activator. Finally, we provide definitive evidence for the role of both TGF-β and TNF-α response elements as enhancer sequences, functioning in the context of a heterologous promoter in an additive manner in response to TGF-β and TNF-α. This study provides the first identification of a functional interaction between the two immediate-early transcription factors, SMAD and NF-κB, to activate the expression of an extracellular matrix-related gene, COL7A1.

Diagnostic dilemma of “sporadic” cases of dystrophic epidermolysis bullosa: a new dominant or mitis recessive mutation?

Abstract: Dystrophic forms of epidermolysis bullosa (DEB), characterized by mutations in the type VII collagen gene (COL7A1), are inherited either in an autosomal dominant or autosomal recessive fashion, and sporadic, de novo cases have also been reported. Clinically, the dominant forms (DDEB) can be indistinguishable from the mild, mitis forms of recessively inherited DEB (M‐RDEB). This situation poses a dilemma in case of families with 1 mildly affected individual and clinically normal parents: Is it a new dominant or mitis recessive DEB? In this study we review 2 cases with mild DEB, the parents being clinically normal. One of the cases was shown to be a compound heterozygote for 2 silent missense mutations (R2063W/G2366S), thus being diagnosed as M‐RDEB. The second case had a single glycine substitution mutation (G2079E) in COL7A1 and had therefore DDEB. These findings have implications for the genetic counseling of these families concerning the risk of recurrence of the disease in subsequent pregnancies in the present and future generations.

Immunohistochemical analysis of the skin in junctional epidermolysis bullosa using laminin 5 chain specific antibodies is of limited value in predicting the underlying gene mutation.

The anchoring filament protein laminin 5 is composed of three polypeptide chains (α3, β3 and γ2) each encoded by separate genes (LAMA3, LAMB3 and LAMC2, respectively). Mutations in any of these three genes may give rise to the autosomal recessive blistering skin disease, junctional epidermolysis bullosa. At present, there is no easy way of predicting which of these three genes might harbour the pathogenetic laminin 5 mutations in a case of junctional epidermolysis bullosa. In this study, we assessed whether immunohistochemistry might be helpful in this regard. We performed immunohistochemical labelling of the dermal‐epidermal junction using α3, β3 and γ2 chain‐specific antibodies in 11 patients with junctional epidermolysis bullosa, in whom the laminin 5 mutations had been previously delineated. Although, labelling for the laminin 5 chain bearing the mutations was attenuated or undetectable in all cases, a complete absence of labelling or a reduction in the staining intensity for the other two chains was also seen in all cases. The results showed that immunohistochemical labelling of the dermal‐epidermal junction using α3, β3 and γ2 chainspecific antibodies is not a specific indicator for which of the laminin 5 chain genes contains the pathogenetic mutations, and is therefore unreliable in screening for individual laminin 5 gene mutations in cases of junctional epidermolysis bullosa.

Keratinocyte‐specific modulation of type VII collagen gene expression by pro‐inflammatory cytokines (tumor necrosis factor‐α and interleukin‐1β)

Abstract:  Previous studies have shown that pro‐inflammatory cytokines such as tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β up‐regulate type VII collagen gene (COL7A1) expression in cultured dermal fibroblasts. The present study was designed to investigate the effects of TNF‐α and IL‐1β on COL7A1 expression in epidermal keratinocytes. We demonstrated that both TNF‐α and IL‐1β reduced COL7A1 expression in epidermal keratinocytes in an additive manner, whereas they increased COL7A1 expression in dermal fibroblasts. Thus, regulation of COL7A1 by pro‐inflammatory cytokines is cell type specific. In particular, the inhibitory effects of TNF‐α and IL‐1β occurred, at least in part, at the transcriptional level. Finally, we demonstrated that TNF‐α and IL‐1β enhanced the TGF‐β‐mediated up‐regulation of COL7A1 expression in HaCaT keratinocytes, suggesting that the combination of TGF‐β and TNF‐α or IL‐1β induces a signaling pathway that is completely different from that induced by either pro‐inflammatory cytokine alone.

Interferon-gamma down-regulates expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1) in epidermal keratinocytes via novel chimeric sequences of ISRE and GAS.

Abstract:  The 230‐kDa bullous pemphigoid antigen (BPAG1) is an integral component of hemidesmosomes. We have previously reported that interferon‐γ (IFNγ) inhibits the transcription of the BPAG1 gene ( 1 ). Here we investigated the target sequences of IFNγ‐signal transduction pathway in the BPAG1 promoter in epidermal keratinocytes. Transient transfections with 5′‐deletion constructs of BPAG1 promoter‐luciferase reporter gene plasmids in cultured normal human epidermal keratinocytes (NHEK) allowed us to narrow the DNA region containing IFNγ inhibitory element (IGIE) to between −1 and −89, upstream from the transcription initiation site (+1). Homology search in this region identified a chimeric sequence, consisting of IFN‐stimulated responsive element (ISRE) with a partial 7‐bp sequence of IFNγ activation site (GAS), as identified in the guanylate‐binding protein (GBP) gene, inserted at its center. Functional analysis of IGIE, inserted in front of the heterologous thymidine kinase promoter, indicated that IGIE acts as a down‐regulatory element of the promoter through IFNγ‐dependent signal pathway. Transient transfection studies with BPAG1 promoter‐reporter gene constructs containing mutated IGIE (with TT to GG transversions in the region of 5′ISRE, GAS, and 3′ISRE) demonstrated that disruption of the ISRE sequences, but not GAS, markedly suppressed the BPAG1 basal promoter activity and resulted in attenuated IFNγ response in keratinocytes. Our findings provide novel insight into the mechanism of IFNγ regulation in keratinocyte differentiation and proliferation.

Novel proteoglycan glycotechnology: chemoenzymatic synthesis of chondroitin sulfate-containing molecules and its application

Proteoglycans consist of a protein core, with one or more glycosaminoglycan chains (i.e., chondroitin sulfate, dermatan sulfate and heparin sulfate) bound covalently to it. The glycosaminoglycan chains account for many of the functions and properties of proteoglycans. The development of proteoglycan glycotechnology to exploit the functionality of glycosaminoglycan chains is an extremely important aspect of glycobiology. Here we describe an efficient and widely applicable method for chemoenzymatic synthesis of conjugate compounds comprising intact long chondroitin sulfate (ChS) chains. An alkyne containing ChS was prepared by an enzymatic transfer reaction and linked with a chemically synthesized core compound containing an azido group using click chemistry. This method enabled highly efficient introduction of ChS into target materials. Furthermore, the ChS-introduced compounds had marked stability against proteolysis, and the chemically linked ChS chain contributed to the stability of these core compounds. We believe the present method will contribute to the development of proteoglycan glycobiology and technology.

Ubiquitination of molluscum body and its implications for pathophysiology

frequent causative agents. In summary, our study showed that symptomatic tinea capitis largely depended on the school that was investigated. Additionally, there were important variations in the causative species described between the different schools in our study group, and the causative species were also different in comparison with studies conducted in other African countries. Minimal training of dermatological nurse-practitioners for early recognition, diagnosis and treatment of tinea capitis could reduce the disease burden in sub-Saharan Africa.

Preparation and application of a fluorogenic substrate for endo-β-xylosidase

The present paper describes a fluorometric assay for galactosaminoglycan-degrading endo-beta-xylosidase, utilizing glycosaminoglycan chains bearing a 4-methylumbelliferyl group at the reducing terminus as a substrate. This fluorogenic substrate is synthesized by human skin fibroblasts cultured in the presence of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside. The assay is based on measurement of the fluorescence of 4-methylumbelliferone, enzymatically liberated from the synthetic substrate by endo-beta-xylosidase. We examined the applicability of the assay for analysis of endo-beta-xylosidase activity.