Hironori Edamatsu

Crucial Role of Phospholipase Cε in Chemical Carcinogen-Induced Skin Tumor Development

Mutational activation of the ras proto-oncogenes is frequently found in skin cancers. However, the nature of downstream signaling pathways from Ras involved in skin carcinogenesis remains poorly understood. Recently, we and others identified phospholipase C (PLC) ε as an effector of Ras. Here we have examined the role of PLCε in de novo skin chemical carcinogenesis by using mice whose PLCε is genetically inactivated. PLCε−/− mice exhibit delayed onset and markedly reduced incidence of skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the papillomas formed in PLCε−/− mice fail to undergo malignant progression into carcinomas, in contrast to a malignant conversion rate of approximately 20% observed with papillomas in PLCε+/+ mice. In all of the tumors analyzed, the Ha-ras gene is mutationally activated irrespective of the PLCε background. The skin of PLCε−/− mice fails to exhibit basal layer cell proliferation and epidermal hyperplasia in response to TPA treatment. These results indicate a crucial role of PLCε in ras oncogene-induced de novo carcinogenesis and downstream signaling from TPA, introducing PLCε as a candidate molecular target for the development of anticancer drugs.

Differential roles of Ras and Rap1 in growth factor-dependent activation of phospholipase Cε

Phospholipase Cε is a phosphoinositide-specific phospholipase C that selectively associates with Ras and Rap small GTPases as a target. Here we explored the molecular basis of the Rap1- as well as Ras-mediated regulation of phospholipase Cε upon platelet-derived growth factor stimulation by using a receptor mutant deficient in its ability to phosphorylate and activate phospholipase Cγ. Following platelet-derived growth factor treatment, this receptor induces persistent activation of ectopically expressed PLCε through activation of Ras and Rap1. The rapid and initial phase of the activation is mediated by Ras, whereas Rap1 is responsible for the prolonged activation. We further demonstrate that the CDC25 homology domain, which exhibits guanine nucleotide exchange factor activity toward Rap1, but not Ras, is critical for the prolonged activation of phospholipase Cε. Platelet-derived growth factor prevented the hematopoietic BaF3 cells containing the mutant receptor from undergoing apoptosis, and enabled these cells to proliferate, only when phospholipase Cε was expressed. Therefore, the phospholipase C signal is suggested to be critical for survival and growth of BaF3 cells.

Phospholipase Cε promotes intestinal tumorigenesis of ApcMin/+ mice through augmentation of inflammation and angiogenesis

Apc(Min/+) mice, carrying an inactivated allele of the adenomatous polyposis coli gene, are widely used as an animal model for human colorectal tumorigenesis, where tumor environment, such as inflammation, is known to play a critical role in tumor progression. We previously demonstrated that phospholipase C (PLC)epsilon, an effector of Ras and Rap small GTPases, plays a crucial role in two-stage skin chemical carcinogenesis using 12-O-tetradecanoyl-phorbor-13-acetate (TPA) as a promoter through augmentation of TPA-induced inflammation. Here, we show that Apc(Min/+) mice lacking PLCepsilon (PLCepsilon(-/-)) exhibit marked resistance to spontaneous intestinal tumorigenesis compared with those with the PLCepsilon(+/+) background. Time course of the development of tumors, which are histopathologically classified into low- and high-grade adenomas with increasing dysplasia and size, and adenocarcinomas indicates that not only the low-grade adenoma formation but also the progression to high-grade adenoma are suppressed in PLCepsilon(-/-);Apc(Min/+) mice. Low-grade adenomas of PLCepsilon(-/-);Apc(Min/+) mice exhibit accelerated apoptosis and reduced cellular proliferation. They also show marked attenuation of tumor angiogenesis and reduction in expression of vascular endothelial growth factor. In contrast, high-grade adenomas of PLCepsilon(-/-);Apc(Min/+) mice exhibit marked attenuation of tumor-associated inflammation without significant differences in apoptosis and proliferation. These results suggest that PLCepsilon plays crucial roles in intestinal tumorigenesis through two distinct mechanisms, augmentation of angiogenesis and inflammation, depending on the tumor stage.

Crucial Role of Phospholipase CE in Skin Inflammation Induced by Tumor-Promoting Phorbol Ester

In two-stage skin chemical carcinogenesis, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) acts as a promoter essential for clonal expansion of the initiated cells carrying the activated ras oncogenes. Although protein kinase C (PKC) isozymes are the main targets of TPA, their role in tumor promotion remains controversial. We previously reported that mice lacking a Ras/Rap effector phospholipase C epsilon (PLC epsilon(-/-) mice) exhibited marked resistance to tumor formation in the two-stage skin carcinogenesis. PLC epsilon(-/-) mice also failed to exhibit basal layer cell proliferation and epidermal hyperplasia induced by TPA, suggesting a role of PLC epsilon in tumor promotion. Here, we show that PLC epsilon(-/-) mice exhibit resistance to TPA-induced skin inflammation as assessed by reduction in edema, granulocyte infiltration, and expression of a proinflammatory cytokine, interleukin-1 alpha (IL-1 alpha). On the other hand, the proliferative potentials of keratinocytes or dermal fibroblasts in culture remain unaffected by the PLC epsilon background, suggesting that the PLC epsilons role in tumor promotion may be ascribed to augmentation of inflammatory responses. In dermal fibroblast primary culture, TPA can induce activation of the PLC epsilon lipase activity, which leads to the induction of IL-1 alpha expression. Experiments using small interfering RNA-mediated knockdown indicate that this activation is mediated by Rap1, which is activated by a TPA-responsive guanine nucleotide exchange factor RasGRP3. Moreover, TPA-induced activation of Rap1 and PLC epsilon is inhibited by a PKC inhibitor GF109203X, indicating a crucial role of PKC in signaling from TPA to PLC epsilon. These results imply that two TPA targets, RasGRP3 and PKC, are involved in TPA-induced inflammation through PLC epsilon activation, leading to tumor promotion.

Congenital Semilunar Valvulogenesis Defect in Mice Deficient in Phospholipase Cε

ABSTRACT Phospholipase Cε is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cε is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cε. These results suggest a crucial role of phospholipase Cε downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.

Neuronal lineage‐specific induction of phospholipase Cε expression in the developing mouse brain

Phospholipase C is a key enzyme of intracellular signal transduction in the central nervous system. We and others recently discovered a novel class of phospholipase C, phospholipase Cε, which is regulated by Ras and Rap small GTPases. As a first step toward analysis of its function, we have examined the spatial and temporal expression patterns of phospholipase Cε during mouse development by in situ hybridization and immunohistochemistry. Around embryonic day 10.5, abundant expression of phospholipase Cε is observed specifically in the outermost layer of the neural tube. On embryonic day 12 and later, it is observed mainly in the marginal zone of developing brain and spinal cord as well as in other regions undergoing neuronal differentiation, such as the retina and olfactory epithelium. The phospholipase Cε‐expressing cells almost invariably express microtubule‐associated protein 2, but hardly express nestin or glial fibrillary acidic protein, indicating that the expression of phospholipase Cε is induced specifically in cells committed to the neuronal lineage. The expression of phospholipase Cε persists in the terminally differentiated neurons and exhibits no regional specificity. Further, an in vitro culture system of neuroepithelial stem cells is employed to show that abundant expression of phospholipase Cε occurs in parallel with the loss of nestin expression as well as with the induction of microtubule‐associated protein 2 expression and neuronal morphology. Also, glial fibrillary acidic protein‐positive glial lineage cells do not exhibit the high phospholipase Cε expression. These results suggest that the induction of phospholipase Cε expression may be a specific event associated with the commitment of the neural precursor cells to the neuronal lineage.

Crucial Role of Phospholipase Cε in Induction of Local Skin Inflammatory Reactions in the Elicitation Stage of Allergic Contact Hypersensitivity

Phospholipase Cε (PLCε) is an effector of Ras/Rap small GTPases. We previously demonstrated that PLCε plays a crucial role in development of phorbor ester-induced skin inflammation, which is intimately involved in the promotion of skin carcinogenesis. In this study, we have examined its role in local skin inflammatory reactions during development of contact hypersensitivity toward a hapten 2,4-dinitrofluorobenzene (DNFB). PLCε+/+ and PLCε−/− mice were sensitized with DNFB, followed by a DNFB challenge on the ears. PLCε−/− mice exhibited substantially attenuated inflammatory reactions compared with PLCε+/+ mice as shown by suppression of ear swelling, neutrophil infiltration, and proinflammatory cytokine production. In contrast, the extent and kinetics of CD4+ T cell infiltration showed no difference depending on the PLCε background. Adoptive transfer of CD4+ T cells from the sensitized mice to naive mice between PLCε+/+ and PLCε−/− backgrounds indicated that PLCε exerts its function in cells other than CD4+ T cells, presumably fibroblasts or keratinocytes of the skin, to augment inflammatory reactions during the elicitation stage of contact hypersensitivity. Moreover, dermal fibroblasts and epidermal keratinocytes cultured from the skin expressed proinflammatory cytokines in a PLCε-dependent manner on stimulation with T cell-derived cytokines such as IL-17, IFN-γ, TNF-α, and IL-4. These results indicate that PLCε plays a crucial role in induction of proinflammatory cytokine expression in fibroblasts and keratinocytes at the challenged sites, where infiltrated CD4+ T cells produce their intrinsic cytokines, thereby augmenting the local inflammatory reactions.

p53 transactivation is involved in the antiproliferative activity of the putative tumor suppressor RBM5

RBM5 (RNA‐binding motif protein 5) is a nuclear RNA binding protein containing 2 RNA recognition motifs. The RBM5 gene is located at the tumor suppressor locus 3p21.3. Deletion of this locus is the most frequent genetic alteration in lung cancer, but is also found in other human cancers. RBM5 is known to induce apoptosis and cell cycle arrest but the molecular mechanisms of RBM5 function are poorly understood. Here, we show that RBM5 is important for the activity of the tumor suppressor protein p53. Overexpression of RBM5 enhanced p53‐mediated inhibition of cell growth and colony formation. Expression of RBM5 augmented p53 transcriptional activity in reporter gene assays and resulted in increased mRNA and protein levels for endogenous p53 target genes. In contrast, shRNA‐mediated knockdown of endogenous RBM5 led to decreased p53 transcriptional activity and reduced levels of mRNA and protein for endogenous p53 target genes. RBM5 affected protein, but not mRNA, levels of endogenous p53 after DNA damage suggest that RBM5 contributes to p53 activity through post‐transcriptional mechanisms. Our results show that RBM5 contributes to p53 transcriptional activity after DNA damage and that growth suppression and apoptosis mediated by RBM5 are linked to activity of the tumor suppressor protein p53.

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.