Kanae Kitatani

Notch signaling may be involved in the abnormal differentiation of epidermal keratinocytes in psoriasis.

Localization of each keratin isoform differs among epidermal layers. Proliferating basal cells synthesize keratin 14 (K14) and suprabasal cells express keratin 10 (K10) in normal skin. Notch signaling is essential for keratinocyte differentiation. Notch1 is expressed in all epidermal layers, Notch2 in the basal cell layer and Notch3 in basal cell and spinous cell layers in normal epidermis. It has been poorly elucidated how localization and expression levels of Notch molecules are related to epidermal molecular markers K10 and K14 in psoriatic skin with abnormal differentiation of epidermal tissue. This study aimed to investigate the relationship between abnormal differentiation of epidermal cells in psoriatic skin and expression of Notch molecules. We investigated keratins (K14 and K10) and Notches (1, 2, 3 and 4) using immunohistochemistry in psoriatic skin (n=30) and normal skin (n=10). In normal skin, K14 and K10 were discretely observed in the basal cell layer and suprabasal layer, respectively. In psoriatic skin, K14 was expressed in the pan epidermal layer while it and K10 were co-expressed in some middle suprabasal layer cells. Notch1, 2, 3, and 4 localized in all epidermal layers in normal skin. In psoriatic skin, Notch1, 2, and 4 mainly localized in suprabasilar layers and Notch3 is lacalized in pan epidermal, suprabasilar, and basilar layers. Protein and mRNA of Notch1, 2, and 3 isoforms decreased in psoriatic epidermis compared with normal epidermis. These data suggest that decrements in these Notch molecules might cause aberrant expression of K10 and K14 leading to anomalous differentiation of the epidermis in psoriatic lesions.

An Up-to-Date Anti-Cancer Treatment Strategy Focusing on HIF-1α Suppression: Its Application for Refractory Ovarian Cancer

Hypoxia inducible factor-1α (HIF-1α) predominantly determines the transcriptional activity of HIF-1, which induces the certain genetic expressions to participate in the proliferation and progression of the tumor. It is supposed that HIF-1α is also an extremely important factor in cancer treatment. Based on the results of our recent analyses using ovarian tumors, which indicated the close association of HIF-1α expression with the acquisition of malignancy and the characterization of histology, we further investigated the possibility of a new strategy of cancer therapy that targeted HIF-1α inhibition in the ovarian carcinoma. The cell line HUOCA-II, which originates from the refractory ovarian clear cell adenocarcinoma, was treated with rapamycin. The inhibitory effect of HIF-1α was analyzed by immunohistochemistry and western blotting. It was demonstrated that inhibition of HIF-1α and vascular endothelial growth factor (VEGF) expressions would lead to the down-regulation of tumor cell proliferation. Interestingly, there was little or no change in GLUT-1 expression by rapamycin administration. Thus, the inhibition of GLUT-1 may also be a key for the new strategy of cancer therapy as well as HIF-1α and VEGF.

Aberrant Activation of Atypical Protein Kinase C in Carbon Tetrachloride–Induced Oxidative Stress Provokes a Disturbance of Cell Polarity and Sealing of Bile Canalicular Lumen

Polarized hepatocytes contain tight junctions (TJs), which are among the most important junctions for sealing the bile canalicular lumen from the sinusoidal space. Alterations in TJs are implicated in chronic cholestatic liver diseases, such as primary biliary cirrhosis and primary sclerosing cholangitis, which have lipid peroxidation marker elevations or antioxidant vitamin decreases. However, the effect of oxidative stress on hepatocyte polarity or liver morphology is unknown. We found that carbon tetrachloride (CCl4)-induced oxidative stress resulted in disassembly of TJs. Ultrastructural analysis revealed disruption in TJs, Golgi morphology, and expansion of the bile canalicular lumen size in CCl4-treated hepatocytes. The Par complex [Par-3-atypical protein kinase C (aPKC) and Par-6 ternary complex] regulates TJs and lumen formation, and the Par-3-aPKC complex formation was inhibited by CCl4 treatment. Moreover, the antioxidant compound vitamin E prohibited a CCl4-induced disturbance in TJs and Par-3-aPKC complex formation. aPKC phosphorylates Par-3 and down-regulates its own affinity with Par-3. Importantly, aPKC kinase activity and Par-3 phosphorylation were significantly increased in CCl4-treated rat livers. These results indicate that the Par-3-aPKC complex plays a crucial role in the maintenance of hepatocyte polarity and sealing of the bile canalicular lumen. Our findings suggest that bile canalicular lumen expansion might explain the presence of cholestasis in patients with primary biliary cirrhosis and primary sclerosing cholangitis.

Roles of Oxidized Diacylglycerol for Carbon Tetrachloride-induced Liver Injury and Fibrosis in Mouse.

Since there is a report that an inhibitor of protein kinase C (PKC) effectively suppresses the development of hepatic fibrosis, it is suggested that the PKC signaling pathway plays an important role in the pathogenesis of hepatic fibrosis. We reported that oxidized diacylglycerol (DAG), which is an activator of PKC, had a remarkably stronger PKC-activating action than un-oxidized DAG. In the present study, we explored the roles of oxidized DAG in hepatic fibrogenesis using mice, the livers of which developed fibrosis by long-term administration of carbon tetrachloride (CCl4). Liver fibrosis models were created by 4- or 8-week repetitive subcutaneous injections of CCl4 to the backs of C57BL/6J mice. The amount of oxidized DAG was significantly increased in the CCl4-treated group. Moreover, it was found that PKCα, βI, βII and δ were activated. In the CCl4-treated group, phosphorylation of ERK and JNK, which are downstream signal transmitters in the PKC pathway, was increased. It was also found in this group that there was an increase in TIMP-1, which is a fibrogenesis-promoting factor whose expression is enhanced by activated JNK, and of TNF-α, an inflammatory cytokine. Analysis by quantitative real-time RT-PCR showed that expressions of αSMA, collagen I, TNF-α and IL-10 were remarkably increased in the 8-week CCl4-treated group. The above results strongly suggested that oxidized DAG, which is increased by augmented oxidative stress, activated PKCα, βI, βII and δ molecular species and that these molecular species in turn stimulated the phosphorylation of MAP kinases including ERK and JNK, resulting in enhancement of hepatic fibrogenesis.

A Histopathological Study of Multi-hormone Producing Proliferative Lesions in Estrogen-induced Rat Pituitary Prolactinoma

Rats with estrogen-induced prolactin-producing pituitary adenoma (E2-PRLoma) have been employed as an animal model of human PRL-producing pituitary adenoma in a large number of studies. Presently, we found that long-term administration of estrogen to SD rats resulted in the development of E2-PRLomas, some of which included multi-hormone producing nodules. We herein report results of histopathological analyses of these lesions. PRLoma models were created in female SD rats by 22 weeks or longer administration of a controlled-release preparation of estradiol at a dose of 10 mg/kg/2 weeks. Ten of the 11 PRLoma model rats had proliferative nodular lesions composed of large eosinophilic cells like gonadotrophs inside the PRLoma. These lesions were positive for PRL, TSHβ, and α subunits and were negative for GH, LHβ, ACTH, and S-100. Double immunostaining revealed that these large eosinophilic cells showed coexpression of PRL and TSHβ, PRL and α subunits, and TSHβ and α subunits. Those results clarified that long-term estrogen administration to female SD rats induced multi-hormone producing neoplastic pituitary nodules that expressed PRL, TSHβ, and α subunits. We studied these neoplastic nodules obtained by laser microdissection to acquire findings similar to those of the immunohistochemical analysis. We consider that this animal model is useful for pathogenesis analyses and therapeutic agent development concerning human multi-hormone producing pituitary adenomas.

Stimulation of melanogenesis by nordihydroguaiaretic Acid in human melanoma cells.

Nordihydroguaiaretic acid (NDGA), a lignan found in vegetables, fruits and legumin, has been shown to possess antineoplastic, antiviral and antioxidant characteristics. In this study, we examined the effect of NDGA on melanogenesis in human melanoma cells (HMVII). In vitro, NDGA does not alter mushroom tyrosinase activity. However, in NDGA-treated HMVII cells, cellular tyrosinase activity increased in both a time- and dose-dependent manner. The concomitant increases in melanin content in NDGA-treated cells indicated an elevation of melanin synthesis by tyrosinase activation. In addition, after a 7-day incubation, melanin content in 20 μM NDGA-treated cells increased 5.02 fold. Tyrosinase protein also increased by treatment with NDGA. Nevertheless, tyrosinase mRNA was not altered in NDGA-treated cells. Our results suggest that NDGA can increase tyrosinase activity and de novo synthesis of melanin in human melanoma cells. We found that NDGA is a novel potent stimulator of melanogenesis in human melanoma cells.