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1.
Mol Cell Neurosci ; 52: 20-30, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23085403

RESUMO

Adult neurogenesis relies on EGF and FGF receptor (EGFR/FGFR) function and endocannabinoid (eCB) signalling. Here we have used a neural stem cell (NSC) line to determine how these systems cooperate to regulate neurogenesis. The results show the EGFR to be solely responsible for maintaining PI3K activation explaining its dominant role in promoting NSC survival. The EGFR and FGFR synergistically regulate the ERK/MAPK pathway, and this explains the requirement for both for optimal cell proliferation. The eCB receptors did not contribute to activation of the PI3K or ERK/MAPK pathways, highlighting the importance of another major proliferation pathway. The EGFR plays the dominant role in maintaining the transcriptome, with significant changes in the expression of over 3500 transcripts seen within hours of inhibition or activation of this receptor. The FGFR has a more modest effect on transcription with evidence for nodal integration with EGFR signalling at the level of the ERK/MAPK pathway. A common set of transcripts are regulated by the CB1 and CB2 receptors, with cooperation between these receptors and the EGFR apparent in the regulation of a pool of transcripts, most likely representing signal integration downstream from an as yet to be identified node. Finally, a first level molecular analysis of the transcriptional response shows regulation of a number of key growth factors, growth factor receptors and GPCRs to be under the control of the EGFR.


Assuntos
Endocanabinoides/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Western Blotting , Linhagem Celular , Camundongos , Células-Tronco Neurais/citologia , Análise Serial de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
2.
Cancer Res ; 69(15): 6171-8, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19622772

RESUMO

Uterine leiomyomata, or fibroids, are benign tumors of the uterine myometrium that significantly affect up to 30% of reproductive-age women. Despite being the primary cause of hysterectomy in the United States, accounting for up to 200,000 procedures annually, the etiology of leiomyoma remains largely unknown. As a basis for understanding leiomyoma pathogenesis and identifying targets for pharmacotherapy, we conducted transcriptional profiling of leiomyoma and unaffected myometrium from humans and Eker rats, the best characterized preclinical model of leiomyomata. A global comparison of mRNA from leiomyoma versus myometrium in human and rat identified a highly significant overlap of dysregulated gene expression in leiomyomata. An unbiased pathway analysis using a method of gene-set enrichment based on the sigPathway algorithm detected the mammalian target of rapamycin (mTOR) pathway as one of the most highly up-regulated pathways in both human and rat tumors. To validate this pathway as a therapeutic target for uterine leiomyomata, preclinical studies were conducted in Eker rats. These rats develop uterine leiomyomata as a consequence of loss of Tsc2 function and up-regulation of mTOR signaling. Inhibition of mTOR in female Eker rats with the rapamycin analogue WAY-129327 for 2 weeks decreased mTOR signaling and cell proliferation in tumors, and treatment for 4 months significantly decreased tumor incidence, multiplicity, and size. These results identify dysregulated mTOR signaling as a component of leiomyoma etiology across species and directly show the dependence of uterine leiomyomata with activated mTOR on this signaling pathway for growth.


Assuntos
Leiomioma/metabolismo , Proteínas Quinases/metabolismo , Neoplasias Uterinas/metabolismo , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leiomioma/genética , Miométrio/metabolismo , Miométrio/fisiologia , Análise Serial de Proteínas , Proteínas Quinases/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Neoplasias Uterinas/genética
3.
J Neurochem ; 108(6): 1539-49, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19183261

RESUMO

The molecular determinants of Sonic Hedgehog (Shh) signaling in mammalian cells and, in particular, those of the CNS are unclear. Here we report that primary cortical astrocyte cultures are highly responsive to both Shh protein and Hh Agonist 1.6, a selective, small molecule Smoothened agonist. Both agonists produced increases in mRNA expression of Shh-regulated gene targets, Gli-1 and Patched in a cyclopamine- and forskolin-sensitive manner. Using this model we show for the first time that Shh pathway activation mediates rapid increases in p38 MAPK phosphorylation, without altering phosphorylation of either extracellular-signal-regulated kinases or c-jun N-terminal kinases. Selective inhibition of p38 MAPK significantly attenuated Shh-dependent up-regulation of Gli-1, inter-alpha trypsin inhibitor and thrombomodulin mRNA, however did not affect expression of insulin-like growth factor 2 or a novel Shh target, membrane-associated guanylate kinase p55 subfamily member 6. Using RNAi and a constitutively-active mutant we show that Shh signaling to p38 MAPK and subsequent Gli-1 transcription requires G-protein receptor kinase 2. Taken together, these findings provide evidence for a central role of G-protein receptor kinase 2-dependent p38 MAPK activity in regulating Shh-mediated gene transcription in astrocytes.


Assuntos
Astrócitos/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção/métodos
4.
J Med Chem ; 51(14): 4115-21, 2008 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-18578470

RESUMO

The JAK-STAT3 pathway regulates genes that are important in cell proliferation and thus is a promising target for cancer therapy. A high-throughput screening (HTS) campaign using an Apo-ONE Homogenous Caspase 3/7 assay in U266 cells identified 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid ethyl ester 4 as a potential STAT3 pathway inhibitor. Optimization of this HTS hit led to the identification of the 7-cyano analogue 8, which inhibited STAT3-Y705 phosphorylation with an EC 50 of 170 nM. Compound 8 also inhibited cytokine induced JAK activation but did not inhibit BCR-ABL activated STAT5 phosphorylation in K562 cells.


Assuntos
Quinolonas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Ésteres , Humanos , Espectroscopia de Ressonância Magnética , Fosforilação , Fator de Transcrição STAT3/metabolismo
5.
BMC Med Genomics ; 1: 27, 2008 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-18578861

RESUMO

BACKGROUND: Vaginal atrophy (VA) is the thinning of the vaginal epithelial lining, typically the result of lowered estrogen levels during menopause. Some of the consequences of VA include increased susceptibility to bacterial infection, pain during sexual intercourse, and vaginal burning or itching. Although estrogen treatment is highly effective, alternative therapies are also desired for women who are not candidates for post-menopausal hormone therapy (HT). The ovariectomized (OVX) rat is widely accepted as an appropriate animal model for many estrogen-dependent responses in humans; however, since reproductive biology can vary significantly between mammalian systems, this study examined how well the OVX rat recapitulates human biology. METHODS: We analyzed 19 vaginal biopsies from human subjects pre and post 3-month 17beta-estradiol treated by expression profiling. Data were compared to transcriptional profiling generated from vaginal samples obtained from ovariectomized rats treated with 17beta-estradiol for 6 hrs, 3 days or 5 days. The level of differential expression between pre- vs. post- estrogen treatment was calculated for each of the human and OVX rat datasets. Probe sets corresponding to orthologous rat and human genes were mapped to each other using NCBI Homologene. RESULTS: A positive correlation was observed between the rat and human responses to estrogen. Genes belonging to several biological pathways and GO categories were similarly differentially expressed in rat and human. A large number of the coordinately regulated biological processes are already known to be involved in human VA, such as inflammation, epithelial development, and EGF pathway activation. CONCLUSION: At the transcriptional level, there is evidence of significant overlap of the effects of estrogen treatment between the OVX rat and human VA samples.

6.
J Immunol ; 180(5): 3520-34, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18292579

RESUMO

TREM-1 (triggering receptor expressed on myeloid cells-1) is an orphan immunoreceptor expressed on monocytes, macrophages, and neutrophils. TREM-1 associates with and signals via the adapter protein DAP12/TYROBP, which contains an ITAM. TREM-1 activation by receptor cross-linking has been shown to be proinflammatory and to amplify some cellular responses to TLR ligands such as bacterial LPS. To investigate the cellular consequences of TREM-1 activation, we have characterized global gene expression changes in human monocytes in response to TREM-1 cross-linking in comparison to and combined with LPS. Both TREM-1 activation and LPS up-regulate chemokines, cytokines, matrix metalloproteases, and PTGS/COX2, consistent with a core inflammatory response. However, other immunomodulatory factors are selectively induced, including SPP1 and CSF1 (i.e., M-CSF) by TREM-1 activation and IL-23 and CSF3 (i.e., G-CSF) by LPS. Additionally, cross-talk between TREM-1 activation and LPS occurs on multiple levels. Although synergy in GM-CSF protein production is reflected in commensurate mRNA abundance, comparable synergy in IL-1beta protein production is not. TREM-1 activation also attenuates the induction of some LPS target genes, including those that encode IL-12 cytokine family subunits. Where tested, positive TREM-1 outputs are greatly reduced by the PI3K inhibitor wortmannin, whereas this attenuation is largely PI3K independent. These experiments provide a detailed analysis of the cellular consequences of TREM-1 activation and highlight the complexity in signal integration between ITAM- and TLR-mediated signaling.


Assuntos
Regulação para Baixo/imunologia , Perfilação da Expressão Gênica , Imunidade Inata , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Receptor Cross-Talk/imunologia , Receptores Imunológicos/metabolismo , Regulação para Cima/imunologia , Separação Celular , Células Cultivadas , Regulação para Baixo/genética , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata/genética , Monócitos/imunologia , Monócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Imunológicos/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/genética
7.
J Neurol Sci ; 239(1): 81-93, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16214174

RESUMO

Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human autoimmune disease multiple sclerosis (MS) and is primarily driven by T helper type 1 (Th1) cells. Interleukin (IL)-12 and interferon (IFN)-gamma are important cytokines involved in the differentiation and amplification of Th1 cells, however mice deficient in either IFN-gamma or IL-12 still develop EAE. We have used microarray analysis of EAE-affected CNS tissues in wild-type, IFN-gamma -/- and IL-12 -/- animals to identify genes critical for development of EAE. Over 500 genes were regulated in at least one genotype and over 94 genes were regulated in all three. Of those, 17 were also upregulated in spleen during the disease. We show that a majority of the genes regulated in EAE are also regulated in diseased regions of human MS tissues. The genes in the pool of 94 are more likely to be found regulated in MS patients than the genes regulated in only one or two of the mouse strains suggesting that analyzing gene expression under these multiple genetic conditions may lead to better identification of the genes critical for disease development.


Assuntos
Encefalomielite Autoimune Experimental/genética , Perfilação da Expressão Gênica , Predisposição Genética para Doença/genética , Interferon gama/genética , Interleucina-12/genética , Células Th1/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Genótipo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para Cima/genética , Regulação para Cima/imunologia
8.
J Steroid Biochem Mol Biol ; 97(4): 328-41, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16157482

RESUMO

Progesterone (P4) is an essential reproductive steroid hormone required for many aspects of female reproductive physiology. Progestins are compounds that demonstrate progesterone-like activity and are used in oral contraception, hormone therapy, and treatment of some reproductive disorders, but differ widely in their chemical structures, potency, and pharmacokinetics. While numerous studies have assessed progestins on specific endpoints, little is known about the activation of global gene expression by progestins. We used Affymetrix GeneChip U133A expression arrays to examine the action of P4 and six clinically relevant synthetic progestins (3-ketodesogestrel, drospirenone, levonorgestrel, medroxyprogesterone acetate, norethindrone acetate, and trimegestone) on the progesterone receptor (PR)-positive T47Dco and the PR-negative T47D-Y breast cancer cell lines. Excluding drospirenone, one or more of the progestins-regulated 329 genes, with 30 genes regulated by at least 2.0-fold by all progestins in the T47Dco cells. The synthetic progestins show a high degree of similarity in their transcriptional responses, and each progestin regulates between 77 and 91% of the genes regulated by P4. Independent quantitative RT-PCR analysis confirmed a similar regulation for S100P, PPL, IL20RA, NET1, ATP1A1, HIG2, and CXCL12 (SDF-1) by all seven progestins. Attempts to find differentially regulated genes by any progestin compared to all other treatments failed, suggesting any differences are quantitative, not qualitative. This analysis demonstrates a high degree of similarity among these progestins on PR-regulated gene expression in T47D cells, suggesting a similar and fairly specific mode of action.


Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Fosfatase Alcalina/metabolismo , Androstenos/química , Linhagem Celular Tumoral , Análise por Conglomerados , Desogestrel/química , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Humanos , Levanogestrel/química , Ligantes , Acetato de Medroxiprogesterona/química , Análise de Sequência com Séries de Oligonucleotídeos , Progesterona/química , Progestinas/química , Promegestona/análogos & derivados , Promegestona/química , RNA/metabolismo , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica
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