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1.
Odontology ; 111(3): 565-572, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36396928

RESUMO

In the present study, expression and localization of PLD2 were examined in mouse submandibular glands in situ in comparison with those of PLD1 previously reported by the present authors. In immunoblots, PLD2-expression was high at postnatal 0 week (P0W) and P2W, and at P4W decreased considerably with the decrease of the male gland more marked, and at P8W it was undetectable in the male gland, but remained faint in the female. In the male gland at P8W after castration at P6W, PLD2-expression reappeared faintly. In the female glands with daily injections of testosterone started at P6W, at P8W the expression was undetectable. In immunohistochemistry, PLD2 was localized throughout immature duct epithelia without distinct regional differences in its intensity at P0W and P2W. PLD2-localization was weak and diffuse in granular convoluted tubules at P4W and negligible there at P8W in the female gland, while it was at negligible levels in the tubules at P4W and at undetectable levels there at P8W in the male gland. The expression changes detected in immunoblots after castration or testosterone injection were duplicated in granular convoluted tubules in immunohistochemistry. The present findings suggest that PLD2 is dominantly involved in proliferation/differentiation/apoptosis of most immature ductal cells at early postnatal and at least perinatal stages, different from PLD1. This also indicates that the female-dominant sexual dimorphism of PLD2-expression occurs in synchrony of differentiation of granular convoluted tubules at puberty and at young adulthood, similar to PLD1 though at much lower levels.


Assuntos
Fosfolipase D , Camundongos , Animais , Masculino , Feminino , Fosfolipase D/metabolismo , Glândula Submandibular/metabolismo , Caracteres Sexuais , Testosterona/farmacologia , Testosterona/metabolismo , Diferenciação Celular
2.
J Anat ; 235(6): 1125-1136, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31402458

RESUMO

To clarify the signal transduction mechanism in the differentiation and secretion of salivary glandular cells, the present study was attempted to examine in the submandibular gland (SMG) of mice, the expression and localization of phospholipase D1 (PLD1), one of the important effector molecules working in response to the activation of intramembranous receptors by first messengers. In immunoblotting analysis, the expression of PLD1 was high at postnatal 4 weeks (P4W) and decreased at P8W, and it was at negligible levels at newborn stage (P0W) and postnatal 2 weeks (P2W). The expression of PLD1 was greater in females, and it was suppressed by administration of testosterone to female mice. In immuno-light microscopy, immunoreactivity for PLD1 at P4W was moderate to intense, in the forms of dots and globules mainly in the apical domains of immature granular convoluted tubule (GCT)-cells localized largely in the proximal portion of the female GCT. By P8W, it decreased in intensity and remained weak to moderate along the apical plasmalemma of cells throughout the course of the female GCT, whereas it was faint throughout the GCT of the male SMG at P4W and negligible at P8W. In immuno-electron microscopy, immature GCT-cells characterized by electron-lucent granules were immunoreactive and the immunoreactive materials were deposited close to, but not within, those granules. Typical GCT cells, characterized by electron-dense granules, were immunonegative. No significant immunoreaction for PLD1 was seen in acini of SMGs of either sex at any time point examined. It is suggested that PLD1 is involved in the signaling for secretion of immature GCT cells and influences differentiation of these cells, probably through their own secretory substances.


Assuntos
Fosfolipase D/metabolismo , Glândula Submandibular/metabolismo , Testosterona/farmacologia , Animais , Diferenciação Celular/fisiologia , Feminino , Masculino , Camundongos , Fatores Sexuais , Transdução de Sinais/fisiologia
3.
J Immunol Res ; 2019: 3030268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838224

RESUMO

IFN-γ is detected in chronic lesions of atopic dermatitis (AD); however, its specific role remains to be elucidated. An impaired stratum corneum barrier function is a hallmark of AD, and it is associated with a reduction in ceramides with long-chain fatty acids (FAs) in the stratum corneum. FA elongases, ELOVL1 and ELOVL4, are essential for the synthesis of these ceramides, together with ceramide synthase 3 (CerS3). We have previously shown that IFN-γ, but not other cytokines, induced the downregulation of these enzymes in cultured keratinocytes. Our aim was to investigate the in vivo role of IFN-γ in the lesional skin of AD by analyzing mouse dermatitis models. The local mRNA expression of IFN-γ increased in mite fecal antigen-induced AD-like dermatitis in NC/Nga mice but not in imiquimod-induced psoriasis-like dermatitis in BALB/c mice. The mRNA expression of ELOVL1 and ELOVL4 significantly decreased in AD-like dermatitis, whereas ELOVL1 increased in psoriasis-like dermatitis. The expression of CerS3 increased slightly in AD-like dermatitis, but it increased by 4.6-fold in psoriasis-like dermatitis. Consistently, the relative amount of ceramides with long-chain FAs decreased in AD-like dermatitis but not in psoriasis-like dermatitis. These results suggest that IFN-γ in the lesional skin may reduce ceramides with long-chain FAs by decreasing the expression of ELOVL. Thus, IFN-γ may contribute to the chronicity of AD by impairing barrier function.


Assuntos
Ceramidas/análise , Dermatite Atópica/imunologia , Interferon gama/imunologia , Pele/imunologia , Acetiltransferases/genética , Animais , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Dermatite Atópica/induzido quimicamente , Modelos Animais de Doenças , Proteínas do Olho/genética , Elongases de Ácidos Graxos , Ácidos Graxos/análise , Feminino , Imiquimode , Interferon gama/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Psoríase/induzido quimicamente , Psoríase/imunologia , Pele/patologia , Esfingosina N-Aciltransferase/genética
4.
Mol Med Rep ; 18(6): 5151-5158, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30320355

RESUMO

Vemurafenib, a selective inhibitor of mutated BRAF, is used to treat late­stage melanoma. However, resistance to vemurafenib is urgently required as it can have fatal consequences. Fingolimod (FTY720), a sphingosine­1­phosphate receptor modulator, has been used for the treatment of several malignant neoplasms in clinical trials. The present study investigated the effects of FTY720 and vemurafenib combination treatment on cell death induction, and defined the molecular mechanisms in vemurafenib­resistant melanoma cells. The combination treatment with FTY720 and vemurafenib reduced cell viability, and the expression of apoptosis­associated cleaved poly (adenosine diphosphate­ribose) polymerase (PARP) was increased when compared with treatment with vemurafenib alone in WM­115 cells, a vemurafenib­resistant human melanoma cell line. In addition, the protein expression of phosphorylated extracellular signal­related kinase (ERK) in WM­115 cells was decreased by this combination treatment. Vemurafenib­resistant SK­Mel­28 cells (R­SK­Mel) were established by culturing SK­Mel­28 cells, which are the most sensitive to vemurafenib, in the presence of vemurafenib. Similar to WM­155 cells, the viability of R­SK­Mel cells was reduced and the expression of cleaved PARP was increased by the combination treatment with FTY720 and vemurafenib. In addition, the expression of phosphorylated ERK and Akt was also reduced by this treatment. These results suggested that FTY720 and vemurafenib synergistically induced cell death by downregulating proliferation and survival signalling pathways in vemurafenib­resistant melanoma cells.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Vemurafenib/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Cell Death Dis ; 7(11): e2448, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27809301

RESUMO

Cancer cells utilize flexible metabolic programs to maintain viability and proliferation under stress conditions including nutrient deprivation. Here we report that phospholipase D1 (PLD1) participates in the regulation of metabolic plasticity in cancer cells. PLD1 activity is required for cancer cell survival during prolonged glucose deprivation. Blocking PLD1 sensitizes cancer cells to glycolysis inhibition by 2-deoxy-D-glucose (2-DG) and results in decreased autophagic flux, enlarged lysosomes, and increased lysosomal pH. Mechanistically, PLD1-regulated autophagy hydrolyzes bulk membrane phospholipids to supply fatty acids (FAs) for oxidation in mitochondria. In low glucose cultures, the blockade of fatty acid oxidation (FAO) by PLD1 inhibition suppresses adenosine triphosphate (ATP) production and increases reactive oxygen species (ROS), leading to cancer cell death. In summary, our findings reveal a novel role of PLD1 in sustaining cancer cell survival during metabolic stress, and suggest PLD1 as a potential target for anticancer metabolism therapy.


Assuntos
Autofagia , Ácidos Graxos não Esterificados/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosfolipase D/metabolismo , Estresse Fisiológico , Álcalis/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glucose/deficiência , Glucose/farmacologia , Homeostase/efeitos dos fármacos , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Fosfolipase D/antagonistas & inibidores , Fosfolipídeos/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
6.
Endocrinology ; 157(5): 1839-51, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26943364

RESUMO

Sphingosine 1-phosphate (S1P) is known to regulate insulin resistance in hepatocytes, skeletal muscle cells, and pancreatic ß-cells. Among its 5 cognate receptors (S1pr1-S1pr5), S1P seems to counteract insulin signaling and confer insulin resistance via S1pr2 in these cells. S1P may also regulate insulin resistance in adipocytes, but the S1pr subtype(s) involved remains unknown. Here, we investigated systemic glucose/insulin tolerance and phenotypes of epididymal adipocytes in high-fat diet (HFD)-fed wild-type and S1pr2-deficient (S1pr2(-/-)) mice. Adult S1pr2(-/-) mice displayed smaller body/epididymal fat tissue weights, but the differences became negligible after 4 weeks with HFD. However, HFD-fed S1pr2(-/-) mice displayed better scores in glucose/insulin tolerance tests and had smaller epididymal adipocytes that expressed higher levels of proliferating cell nuclear antigen than wild-type mice. Next, proliferation/differentiation of 3T3-L1 and 3T3-F442A preadipocytes were examined in the presence of various S1pr antagonists: JTE-013 (S1pr2 antagonist), VPC-23019 (S1pr1/S1pr3 antagonist), and CYM-50358 (S1pr4 antagonist). S1P or JTE-013 treatment of 3T3-L1 preadipocytes potently activated their proliferation and Erk phosphorylation, whereas VPC-23019 inhibited both of these processes, and CYM-50358 had no effects. In contrast, S1P or JTE-013 treatment inhibited adipogenic differentiation of 3T3-F442A preadipocytes, whereas VPC-23019 activated it. The small interfering RNA knockdown of S1pr2 promoted proliferation and inhibited differentiation of 3T3-F442A preadipocytes, whereas that of S1pr1 acted oppositely. Moreover, oral JTE-013 administration improved glucose tolerance/insulin sensitivity in ob/ob mice. Taken together, S1pr2 blockade induced proliferation but suppressed differentiation of (pre)adipocytes both in vivo and in vitro, highlighting a novel therapeutic approach for obesity/type 2 diabetes.


Assuntos
Adipócitos/metabolismo , Crescimento Celular/efeitos dos fármacos , Dieta Hiperlipídica , Intolerância à Glucose/metabolismo , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais/genética , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/patologia , Adipogenia/efeitos dos fármacos , Animais , Peso Corporal/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Intolerância à Glucose/genética , Masculino , Camundongos , Camundongos Knockout , Fosfosserina/análogos & derivados , Fosfosserina/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores de Lisoesfingolipídeo/genética , Receptores de Esfingosina-1-Fosfato
8.
Int J Mol Med ; 34(4): 1169-74, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25109763

RESUMO

Sphingosine kinase (SK), a key enzyme in sphingosine-1-phosphate (S1P) synthesis, is known to be overexpressed in various types of cancer cells. The effects of anticancer agents on SK1/S1P signaling have not yet been fully assessed in melanoma cells. In the present study, we investigated the effects of the combination of FTY720, an S1P receptor antagonist, and cisplatin, a DNA-damaging agent, on the induction of the death of human melanoma cells, as well as the molecular mechanisms involved. The viability of various human melanoma cell lines was examined following treatment with anticancer drugs. The cisplatin-resistant SK-Mel-28 and cisplatin-sensitive A375 cell lines were selected for this analysis. Protein expression and apoptotic rates were evaluated by western blot analysis following treatment with cisplatin and/or FTY720. Following treatment with a combination of FTY720 and cisplatin, cell viability significantly decreased and the expression of apoptosis-associated cleaved poly(ADP-ribose) polymerase (PARP) was significantly higher in comparison to treatment with cisplatin alone in the SK-Mel-28 cells. In addition, the combination of FTY720 and cisplatin reduced the protein expression of SK1 and the phosphorylation levels of phosphoinositide 3-kinase (PI3K), Akt and mTOR in the SK-Mel-28 cells; the expression of epidermal growth factor receptor (EGFR) was also markedly reduced. These findings suggest that FTY720 and cisplatin synergistically induce cell death through the downregulation of the PI3K/Akt/mTOR pathway and the decrease in EGFR expression in SK-Mel-28 cells. Thus, the combination of FTY720 and cisplatin may have therapeutic potential for chemotherapy-resistant melanoma, and the effects are likely exerted through the downregulation of S1P signaling.


Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Melanoma/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Cloridrato de Fingolimode , Humanos , Concentração Inibidora 50 , Lisofosfolipídeos , Melanoma/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologia
9.
J Invest Dermatol ; 134(3): 712-718, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24008422

RESUMO

Ceramide (CER) with long-chain fatty acids (FAs) in the human stratum corneum (SC) is important for the skin barrier functions. Changes in the CER profile have been associated with abnormal permeability of dermatoses such as atopic dermatitis (AD) and psoriasis. In addition, interferon-γ (IFN-γ) has been known to be abundant in both AD and psoriatic skin lesions. In this study, we aimed to identify the mechanism underlying the alteration of FA chain length of CERs in these diseases. Mass spectrometry analysis of CERs in the SC showed that the proportion of CERs with long-chain FAs was significantly lower in AD and psoriasis patients than in healthy controls, and this reduction was more pronounced in psoriasis than in AD. Using cultured human keratinocytes and epidermal sheets, we found that only IFN-γ among various cytokines decreased the mRNA expression of elongase of long-chain fatty acids (ELOVL) and ceramide synthase (CerS), enzymes involved in FA chain elongation. Furthermore, quantitative analysis showed that IFN-γ decreased the levels of CERs with long-chain FAs. These results suggest that IFN-γ decreases CERs with long-chain FAs through the downregulation of ELOVL and CerS and that this mechanism may be involved in the CER profile alteration observed in psoriasis and AD.


Assuntos
Ceramidas/metabolismo , Dermatite Atópica/metabolismo , Ácidos Graxos/metabolismo , Interferon gama/metabolismo , Psoríase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Ceramidas/imunologia , Criança , Pré-Escolar , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Células Epidérmicas , Epiderme/imunologia , Epiderme/metabolismo , Ácidos Graxos/imunologia , Feminino , Humanos , Lactente , Interferon gama/imunologia , Queratinócitos/citologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Psoríase/genética , Psoríase/imunologia , Adulto Jovem
10.
Biol Pharm Bull ; 36(7): 1204-7, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23811570

RESUMO

FLJ00018, a heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein (G protein) Gßγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, regulates cellular responses, including cell morphological changes and gene transcriptional regulation, and targets the cellular membranes. FLJ00018 contains a Dbl homology (DH) domain in addition to a pleckstrin homology (PH) domain. Here we show that the PH domain of FLJ00018 is required for FLJ00018-induced, serum response element-dependent gene transcription. Although the PH domain of KIAA1415/P-Rex1, another Gßγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, binds to phosphatidylinositol 3,4,5-triphosphate and phosphatidylinositol 3,4-bisphosphate, the PH domain of FLJ00018 binds to polyphosphoinositides including phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid. These results suggest that FLJ00018 is targeted via its PH domain to cellular membranes.


Assuntos
Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Lipídeos de Membrana/metabolismo , Fracionamento Celular , DNA Complementar/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Luciferases/genética , Plasmídeos , Ligação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho , Elemento de Resposta Sérica/fisiologia , Transcrição Gênica
11.
Anticancer Drugs ; 24(5): 473-83, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23426175

RESUMO

Our previous study has shown that the activity and expression of sphingosine kinase (SPHK) regulated the sensitivity of human colon cancer cells to the chemotherapeutic oxaliplatin (L-OHP). In addition, the cancer stem cell marker CD44 increases cell resistance to anticancer drugs. Here, we use colon cancer cell lines to examine the relationship between SPHK1 activity and CD44 expression.CD44 expression was measured by western blotting and quantitative PCR in two human colon cancer cell lines: L-OHP-resistant RKO and L-OHP-sensitive HCT116. The regulation of CD44 by SPHK1 was examined by either blocking or overexpressing SPHK1 and by using an L-OHP-resistant HCT116 clone (HCT116-R).The levels of SPHK1, CD44, phosphorylated-Akt, and phosphorylated-extracellular signal-regulated kinase (ERK) were much higher in the RKO cells than in the HCT116 cells. The treatment of RKO cells with the SPHK inhibitor or SPHK1 silencing by RNA interference suppressed CD44 protein expression. SPHK1 and CD44 levels were much higher in HCT116-R cells compared with the parental HCT116 cells. Transfection of HCT116 cells with SPHK1 cDNA enhanced the expression of both CD44 and phosphorylated-ERK. The increase in the CD44 protein level was abolished by the inhibition of ERK phosphorylation. Treatment of RKO cells with the sphingosine-1-phosphate (S1P)2 receptor antagonist suppressed ERK phosphorylation and the expression of CD44 mRNA and protein. Exogenous stimulation with S1P increased ERK phosphorylation and CD44 protein expression in HCT116 cells, but treatment with an MEK inhibitor and S1P2 receptor antagonist blocked this effect.These findings indicate that SPHK1 and its product, S1P, contribute toward the regulation of CD44 protein expression through the ERK signaling pathway through S1P2 in human colon cancer cells.


Assuntos
Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores de Hialuronatos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HCT116/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/genética , Lisofosfolipídeos/metabolismo , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Regulação para Cima
12.
J Biochem ; 151(6): 599-610, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22496486

RESUMO

Effects of all-trans retinoic acid (ATRA) on sphingomyelinase expression were examined using MCF-7 (ATRA-sensitive) and MDA-MB-231 (ATRA-resistant) breast cancer cells. Increased NSMase activity, NSMase2 mRNA and protein were observed in ATRA-treated MCF-7 but not in ATRA-treated MDA-MB-231. Increased NSMase2 mRNA of ATRA-treated MCF-7 was mostly due to enhanced transcription. Promoter analysis revealed the important 5'-promoter region of NSMase2 between -148 and -42 bp containing three Sp1 sites but no retinoic acid responsive elements. Experiments using mutated Sp1 sites of the NSMase2 promoter, Mithramycin A (a Sp inhibitor) and Sp family over-expression demonstrated the importance of Sp family protein and the three Sp1 sites for ATRA-induced NSMase2 transcription of MCF-7 cells. Although no quantitative change of bound Sp1 on NSMase2 promoter region after ATRA treatment was detected, Sp1 phosphorylation (activation) by ATRA was observed. Interestingly, PKCδ was involved in ATRA-induced increased NSMase2 transcription. ATRA-induced PKCδ phosphorylation and then activated PKCδ phosphorylated Sp1. Chromatin immunoprecipitation (ChIP) assay showed Sp1, RARα and RXRα complex formation in MCF-7 cells regardless of ATRA treatment and ATRA-induced acetylated histone H3 of the 5'-promoter. Thus, NSMase2 mRNA expression enhanced by ATRA was due to increased transcription via phosphorylated Sp1 caused by PKCδ activation, followed by chromatin remodelling with histone H3 acetylation.


Assuntos
Esfingomielina Fosfodiesterase/genética , Transcrição Gênica , Tretinoína/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Células MCF-7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , Tretinoína/uso terapêutico
13.
J Biochem ; 151(6): 611-20, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22451680

RESUMO

Neutral ceramidase (NCDase) is considered to be a critical enzyme for controlling the turnover of ceramide, an important bioactive lipid, which determines cell's fate. All-trans retinoic acid (ATRA) has been reported to induce neuronal differentiation and cell-cycle arrest [Lopez-Carballo, Moreno, Masia, Perez, and Barettino (Activation of the phosphatidylinositol 3-kinase/Akt signalling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 2002:277:25297-304.)]. In this study, we observed that ATRA-induced cellular ceramide accumulation, cell-growth arrest and differentiation accompanied with down-regulation of NCDase in SH-SY5Y cells, without a decrease in sphingosine or sphingosine 1-phosphate. We examined whether the down-regulation of NCDase was involved in the increase in ceramide and cell differentiation. ATRA was found to down-regulate mRNA, protein and the enzyme activity of NCDase. Interestingly, GATA-2 was also decreased with ATRA treatment, and experiments using its expression vector and siRNA and chromatin immunoprecipitation assay demonstrated GATA-2 acted as transcription-factor of NCDase gene expression. By establishing stable transfectants with decreased NCDase expression and activity, we clarified the significance of NCDase down-regulation for ATRA-induced neuronal differentiation. Those sub-clones showed both increased cellular ceramide and reduced cell growth as well as neuronal differentiation phenotypes. These results demonstrate that down-regulation of NCDase through ATRA-induced GATA-2 decrease plays an important role in induction of ceramide accumulation and neuronal differentiation in SH-SY5Y cells.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Ceramidase Neutra/metabolismo , Tretinoína/farmacologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neurônios/patologia , Ceramidase Neutra/biossíntese , Ceramidase Neutra/genética , Relação Estrutura-Atividade
14.
Chem Res Toxicol ; 24(11): 1845-52, 2011 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-21981408

RESUMO

The antineoplastic efficacy of oxaliplatin, a widely used anticancer drug, is restricted by its adverse effects such as peripheral neuropathy. Infusing a combination of calcium gluconate and magnesium sulfate (Ca/Mg) suppresses the acute neurotoxic side effects of oxaliplatin, although the mechanism is unclear. To elucidate the molecular mechanisms of oxaliplatin-induced neurotoxicity and the effects of Ca/Mg against this toxicity, we examined the effect of Ca/Mg on oxaliplatin-induced inhibition of neurite outgrowth in PC12 cells, a commonly used neuronal cell model. Oxaliplatin and oxalate suppressed nerve growth factor (NGF)-induced neurite outgrowth and reduced the NGF-mediated increase in the intracellular calcium concentration [Ca(2+)](i). A calcium-chelating agent, BAPTA/AM, also exhibited similar inhibitory effects on neurite outgrowth and [Ca(2+)](i). The addition of Ca/Mg attenuated these inhibitions induced by oxaliplatin and oxalate. The NGF-induced upregulation of growth-associated protein-43 (GAP-43) was suppressed by oxaliplatin and oxalate. Oxaliplatin, but not oxalate, suppressed NGF-stimulated extracellular signal-regulated kinase activation, and this inhibition was not affected by Ca/Mg. Ca/Mg did not modify the oxaliplatin-induced loss of cell viability or apoptosis in PC12 or HCT-116 cells, a human colorectal cancer cell line. These results suggest that the inhibition of neurite outgrowth but not tumor cell death induced by oxaliplatin is partly associated with reductions in [Ca(2+)](i) and GAP-43 expression, and this inhibition was suppressed by the addition of Ca/Mg. Therefore, it may be assumed that Ca/Mg is useful for protecting against oxaliplatin-induced neurotoxicity without reducing the antitumor activity of oxaliplatin.


Assuntos
Gluconato de Cálcio/uso terapêutico , Cálcio/metabolismo , Sulfato de Magnésio/uso terapêutico , Neoplasias/metabolismo , Neuritos/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Gluconato de Cálcio/metabolismo , Gluconato de Cálcio/farmacologia , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Quelantes/metabolismo , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Ácido Egtázico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Expressão Gênica , Células HCT116 , Humanos , Sulfato de Magnésio/metabolismo , Sulfato de Magnésio/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fator de Crescimento Neural/farmacologia , Neuritos/metabolismo , Compostos Organoplatínicos/efeitos adversos , Ácido Oxálico/metabolismo , Oxaliplatina , Células PC12 , Ratos , Transdução de Sinais
15.
Biochem Biophys Res Commun ; 415(1): 168-73, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-22033413

RESUMO

Rho family GTPase-specific guanine nucleotide exchange factors of the Dbl family regulate a variety of cellular events including cytoskeletal arrangement, signal transduction, and gene expression through activation of Rho family GTPases. In this study, we show that hPEM-2 is a downstream effector of G(s) and G(q) signaling in Neuro-2a neuroblastoma cells. Co-expression with hPEM-2 and GTPase-deficient (constitutively active) mutants of Gαs (Gα(s)Q213L) or Gα(q) (Gα(q)Q209L), but not other GTPase-deficient mutants of Gα subunit and Gßγ subunits, activated serum response element (SRE)-dependent gene transcription, which is known to be induced by Rho family activation. Although a dominant negative mutant of Rac1 strongly blocks Gα(s)Q213L or Gα(q)Q209L/hPEM-2 activated SRE-dependent gene transcription, those of Cdc42 or RhoA are marginally affected. A PKA inhibitor, H-89, attenuated Gα(s)/hPEM-2-activated SRE-dependent gene transcription. And a dominant negative mutant of c-Src and an Src inhibitor attenuated Gα(q)Q209L/hPEM-2-activated SRE-dependent gene transcription. Experiments using hPEM-2 deletion mutants indicate that some regions of hPEM-2 play an important role in enhancing SRE activation by G(s) and G(q) signalings. These results reveal that G(s) and G(q) signalings regulate hPEM-2 functions through PKA and c-Src in Neuro-2a neuroblastoma cells, respectively.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Camundongos , Proteínas Tirosina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transcrição Gênica , Quinases da Família src
16.
J Cell Biochem ; 112(11): 3449-58, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21769916

RESUMO

Glial cell line-derived neurotrophic factor (GDNF) is important for the development and maintenance of dopamine neurons (Lin et al. [1993] Science 260: 1130-1132). GDNF is neuroprotective in animal models of Parkinson disease, where dopamine neurons show selective degeneration. We previously reported GDNF-induced SPHK1 gene expression in a neuroblastoma cell line, TGW (Murakami et al. [2007] J Neurochem 102: 1585-1594). In the present study, we focused on the regulatory mechanism of GAP43 (GDNF-induced neuronal phenotype) transcription to further elucidate physiological roles of GDNF-induced SPHK1 expression and activity. Stable wild-type (SPHK1-WT) but not dominant-negative SPHK1 (SPHK1-DN) overexpression increased both control- and GDNF-induced GAP43 expression. SPHK1-WT cells showed enhanced GDNF-induced sphingosine 1-phosphate (S1P) secretion compared with mock- and SPHK1-DN cells. Exogenous S1P also increased GAP43 expression. In TGW cells, PD98059, a MEK inhibitor, but not SB203580 (a p38 MAPK inhibitor) and LY294002 (a PI3K inhibitor) inhibited GDNF-induced GAP43 expression, suggesting the MEK/ERK pathway has a major role in GDNF-induced GAP43 transcription. A G-protein-coupled receptor inhibitor, pertussis toxin, and S1P(1) and S1P(3) receptor antagonists (VPC23019 and CAY10444) also inhibited ERK activation. Moreover, both S1P1 and S1P3 were serine-phosphorylated by GDNF, suggesting their activated states. C/EBPß transcription factor was induced by GDNF, and DNA pull-down and chromatin immunoprecipitation assays revealed the C/EBP binding site between -131 bp and -98 bp from the first exon of GAP43. Taken together, our results showed that in TGW cells, GDNF increased SPHK1 transcription, leading to the production and secretion of S1P. Through MEK/ERK pathway, S1P stimulates GAP43 transcription with increased binding of C/EBPß to the 5'-promoter.


Assuntos
Proteína GAP-43/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Transcrição Gênica , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Clonagem Molecular , Primers do DNA , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Transdução de Sinais , Esfingosina/metabolismo
17.
Neurochem Res ; 36(7): 1304-13, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21369758

RESUMO

Caspase plays an important role in apoptosis and physiological processes such as synaptic plasticity. However, the caspase substrate at the synapse is still unknown. Here we used an in vitro cleavage assay with a small-pool human brain cDNA library. We identified the presynaptic protein Caytaxin as a substrate of caspase-3 and caspase-7. Deficiency in Caytaxin causes Cayman ataxia, a disorder characterized by cerebellar dysfunction and mental retardation. Caytaxin cleavage in cerebellar granule neurons is dependent on caspase-3 activation. The cleavage site is upstream of the cellular retinal and the TRIO guanine exchange factor domain, producing a C-terminal fragment that may play an alternative role in inhibiting MEK2 signaling. Thus, we concluded that Caytaxin is a novel substrate of caspase-3 at the presynapse.


Assuntos
Caspase 3/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Caspase 7/metabolismo , Linhagem Celular , Humanos , MAP Quinase Quinase 2/fisiologia , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Transdução de Sinais , Sinapses/metabolismo
18.
Anticancer Drugs ; 22(5): 402-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21317765

RESUMO

The human aldo-keto reductase (AKR) 1B10 is suggested as a tumor marker in various solid tumors. Using colon cancer cells, we found that AKR1B10 was induced with acquisition of resistance to the anticancer drug mitomycin-c (MMC). In the resistant cells, treatment with an AKR1B10 inhibitor decreased their MMC tolerance. In the nonresistant cells, overexpression and silencing of AKR1B10 decreased and increased, respectively, susceptibility to cytotoxic effects of MMC and 4-hydroxy-2-nonenal, which was formed as a product of lipid peroxidation by MMC treatment. These results suggest a role of AKR1B10 in the development of MMC resistance, which may be mediated by its ability to detoxify cytotoxic aldehydes including 4-hydroxy-2-nonenal.


Assuntos
Aldeído Redutase/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Mitomicina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/biossíntese , Aldeído Redutase/genética , Aldeídos/metabolismo , Aldeídos/farmacologia , Aldo-Ceto Redutases , Antibióticos Antineoplásicos/farmacologia , Caspase 3/metabolismo , Neoplasias do Colo/genética , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Células HT29 , Humanos , Peroxidação de Lipídeos , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Regulação para Cima
19.
Biochim Biophys Acta ; 1811(3): 119-28, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21184844

RESUMO

The role of sphingolipid metabolic pathway has been recognized in determining cellular fate. Although sphingolipid degradation has been extensively studied, gene expression of human sphingosine 1-phosphate lyase (SPL) catalyzing sphingosine 1-phosphate (S1P) remains to be determined. Among 5 human lung cancer cell lines examined, SPL protein levels paralleled the respective mRNA and enzyme activities. Between H1155 and H1299 cells used for further experiments, higher cellular S1P was observed in H1155 with higher SPL activity compared with H1299 with low SPL activity. GATA-4 has been reported to affect SPL transcription in Dictyostelium discoideum. GATA-4 was observed in H1155 but not in other cell lines. Overexpression of GATA-4 in H1299 increased SPL expression. However, promoter analysis of human SPL revealed that the most important region was located between -136bp and -88bp from the first exon, where 2 Sp1 sites exist but no GATA site. DNA pull-down assay of H1155 showed increased DNA binding of Sp1 and GATA-4 within this promoter region compared with H1299. Electrophoresis mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, reporter assay using mutated binding motif, and mithramycin A, a specific Sp1 inhibitor, suggest the major role of Sp1 in SPL transcription and no direct binding of GATA-4 with this 5' promoter region. The collaborative role of GATA-4 was proved by showing coimmunoprecipitation of Sp1 and GATA-4 using GST-Sp1 and overexpressed GATA-4. Thus, high SPL transcription of H1155 cells was regulated by Sp1 and GATA-4/Sp1 complex formation, both of which bind to Sp1 sites of the 5'-SPL promoter.


Assuntos
Aldeído Liases/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/enzimologia , Proteínas de Neoplasias/biossíntese , Transcrição Gênica , Aldeído Liases/genética , Linhagem Celular Tumoral , Dictyostelium/genética , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Elementos de Resposta , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo
20.
FASEB J ; 25(4): 1133-44, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21163859

RESUMO

Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine-1-phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild-type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase-3ß. In addition, ASM induced up-regulation of glucose transporter 2 accompanied by suppression of AMP-activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase-1 (SphK1) diminished ASM-mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM-mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high-dose glucose increased glycogen deposition and lipid accumulation in wild-type hepatocytes but not in ASM(-/-) cells. This result is consistent with glucose intolerance in ASM(-/-) mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingomielina Fosfodiesterase/fisiologia , Animais , Glicemia/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 2/biossíntese , Glicogênio/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Ratos
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