Eriko Aizu-Yokota

FAK Is the Upstream Signal Protein of the Phosphatidylinositol 3-Kinase-Akt Survival Pathway in Hydrogen Peroxide-induced Apoptosis of a Human Glioblastoma Cell Line

Protein phosphorylation in a human glioblastoma cell line, T98G, was examined after exposure to oxidative stressin vitro. Hydrogen peroxide (1 mm) markedly induced tyrosine phosphorylation of focal adhesion kinase (FAK) and serine phosphorylation of Akt at 1 h after stimulation. Concommitantly, the association of FAK with phosphatidylinositide 3′-OH-kinase (PI 3-kinase) was also observed by the hydrogen peroxide stimulation. When T98G cells were incubated with wortmannin, a PI 3-kinase inhibitor, both PI 3-kinase activity and phosphorylation of Akt were inhibited, whereas apoptosis by oxidative stress was accelerated. Concomitant with apoptosis, elevated level of CPP32 protease activity (caspase-3) was observed, with decreases in Bcl-2 protein and increases in Bax protein. These results suggested that in the signal transduction pathway from FAK to PI 3-kinase, Akt promotes survival. Thus, it became apparent that FAK is the upstream signal protein of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis in T98G cells.

Glycyrrhizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line

Abstract Glycyrrhizin (GL) is known to have various immunomodulating activities and has long been used clinically as an anti-allergic and anti-hepatitis agent. While the potency of GL against lung inflammatory diseases has been expected, the effect of GL on the lung has been poorly understood. Lung fibroblasts are known as a potent producer of inflammatory chemokines, IL-8 and eotaxin 1, by which neutrophils and eosinophils are strongly attracted during inflammation. Therefore, we studied the effects of GL on the production of these chemokines using a human fetal lung fibroblast cell line, HFL-1, stimulated with TNF-α and IL-4. Moreover, we examined the structure–activity relationships of GL to explore more beneficial compounds. 18α,β-GL inhibited IL-8 dose-dependently and inhibited eotaxin 1 slightly. 18α,β-Glycyrrhetic acid (GA) did not inhibit IL-8 but inhibited eotaxin 1. The effect of 18α,β-glycyrrhetic acid monoglucuronide (MGA) resembled that of 18α,β-GL but was weaker. Both 3β-[(2-O-β-d-glucopyranuronosyl-β-d-glucopyranuronosyl)oxy]-18β-11-deoxo-olean-12-en-30-oic acid (11-deoxo-GL) and 3β-[(2-O-β-d-glucopyranuronosyl-β-d-glucopyranuronosyl)oxy]-olean-11,13,(18)-dien-30-oic acid (hetero-GL) exhibited inhibitory activity with significant cytotoxicity. 3β-[(2-O-β-d-Glucopyranuronosyl-β-d-glucopyranuronosyl)oxy]-18β-olean-9,12-dien-30-oic acid (homo-GL) did not have cytotoxicity but its activity was mild like that of 18α,β-GL. 3β-[(2-O-β-d-Glucopyranuronosyl-β-d-glucopyranuronosyl)oxy]-olean-11,13(18)-dien-30-ol (hetero-30-OH-GL) and 3β-[(2-O-β-d-glucopyranuronosyl-β-d-glucopyranuronosyl)oxy]-18β-olean-9,12-dien-30-ol (homo-30-OH-GL) showed potent inhibitory effects, at concentrations lower than 18α,β-GL with no significant cytotoxicity. These results suggest that GL-related compounds are effective in reducing chemokine production and that GL-modified compounds including hetero-30-OH-GL and homo-30-OH-GL appear most beneficial in view of their inhibitory capacity with less cytotoxicity.

FAK overexpression upregulates cyclin D3 and enhances cell proliferation via the PKC and PI3-kinase-Akt pathways.

We previously demonstrated that FAK-transfected HL-60 (HL-60/FAK) cells exhibit anti-apoptotic capacity. Here, we report that HL-60/FAK cells proliferate much faster than vector-transfected control (HL-60/Vect) cells with a 1.5-fold faster doubling time. This observation prompted us to investigate the mechanism of how HL-60/FAK cells augment cell proliferation. Since a protein kinase C (PKC) inhibitor, chelerythrine, or a PI3-kinase inhibitor, LY294002, suppressed cell proliferation effectively, both PKC and PI-3-kinase pathways are presumed to be involved in the cell proliferation. Among cyclins and CDKs, cyclin D3 expression was particularly prominent in the HL-60/FAK cells. Among PKC family, particularly PKCalpha, beta and eta isoforms were activated and directly associated with FAK in HL-60/FAK cells. We assumed that FAK activates PKC and PI3-kinase-Akt pathway, which resulted in marked induction of cyclin D3 expression and CDK activity.

Dermatophagoides farinae extract induces severe atopic dermatitis in NC/Nga mice, which is effectively suppressed by the administration of tacrolimus ointment.

Atopic dermatitis (AD) is a chronic inflammatory skin disease, which is accompanied by marked increases in the levels of inflammatory cells, including mast cells and eosinophils as well as T cells and macrophages. To investigate the expression pattern of chemokines in AD, a house dust mite, Dermatophagoides farinae extracts (DfE)-induced NC/Nga AD model was developed in mice, and this model was used to determine the expression levels of chemokines in atopic lesions using DNA microarrays and RT-PCR. When NC/Nga mice were repeatedly treated with DfE for 4 to 7 weeks on the back skin, the mRNA expression levels of CCL20/LARC, CCL24/eotaxin-2, CCL17/TARC, and CCL11/eotaxin-1 were markedly induced and lesser of CCL2/MCP-1, within the inflammatory lesion of the back skin. Immunohistochemical staining revealed the expression of these chemokines in the epidermis and dermis of DfE-treated NC/Nga mice. Interestingly, repeated application of tacrolimus ointment potently inhibited DfE-induced atopic dermatitis in NC/Nga mice concomitant with the inhibition of these changes in chemokine gene and protein expression levels particularly of CCL20/LARC, CCL17/TARC, and CCL11/eotaxin-1. These data indicate that severe atopic dermatitis induced by DfE accompanies elevated chemokine levels, and it was proposed that tacrolimus ointment is beneficial for the treatment of severe AD.

Licochalcones suppress degranulation by decreasing the intracellular Ca2+ level and tyrosine phosphorylation of ERK in RBL-2H3 cells

Mast cells play a key role in allergic inflammation by releasing various mediators, such as histamine, serotonin, leukotrienes and cytokines. A signaling cascade of events activated by stimulation with antigens contributes to the regulation of mast cell degranulation. While various anti-inflammatory and anti-allergic drugs have been developed that inhibit degranulation of mast cells, the inhibitory mechanism has been poorly understood. Licochalcone A (Lico A) is a retrochalcone isolated from the root of Xinjiang liquorice and has been reported to exhibit various biological activities such as anti-inflammatory activity. We examined the effects of Lico A and related chalcones on degranulation in a rat basophilic leukemia cell line, RBL-2H3. Whereas Lico A and licochalcone C (Lico C) exhibited inhibitory activity with cytotoxicity, licochalcone D (Lico D) significantly inhibited the degranulation in RBL-2H3 cells with low cytotoxicity. Moreover, Lico D significantly inhibited the Ca2+ influx and phosphorylation of extracellular signal regulated kinase (ERK) and MEK. These results suggest that Lico D inhibits mast cell degranulation via the inhibition of both extracellular Ca2+ influx and activation of the MEK-ERK pathway.

The polycythemia vera-associated Jak2 V617F mutant induces tumorigenesis in nude mice

The somatic Jak2 mutation (V617F) was identified in most patients with polycythemia vera (PV). Here, we show that the activating Jak2 V617F mutant completely protected Ba/F3 cells from cytokine withdrawal-induced apoptotic cell death. Interestingly, Ba/F3 cells expressing Jak2 V617F mutant induced rapid tumorigenesis in nude mice, leading to rapid death. Whereas an injection of Ba/F3 cells expressing wild-type Jak2 had no effect, an injection of Ba/F3 cells expressing Jak2 V617F mutant promptly invaded and spread into various distinct organs, such as the liver and spleen. Strikingly, Jak2 inhibitor, AG490 potently inhibited cytokine-independent cell growth induced by the Jak2 V617F mutant. Also, treatment with AG490 effectively delayed Jak2 V617F mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo suggest that Jak2 harboring V617F mutation is a potent oncogene able to promote cell transformation and tumorigenesis.

The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice

JAK2 plays important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Recently the L611S point mutation in JAK2 has been identified in a child with acute lymphoblastic leukemia. Here we analyzed the mechanism by which JAK2 exhibits its oncogenicity. In BaF3 murine hematopoietic cells, L611S mutant increased the expression of antiapoptotic proteins including X chromosome-linked inhibitor of apoptosis protein, inhibitor of apoptosis protein, and Bcl-XL. We also showed that JAK2 L611S mutant protects BaF3 cells from cytokine withdrawal-induced apoptotic cell death and leads to cytokine-independent cell growth. Furthermore BaF3 cells expressing JAK2 L611S mutant gained the ability to induce tumorigenesis in nude mice. The L611S mutant also exhibited malignancy, including prompt invasion and spreading into various organs, leading to rapid lethality of the mice. Finally we showed that a specific JAK2 inhibitor, AG490, potently inhibited cytokine-independent cell growth induced by JAK2 L611S mutant via the induction of apoptotic cell death. In addition, treatment with AG490 significantly inhibited the JAK2 L611S mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo strongly suggest that L611S mutant of JAK2 harbors potent oncogenic activity, and this probably requires the antiapoptotic signaling pathway.