Masanobu Sakaguchi

Role and Regulation of STAT3 Phosphorylation at Ser727 in Melanocytes and Melanoma Cells

The transcription factor signal transducer and activator of transcription 3 (STAT3) has two important phosphorylation sites, Tyr705 and Ser727, for its activation. Ser727 phosphorylation has been considered to be a secondary event after Tyr705 phosphorylation. In this study, the role and regulation of Ser727 phosphorylation in STAT3 in melanocytic cells were examined. STAT3 was phosphorylated on Ser727 in the absence of Tyr705 phosphorylation in melanocytes. 12-O-tetradecanoylphorbol-13-acetate-induced increase in cell survival activity and nuclear translocation of STAT3 was associated with Ser727 phosphorylation. Ser727 was constitutively phosphorylated in all melanoma cell lines examined irrespective of Tyr705 phosphorylation. The possible involvement of Ser727 phosphorylation in STAT3 in cell survival activity and nuclear translocation of STAT3 in melanocytes was demonstrated also in melanoma cells. The constitutive Ser727 phosphorylation in melanoma cells was partially mediated by the B-Raf-MEK-ERK1/2 pathway. Immunohistochemical studies on specimens of primary lesions of acral lentiginous melanoma revealed that Ser727 phosphorylation precedes Tyr705 phosphorylation in the early stages of melanoma progression. Our results indicate that Ser727 phosphorylation on STAT3 is not necessarily a secondary event after Tyr705 phosphorylation and suggest that it has a role in the regulation of cell survival activity and nuclear translocation of STAT3 in melanocytic cells.

Enhancement of ultraviolet B-induced skin tumor development in phospholipase Cε knockout mice is associated with decreased cell death

Phospholipase C (PLC) ε is a phosphoinositide-specific PLC regulated by small guanosine triphosphatases including Ras and Rap. Our previous studies revealed that PLCε gene-knockout (PLCε(-/-)) mice exhibit marked resistance to tumor formation in two-stage skin chemical carcinogenesis using 7,12-dimethylbenz(a)anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate as a promoter. In this model, PLCε functions in tumor promotion through augmentation of 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. In this study, we have further assessed the role of PLCε in tumorigenesis using a mouse model of ultraviolet (UV) B-induced skin tumor development. We irradiated PLCε(+/+), PLCε(+/-) or PLCε(-/-) mice with doses of UVB increasing from 1 to 10 kJ/m(2) three times a week for a total of 25 weeks and observed tumor formation for up to 50 weeks. In sharp contrast to the results from the two-stage chemical carcinogenesis study, PLCε(-/-) mice developed a large number of neoplasms including malignant tumors, whereas PLCε(+/+) and PLCε(+/-) mice developed a relatively small number of benign tumors. However, UVB-induced skin inflammation was greatly suppressed in PLCε(-/-) mice, as observed with 12-O-tetradecanoylphorbol-13-acetate-induced inflammation, implying that PLCεs role in the suppression of UVB-induced tumorigenesis is not mediated by inflammation. Studies of the tumor initiation stage revealed that UVB-induced cell death in the skin was markedly suppressed in PLCε(-/-)mice. Our findings identify a novel function for PLCε as a critical molecule regulating UVB-induced cell death and suggest that resistance to UVB-induced cell death conferred by the absence of PLCε is closely related to the higher incidence of skin tumor formation.

Phospholipase Cɛ has a crucial role in ultraviolet B-induced neutrophil-associated skin inflammation by regulating the expression of CXCL1/KC.

Phospholipase C (PLC) ɛ is a phosphoinositide-specific PLC regulated by small GTPases including Ras and Rap. We previously demonstrated that PLCɛ has an important role in the development of phorbol ester-induced skin inflammation. In this study, we investigated the role of PLCɛ in ultraviolet (UV) B-induced acute inflammatory reactions in the skin. Wild-type (PLCɛ+/+) and PLCɛ gene knockout (PLCɛ−/−) mice were irradiated with a single dose of UVB at 1, 2.5, and 10 kJ/m2 on the dorsal area of the skin, and inflammatory reactions in the skin were histologically evaluated up to 168 h after irradiation. In PLCɛ+/+ mice, irradiation with 1 and 2.5 kJ/m2 UVB resulted in dose-dependent neutrophil infiltration in the epidermis at 24 and 48 h after irradiation. When mice were irradiated with 10 kJ/m2 of UVB, most mice developed skin ulcers by 48 h and these ulcers became more severe at 168 h. In PLCɛ−/− mice, UVB (1 or 2.5 kJ/m2)-induced neutrophil infiltration was markedly suppressed compared with PLCɛ+/+ mice. The suppression of neutrophil infiltration in PLCɛ−/− mice was accompanied by attenuation of UVB-induced production of CXCL1/keratinocyte-derived chemokine (KC), a potent chemokine for neutrophils, in the whole skin. Cultured epidermal keratinocytes and dermal fibroblasts produced CXCL1/KC in a PLCɛ-dependent manner after UVB irradiation, and the UVB-induced upregulation of CXCL1/KC in these cells was significantly abolished by a PLC inhibitor. Furthermore, UVB-induced epidermal thickening was noticeably reduced in the skin of PLCɛ−/− mice. These results indicate that PLCɛ has a crucial role in UVB-induced acute inflammatory reactions such as neutrophil infiltration and epidermal thickening by at least in part regulating the expression of CXCL1/KC in skin cells such as keratinocytes and fibroblasts.

Signal transducer and activator of transcription 3 upregulates interleukin-8 expression at the level of transcription in human melanoma cells

Please cite this paper as: Signal transducer and activator of transcription 3 upregulates interleukin‐8 expression at the level of transcription in human melanoma cells. Experimental Dermatology 2010; 19: e50–e55.

Three Cases of Linear IgA/IgG Bullous Dermatosis Showing IgA and IgG Reactivity With Multiple Antigens, Particularly Laminin-332

IMPORTANCE Linear IgA/IgG bullous dermatosis (LAGBD) is a relatively rare autoimmune bullous disease characterized by both IgA and IgG antibodies to epidermal basement membrane zone. The heterogeneity and pathogenesis of the LAGBD autoantigens have not been fully elucidated. OBSERVATIONS We report 3 Japanese cases of LAGBD (ages 81, 88, and 64 years; 1 woman and 2 men). The patients showed bullous and erosive lesions on the trunk and extremities with minimal mucosal lesions. Histopathological analysis revealed a subepidermal blister with neutrophilic infiltration with eosinophils in 2 cases. Direct and indirect immunofluorescence studies disclosed IgG and IgA antibasement membrane zone antibodies. In immunoblot analyses of various antigen sources, all cases showed IgG and IgA antibodies to various subunits of laminin-332, in addition to IgG and IgA reactivity with type VII collagen, laminin-γ1, and BP230 and BP180 recombinant proteins. CONCLUSIONS AND RELEVANCE Our studies revealed that the 3 LAGBD cases showed prominent IgG and IgA reactivity with laminin-332, which was only rarely reported. In addition, all cases showed IgG and IgA reactivity with other multiple antigens, indicating the role of epitope-spreading mechanisms initiated from laminin-332. The significance of IgA antibodies to laminin-332 should be studied in larger cohorts of both LAGBD and linear IgA bullous dermatosis.

12-O-Tetradecanoylphorbol-13-acetate Inhibits Melanoma Growth by Inactivation of STAT3 through Protein Kinase C-activated Tyrosine Phosphatase(s)

The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCα, PKCδ, and PKCϵ. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).

High expression of Mcl-1L via the MEK-ERK-phospho-STAT3 (Ser727) pathway protects melanocytes and melanoma from UVB-induced apoptosis.

Ultraviolet (UV) B is a major factor in melanomagenesis. This fact is linked to the resistance of melanocytes to UVB‐induced apoptosis. In this study, we characterized the involvement of Mcl‐1L in the regulation of UVB‐induced apoptosis in melanocytes and in melanoma cells. In melanocytes, apoptosis was not evident at 24 h after UVB irradiation. The Mcl‐1L expression increased after UVB irradiation, and the high Mcl‐1L expression continued for at least 24 h. This UVB‐dependent increase in Mcl‐1L was mediated by the MEK‐ERK‐pS‐STAT3 (STAT3 phosphorylated at Ser727) pathway. The Ser727 phosphorylation facilitated nuclear localization of STAT3. In melanoma cells, the expression levels of Mcl‐1L varied depending on the cell line. WM39 melanoma cells expressed high levels of Mcl‐1L via the MEK‐ERK‐pS‐STAT3 pathway and were resistant to UVB‐induced apoptosis without up‐regulation of Mcl‐1L. In melanocytes and in WM39 cells, transfection with Mcl‐1 siRNA promoted UVB‐induced apoptosis. Immunohistochemical studies showed that melanoma cells in in situ lesions expressed high amounts of Mcl‐1L. These results indicate that the high expression of Mcl‐1L mediated by the MEK‐ERK‐pS‐STAT3 pathway protects melanocytes and melanoma cells from UVB‐induced apoptosis.

Peristomal skin ulcer with intestinal metaplasia.

tations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern. Proc Natl Acad Sci USA 2007; 104:13450–4. 9 Bourdeaut F, Herault A, Gentien D et al. Mosaicism for oncogenic G12D KRAS mutation associated with epidermal nevus, polycystic kidneys and rhabdomyosarcoma. J Med Genet 2010; 47:859–62. 10 Hafner C, Toll A, Real FX. HRAS mutation mosaicism causing urothelial cancer and epidermal nevus. N Engl J Med 2011; 365:1940–2.

Malignant melanoma with bone marrow involvement diagnosed from hypercalcemia: Development of a neural cell adhesion molecule stain.

Dear Editor, Hypercalcemia is a life-threatening complication in patients with cancer, but rarely in patients with malignant melanoma. We report a case of a malignant melanoma associated with bone marrow involvement, which was referred to us because of hypercalcemia. Neural cell adhesion molecule (NCAM) is a protein expressed in malignant melanoma. Some reports suggested that NCAM potentiates cellular invasion and metastasis of melanoma cells, although its function in melanoma progression remains unclear. In this case, immunohistochemical observation revealed dynamic changes in NCAM expression with strong expression in bone marrow metastases. A 29-year-old man presented with back pain. He had no relevant medical history. A physical skin examination revealed a black nodule on his right abdomen that had persisted for 1 year (Fig. 1a). Laboratory findings revealed mild leukocytosis, lactate dehydrogenase level of 357 IU/L (normal, 115–217), calcium level of 4.01 mmol/L (normal, 2.2–2.5) and phosphorus level of 0.326 mmol/L (normal, 0.77–1.5). His parathyroid hormone (PTH) level was in the low-normal range at 6 ng/L (normal, 15–65); his PTH-related peptide (PTH-rP) level was lower than normal. Other hormone levels were normal. Computed tomography showed multiple spinal fractures. Magnetic resonance imaging showed osteolysis with non-traumatic

Mucosal lichen sclerosus/lichen planus overlap syndrome with cutaneous lesions of lichen sclerosus

In 1994, Marren et al. proposed the new concept of mucosal lichen sclerosus (LS)/lichen planus (LP) syndrome, referring to the simultaneous presence of vulvar LS and oral lichenoid lesions [1]. The two important points of the concept of this syndrome are that: 1) the oral lichenoid lesions should be regarded as LS, or at least a distinct group, when associated with vulvar LS: and 2) that careful attention to malignant transformation is required not only for the vulvar LS but also for the oral LP. [...]