Junichi Yamaoka

Suppressive effect of zinc ion on iNOS expression induced by interferon-γ or tumor necrosis factor-α in murine keratinocytes

Abstract Zinc, an essential metal, is a critical component of zinc binding proteins such as zinc fingers, zinc enzymes and metallothioneins. Recently, evidence for its anti-inflammatory property in skin has been accumulating, as shown in the treatment of acne, alopecia and zinc deficiency. In cutaneous inflammations, a large amount of nitric oxide (NO) is produced through induction of inducible nitric oxide synthase (iNOS) under the influence of proinflammatory cytokines, resulting in tissue damages in skin, as clarified in other organs. Therefore, we asked if the effect of zinc on NO production and/or on iNOS expression in keratinocytes may explain the anti-inflammatory property of zinc in skin. Accordingly, we sought to determine in this study whether zinc ion may have effect on IFN-γ or TNF-α induced NO production and iNOS expression in cultured murine keratinocytes. Ten μM of zinc ion remarkably suppressed cytokine-induced NO production in keratinocytes. Furthermore, zinc ion also suppressed cytokine-induced iNOS expression in the protein level as well as in the messenger RNA level. These results suggest the possibility that the suppressive effect of zinc ion on cytokine-induced NO production in keratinocytes may be in part implicated in the anti-inflammatory property of zinc in some of skin disorders.

The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytes.

Abstract: Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression.

Ultraviolet B radiation downregulates inducible nitric oxide synthase expression induced by interferon-γ or tumor necrosis factor-α in murine keratinocyte Pam 212 cells

Abstract  Ultraviolet radiation causes inflammation characterized by erythema and swelling, but also exhibits antiinflammatory effects which have led to the use of ultraviolet B radiation (UVBR) and psoralen plus ultraviolet A (PUVA) in the treatment of psoriasis, chronic severe atopic dermatitis and uremic pruritus. In inflammatory dermatoses, a pathogenic role of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) has been suggested. To elucidate how UVBR regulates iNOS expression in skin under inflammatory conditions, we investigated the effect of UVBR on NO production and iNOS expression in cultured murine keratinocyte Pam 212 cells stimulated with interferon-Á (IFN-Á) or tumor necrosis factor-· (TNF-·). Low doses of UVBR significantly suppressed IFN-Á- or TNF-·-induced NO production. UVBR also downregulated IFN-Á- or TNF-·-induced iNOS expression at both the mRNA level and the protein level. These findings suggest the possibility that the downregulatory effect of UVBR on IFN-Á- or TNF-·-induced iNOS expression may, in part, explain the antiinflammatory and therapeutic properties of UVBR in inflammatory dermatoses.

Cutaneous tuberculosis and pyoderma gangrenosum

A 59‐year‐old woman with multiple myeloma at stage IA exhibited recurrent pyoderma gangrenosum of 13 years’ duration. She also had a history of mitral regurgitation and cerebral infarction, but no significant family history was present. In September 1994, she noted a painful erythematous papule on her left foot, which was treated with a topical injection of triamcinolone. It responded well to this treatment at the time, but a similar eruption developed in the same place in February 1995, and enlarged to form an irregularly shaped, punched‐out ulcer with surrounding infiltrative erythema despite topical treatment ( Fig. 1 ). Further, since November 1994, she had noted pain in the right dorsal foot.

Analysis of mechanisms of epidermal proliferation induced by intracutaneous injection of cholera toxin by the use of site-specifically mutated cholera toxins

Intracutaneous injection of cholera toxin (CT) into rabbits increases vascular permeability and induces epidermal proliferation. To understand the mechanisms of these effects on the skin, we evaluated the involvement of the ADP-ribosyltransferase activity of the A subunit of CT and receptor-binding interactions between GM1-ganglioside and the B subunit of CT. We constructed two mutant CTs, E112K and W88K, by site-directed mutagenesis. Mutant CT-E112K, in which glutamic acid at position 112 (E112) of the A subunit of CT was replaced by lysine, has been shown to have lost its biological activity on Chinese hamster ovary (CHO) cells because of its abolished ADP-ribosyltransferase activity. Mutant CT-W88K, in which tryptophan at position 88 (W88) of the B subunit of CT was replaced by lysine, has been shown to have lost its binding ability to GM1-ganglioside. Intracutaneous injection of these mutant CTs evoked less vascular permeability and less epidermal proliferation than recombinant wild-type CT. These results suggest that: (1) the ADP-ribosyltransferase activity carried by E112 of the A subunit of CT; and (2) the binding ability to GM1-ganglioside via W88 of the B subunit of CT are essential for these effects of CT on the skin.

Effect of UVB radiation on NO production and NOS expression in epidermal keratinocytes

UV radiation causes inflammation characterized by erythema and swelling, immunosuppression, apoptosis, melanogenesis, photoaging, and photocarcinogenesis, whereas it also exhibits anti-inflammatory or therapeutic effects as seen in the treatment of psoriasis and atopic dermatitis. Nitric oxide (NO), a reactive gaseous molecule with multiple cellular functions, has been speculated to play pathogenic or protective roles in inflammatory dermatoses. Therefore, it is important to elucidate how UVB radiation regulates or modifies NO production and nitric oxide synthase (NOS) expression in skin. To examine this issue, we used murine cultured keratinocyte Pam 212 cells and found that low doses of UVB radiation significantly suppressed NO production and inducible NOS (iNOS) expression in keratinocytes stimulated by IFN-γ or TNF-α. Taking pathogenic roles of excessive NO produced through iNOS induction into consideration, UVB-induced suppression of NO production may explain in part the anti-inflammatory or therapeutic effects of UVB radiation in inflammatory dermatoses. However, in keratinocytes under nonstimulated condition, UVB radiation increased NO production, where brain NOS (bNOS) mRNA and protein expressions were enhanced and iNOS mRNA expression was suppressed. These results suggest that NO thus produced after UVB radiation in non-inflammatory skin may explain in part the subsequent erythema formation, melanogenesis and other UVB-induced skin reactions.