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1.
Immunity ; 45(1): 46-59, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27396959

RESUMO

Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.


Assuntos
Imunidade Inata , Inflamação/imunologia , Macrófagos/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Células Cultivadas , Feminino , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais , Transcriptoma
2.
J Biol Chem ; 287(30): 25030-7, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22665479

RESUMO

Nucleotide-binding domain leucine-rich repeat proteins (NLRs) play a key role in immunity and disease through their ability to modulate inflammation in response to pathogen-derived and endogenous danger signals. Here, we identify the requirements for activation of NLRP1, an NLR protein associated with a number of human pathologies, including vitiligo, rheumatoid arthritis, and Crohn disease. We demonstrate that NLRP1 activity is dependent upon ASC, which associates with the C-terminal CARD domain of NLRP1. In addition, we show that NLRP1 activity is dependent upon autolytic cleavage at Ser(1213) within the FIIND. Importantly, this post translational event is dependent upon the highly conserved distal residue His(1186). A disease-associated single nucleotide polymorphism near His(1186) and a naturally occurring mRNA splice variant lacking exon 14 differentially affect this autolytic processing and subsequent NLRP1 activity. These results describe key molecular pathways that regulate NLRP1 activity and offer insight on how small sequence variations in NLR genes may influence human disease pathogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Imunidade Inata , Inflamassomos/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Proteólise , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células HEK293 , Humanos , Inflamassomos/metabolismo , Proteínas NLR , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional/genética , Estrutura Terciária de Proteína
3.
ACS Med Chem Lett ; 10(6): 857-862, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31223438

RESUMO

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.

4.
J Med Chem ; 62(10): 5096-5110, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31013427

RESUMO

RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.


Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Pirazóis/síntese química , Pirazóis/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Disponibilidade Biológica , Linhagem Celular , Doença Crônica , Desenho de Fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Haplorrinos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Esclerose Múltipla/tratamento farmacológico , Pirazóis/farmacocinética , Ratos , Retinose Pigmentar/tratamento farmacológico , Relação Estrutura-Atividade
5.
Ann N Y Acad Sci ; 1120: 36-46, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18184910

RESUMO

In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome-wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX- XI, XII, XIII-XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, led to the identification of 58 genes that have 1000-fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N-ethyl-N-nitrosourea (ENU) mutagenesis. The microarray data and the qRT-PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).


Assuntos
Ciclo Celular/genética , Anticoncepcionais Masculinos/farmacologia , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Epitélio Seminífero/fisiologia , Testículo/metabolismo , Animais , Anticoncepção , DNA Complementar/isolamento & purificação , Infertilidade Masculina/genética , Masculino , Camundongos , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ratos , Epitélio Seminífero/metabolismo , Testículo/efeitos dos fármacos , Testículo/fisiologia , Transcrição Gênica , Estudos de Validação como Assunto
6.
Ann N Y Acad Sci ; 1120: 16-35, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18184909

RESUMO

In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.


Assuntos
Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/fisiologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/fisiologia , Fatores Etários , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos , Perfilação da Expressão Gênica , Masculino , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Células-Tronco/metabolismo
7.
Asian J Androl ; 9(4): 522-7, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17589790

RESUMO

As part of our efforts to identify novel contraceptive targets in the epididymis we performed transcriptional profiling on each of the 10 and 19 segments of the mouse and rat epididymidis, respectively, using Affymetrix whole genome microarrays. A total of 17 096 and 16 360 probe sets representing transcripts were identified as being expressed in the segmented mouse and rat epididymal transcriptomes, respectively. Comparison of the expressed murine transcripts against a mouse transcriptional profiling database derived from 22 other mouse tissues identified 77 transcripts that were expressed uniquely in the epididymis. The expression of these genes was further evaluated by reverse transcription polymerase chain reaction (RT-PCR) analysis of RNA from 21 mouse tissues. RT-PCR analysis confirmed epididymis-specific expression of Defensin Beta 13 and identified two additional genes with expression restricted only to the epididymis and testis. Comparison of the 16 360 expressed transcripts in the rat epididymis with data of 21 other tissues from a rat transcriptional profiling database identified 110 transcripts specific for the epididymis. Sixty-two of these transcripts were further investigated by qPCR analysis. Only Defensin 22 (E3 epididymal protein) was shown to be completely specific for the epididymis. In addition, 14 transcripts showed more than 100-fold selective expression in the epididymis. The products of these genes might play important roles in epididymal and/or sperm function and further investigation and validation as contraceptive targets are warranted. The results of the studies described in this report are available at the Mammalian Reproductive Genetics (MRG) Database (http://mrg.genetics.washington.edu/).


Assuntos
Epididimo/fisiologia , Perfilação da Expressão Gênica/métodos , Transcrição Gênica , Animais , Masculino , Camundongos , Especificidade de Órgãos , RNA/genética , RNA/isolamento & purificação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Asian J Androl ; 9(4): 565-73, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17589796

RESUMO

The epididymis is divided into caput, corpus and cauda regions, organized into intraregional segments separated by connective tissue septa (CTS). In the adult rat and mouse these segments are highly differentiated. Regulation of these segments is by endocrine, lumicrine and paracrine factors, the relative importance of which remains under investigation. Here, the ability of the CTS to limit signaling in the interstitial compartment is reviewed as is the effect of 15 days of unilateral efferent duct ligation (EDL) on ipsilateral segmental transcriptional profiles. Inter-segmental microperifusions of epidermal growth factor (EGF), vascular endothelial growth factor (VEGFA) and fibroblast growth factor 2 (FGF2) increased phosphorylation of mitogen activated protein kinase (MAPK) in segments 1 and 2 of the rat epididymis and the effects of all factors were limited by the CTS separating the segments. Microarray analysis of segmental gene expression determined the effect of 15 days of unilateral EDL on the transcriptome-wide gene expression of rat segments 1-4. Over 11,000 genes were expressed in each of the four segments and over 2000 transcripts in segment 1 responded to deprivation of testicular lumicrine factors. Segments 1 and 2 of control tissues were the most transcriptionally different and EDL had its greatest effects there. In the absence of lumicrine factors, all four segments regressed to a transcriptionally undifferentiated state, consistent with the less differentiated histology. Deprivation of lumicrine factors could stimulate an individual gene's expression in some segments yet suppress it in others. Such results reveal a higher complexity of the regulation of rat epididymal segments than that is generally appreciated.


Assuntos
Ductos Ejaculatórios/fisiologia , Epididimo/fisiologia , Regulação da Expressão Gênica , Animais , Epididimo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Masculino , Camundongos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
9.
Artigo em Inglês | MEDLINE | ID: mdl-29226625

RESUMO

Therapies that suppress RIPK1 kinase activity are emerging as promising therapeutic agents for the treatment of multiple inflammatory disorders. The ability to directly measure drug binding of a RIPK1 inhibitor to its target is critical for providing insight into pharmacokinetics, pharmacodynamics, safety and clinical efficacy, especially for a first-in-class small-molecule inhibitor where the mechanism has yet to be explored. Here, we report a novel method for measuring drug binding to RIPK1 protein in cells and tissues. This TEAR1 (Target Engagement Assessment for RIPK1) assay is a pair of immunoassays developed on the principle of competition, whereby a first molecule (ie, drug) prevents the binding of a second molecule (ie, antibody) to the target protein. Using the TEAR1 assay, we have validated the direct binding of specific RIPK1 inhibitors in cells, blood and tissues following treatment with benzoxazepinone (BOAz) RIPK1 inhibitors. The TEAR1 assay is a valuable tool for facilitating the clinical development of the lead RIPK1 clinical candidate compound, GSK2982772, as a first-in-class RIPK1 inhibitor for the treatment of inflammatory disease.


Assuntos
Anticorpos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Células HT29 , Humanos , Imunoensaio , Macaca fascicularis , Masculino , Ligação Proteica/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Bibliotecas de Moléculas Pequenas/farmacologia
10.
J Med Chem ; 60(4): 1247-1261, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28151659

RESUMO

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Colite Ulcerativa/tratamento farmacológico , Inflamação/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Animais , Benzazepinas/química , Benzazepinas/farmacologia , Colite Ulcerativa/imunologia , Citocinas/imunologia , Cães , Haplorrinos , Humanos , Inflamação/imunologia , Camundongos , Simulação de Acoplamento Molecular , Coelhos , Ratos , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Suínos , Porco Miniatura , Fator de Necrose Tumoral alfa/imunologia
11.
J Med Chem ; 59(5): 2163-78, 2016 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-26854747

RESUMO

The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.


Assuntos
DNA/química , Isoxazóis/farmacologia , Oxazepinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células HT29 , Humanos , Isoxazóis/síntese química , Isoxazóis/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Oxazepinas/síntese química , Oxazepinas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Células U937
12.
J Med Chem ; 59(10): 4867-80, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27109867

RESUMO

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.


Assuntos
Aminoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Sulfonas/farmacologia , Aminoquinolinas/sangue , Aminoquinolinas/química , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Relação Estrutura-Atividade , Sulfonas/sangue , Sulfonas/química
13.
Cell Host Microbe ; 17(2): 243-51, 2015 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-25674983

RESUMO

Herpes simplex virus (HSV)-1 and HSV-2 are significant human pathogens causing recurrent disease. During infection, HSV modulates cell death pathways using the large subunit (R1) of ribonucleotide reductase (RR) to suppress apoptosis by binding to and blocking caspase-8. Here, we demonstrate that HSV-1 and HSV-2 R1 proteins (ICP6 and ICP10, respectively) also prevent necroptosis in human cells by inhibiting the interaction between receptor-interacting protein kinase 1 (RIP1) and RIP3, a key step in tumor necrosis factor (TNF)-induced necroptosis. We show that suppression of this cell death pathway requires an N-terminal RIP homotypic interaction motif (RHIM) within R1, acting in concert with the caspase-8-binding domain, which unleashes necroptosis independent of RHIM function. Thus, necroptosis is a human host defense pathway against two important viral pathogens that naturally subvert multiple death pathways via a single evolutionarily conserved gene product.


Assuntos
Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Ribonucleotídeo Redutases/metabolismo , Caspase 8/metabolismo , Morte Celular , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 2/fisiologia , Humanos , Proteínas Virais/metabolismo , Replicação Viral
14.
ACS Med Chem Lett ; 4(12): 1238-43, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24900635

RESUMO

Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.

15.
Biol Reprod ; 77(1): 165-71, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17377138

RESUMO

The epididymis has traditionally been divided into the caput, corpus, and cauda regions, which are further organized into intraregional segments. In the rat and mouse, these segments have high degrees of transcriptional differentiation, and what has traditionally been called the initial segment of the rat epididymis actually consists of three transcriptionally different intraregional segments. These segments are regulated by endocrine, lumicrine, and paracrine factors, whose relative importance remains a topic of investigation. In the present study, 15-day unilateral efferent duct ligation (EDL) was used to deprive ipsilateral rat epididymides of lumicrine regulation. Segments 1-4 of EDL epididymides and contralateral, sham-operated tissues were collected individually. Microarray analysis of gene expression was used to determine the effect of lumicrine factor deprivation on the transcriptome-wide gene expression of each segment studied. More than 11 000 genes were detected as being expressed in each of the four segments examined. More than 2000 genes responded significantly to EDL in segment 1, although this number of genes declined in each succeeding segment. Segments 1 and 2 of control tissues were the most different transcriptionally and the most affected by EDL. In the absence of lumicrine factors, the four segments regressed to a transcriptionally undifferentiated state, which was consistent with the less-differentiated histology seen after EDL. Interestingly, for an individual gene, lumicrine factor deprivation could stimulate expression in some segments and suppress expression in other segments. These results reveal a higher complexity to the regulation of rat epididymal segments than heretofore appreciated.


Assuntos
Epididimo/metabolismo , Epididimo/cirurgia , Regulação da Expressão Gênica/fisiologia , Animais , Epididimo/citologia , Perfilação da Expressão Gênica , Ligadura , Masculino , Ratos , Ratos Sprague-Dawley
16.
Biol Reprod ; 76(4): 561-70, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17167166

RESUMO

Regional differences along the epididymis are essential for the establishment of the luminal environment required for sperm maturation. In the current study, 19 morphologically distinct segments of the rat epididymis were identified by microdissection. Total RNA was isolated from each segment and subjected to microarray analysis. Segmental analysis of epididymal gene expression identified more than 16,000 expressed qualifiers, whereas profiling of RNA from whole rat epididymis identified approximately 12,000 expressed qualifiers. Screening a panel of normal rat tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, more than 3500 qualifiers were shown to be present and differentially upregulated or downregulated by more than fourfold between any two segments. The present study complements our previous segment-dependent analysis of gene expression in the mouse epididymis and allows for comparative analyses between datasets. A total of 492 genes was shown to be present on both the MOE430 (mouse) and RAE230_2 (rat) microarrays, expressed in the epididymis of both species, and differentially expressed by more than fourfold in between segments in each species. Moreover, in-depth quantitative RT-PCR analysis of 36 members of the beta defensin gene family showed highly conserved patterns of expression along the lengths of the mouse and rat epididymides. These analyses elucidate global gene expression patterns along the length of the rat epididymis and provide a novel evaluation of conserved and nonconserved gene expression patterns in the epididymides of the two species. Furthermore, these data provide a powerful resource for the research community for future studies of biological factors that mediate sperm maturation and storage.


Assuntos
Epididimo/metabolismo , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Animais , Análise por Conglomerados , Defensinas/genética , Defensinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Família Multigênica/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
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