RESUMEN

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5'-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Células de la Granulosa/metabolismo , Animales , Biomarcadores , Bovinos , Regulación hacia Abajo , Femenino , Folículo Ovárico/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Técnicas del Sistema de Dos Híbridos

RESUMEN

Ankyrin-repeat and SOCS-box protein 9 (ASB9) is a member of the large SOCS-box containing proteins family and acts as the specific substrate recognition component of E3 ubiquitin ligases in the process of ubiquitination and proteasomal degradation. We previously identified ASB9 as a differentially expressed gene in granulosa cells (GC) of bovine ovulatory follicles. This study aimed to further investigate ASB9 mRNA and protein regulation, identify binding partners in GC of bovine ovulatory follicles, and study its function. GC were obtained from small follicles (SF: 2-4 mm), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 hours following hCG injection (OF). Analyses by RT-PCR showed a 104-fold greater expression of ASB9 in GC of OF than in DF. Steady-state levels of ASB9 in follicular walls (granulosa and theca cells) analyzed at 0, 6, 12, 18 and 24 hours after hCG injection showed a significant induction of ASB9 expression at 12 and 18 hours, reaching a maximum induction of 10.2-fold at 24 hours post-hCG as compared to 0 hour. These results were confirmed in western blot analysis showing strongest ASB9 protein amounts in OF. Yeast two-hybrid screening of OF-cDNAs library resulted in the identification of 10 potential ASB9 binding partners in GC but no interaction was found between ASB9 and creatine kinase B (CKB) in these GC. Functional studies using CRISPR-Cas9 approach revealed that ASB9 inhibition led to increased GC proliferation and modulation of target genes expression. Overall, these results support a physiologically relevant role of ASB9 in the ovulatory follicle by targeting specific proteins likely for degradation, contributing to reduced GC proliferation, and could be involved in the final GC differentiation into luteal cells.

Asunto(s)

Gonadotropina Coriónica/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células de la Granulosa/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Células Tecales/metabolismo , Animales , Repetición de Anquirina , Bovinos , Diferenciación Celular , Proliferación Celular , Gonadotropina Coriónica/administración & dosificación , Femenino , Células Lúteas/fisiología , Modelos Animales , Unión Proteica , Mapeo de Interacción de Proteínas , Proteolisis , Proteínas Supresoras de la Señalización de Citocinas/genética

RESUMEN

BACKGROUND: Ovulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles. METHODS: Granulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of human chorionic gonadotropin (hCG). A granulosa cell subtracted cDNA library (OF-DF) was generated using suppression subtractive hybridization and screened. RESULTS: Detection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, 774KNSHNEL780. Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The 774KNSHNEL780 sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression. CONCLUSIONS: We conclude that we have identified novel genes that are upregulated by hCG in bovine GC of OF, thereby providing novel insight into peri-ovulatory regulation of genes that contribute to ovulation and/or luteinization processes.

Asunto(s)

Gonadotropina Coriónica/farmacología , Células de la Granulosa/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Bovinos , Femenino , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Ovulación/efectos de los fármacos , ARN Mensajero/genética

RESUMEN

BACKGROUND: Janus kinase 3 (JAK3) is a member of the membrane-associated non-receptor tyrosine kinase protein family and is considered predominantly expressed in hematopoietic cells. We previously identified JAK3 as a differentially expressed gene in granulosa cells (GC) of bovine preovulatory follicles. The present study aimed to further investigate JAK3 regulation, to identify protein binding partners and better understand its mode of action in bovine reproductive cells. RESULTS: GC were obtained from small follicles (SF), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 h following hCG injection (OF). RT-PCR analyses showed greatest expression of JAK3 in GC of DF, while JAK3 expression was downregulated in OF (P < 0.0001). In addition, there was a 5- and 20-fold reduction of JAK3 steady-state mRNA levels in follicular walls, respectively at 12 and 24 hours post-hCG as compared to 0 h (P < 0.05). Similarly, JAK3 expression was downregulated by the endogenous LH surge. These results were confirmed in western blot analysis showing weakest JAK3 protein amounts in OF as compared to DF. Yeast two-hybrid screening of a DF-cDNA library resulted in the identification of JAK3 partners in GC that were confirmed by co-immunoprecipitation and included leptin receptor overlapping transcript-like 1 (LEPROTL1), inhibin beta A (INHBA) and cyclin-dependent kinase inhibitor 1B (CDKN1B). In functional studies using bovine endometrial cells, JAK3 increased phosphorylation of STAT3 and cell viability, while the addition of JANEX-1 inhibited JAK3 actions. CONCLUSION: These results support a physiologically relevant role of JAK3 in follicular development and provide insights into the mode of action and function of JAK3 in reproductive tissues.

Asunto(s)

Proteínas Portadoras/metabolismo , Células de la Granulosa/metabolismo , Janus Quinasa 3/metabolismo , Folículo Ovárico/metabolismo , Ovulación/metabolismo , Animales , Bovinos , Supervivencia Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Endometrio/metabolismo , Femenino , Regulación de la Expresión Génica , Subunidades beta de Inhibinas/metabolismo , Janus Quinasa 3/genética , Ovulación/genética , Unión Proteica , Mapeo de Interacción de Proteínas , ARN Mensajero/genética , Factores de Transcripción STAT/metabolismo

RESUMEN

BACKGROUND: LAPTM4B is a member of the lysosome-associated transmembrane protein superfamily that is differentially expressed in normal human tissues and upregulated in various types of carcinomas. These proteins are thought to be involved in the regulation of cell proliferation and survival. The objective of this study was to investigate the expression of bovine LAPTM4B during ovarian follicular development and in various bovine tissues. METHODS AND RESULTS: Northern blot analysis revealed a 1.8 kb transcript, with highly variable steady state levels among tissues. RT-PCR analysis showed that LAPTM4B mRNA transcripts were low in granulosa cells of small antral follicles, increased in large dominant follicles, and decreased in ovulatory follicles following injection of human chorionic gonadotropin (hCG; P < 0.003). Ovulatory follicles collected at various times after hCG injection revealed a significant reduction of LAPTM4B mRNA starting at 18 h post-hCG (P < 0.029). Immunoblotting analysis using antibodies generated against bovine LAPTM4B recognized proteins of 26.3 and 31.5 kDa in granulosa cells of developing follicles and corpus luteum. Further analyses of affinity-purified His-tag LAPTM4B overexpressed in HEK cells showed that the 31.5 kDa protein represented the ubiquinated isoform of the 26.3 kDa native protein. The 26.3 kDa protein was differentially expressed showing highest amounts in dominant follicles and lowest amounts in ovulatory follicles 24 h post-hCG. Immunohistochemical analyses of LAPTM4B showed marked heterogeneity of labeling signal among tissues, with LAPTM4B mainly localized to perinuclear vesicles, in keeping with its putative lysosomal membrane localization. CONCLUSION: This study reports for the first time that bovine LAPTM4B in granulosa cells is present in both unubiquinated and ubiquinated forms, and is differentially expressed in developing ovarian follicles, suggesting a possible role in terminal follicular growth.

Asunto(s)

Células de la Granulosa/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas Oncogénicas/biosíntesis , Animales , Northern Blotting , Bovinos , Femenino , Inmunohistoquímica , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma , Ubiquitinación

RESUMEN

The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropin (hCG) injection. Results showed that RGS2 mRNA levels were very low at 0 h but markedly increased 12-39 h post-hCG (P < 0.05). In the bovine species, results revealed that RGS2 mRNA levels were low in small and dominant follicles and in ovulatory follicles obtained at 0 h, but markedly increased in ovulatory follicles 6-24 h post-hCG (P < 0.05). To study the molecular control of RGS2 expression, primary cultures of bovine granulosa cells were used. Stimulation with forskolin induced an up-regulation of RGS2 mRNA in vitro. Studies using 5'-deletion mutants identified a minimal region containing full-length basal and forskolin-inducible RGS2 promoter activities. Site-directed mutagenesis indicated that these activities were dependent on CRE and ETS1 cis-elements. Electrophoretic mobility shift assays confirmed the involvement of these elements and revealed their interactions with CREB1 and ETS1 proteins. Chromatin immunoprecipitation assays confirmed endogenous interactions of these proteins with the RGS2 promoter in granulosa cells. Forskolin-inducible RGS2 promoter activity and mRNA expression were markedly decreased by PKA and ERK1/2 inhibitors, and treatment with an antagonist of PGR (RU486) and inhibitors of PTGS2 (NS398) and EGFR (PD153035) blocked the forskolin-dependent RGS2 transcript expression, suggesting the importance of RGS2 in ovulation. Collectively, this study reports for the first time the gonadotropin-dependent up-regulation of RGS2 in equine and bovine preovulatory follicles and presents some of the regulatory controls involved in RGS2 gene expression in granulosa cells.

Asunto(s)

Bovinos/genética , Células de la Granulosa/metabolismo , Caballos/genética , Folículo Ovárico/metabolismo , Ovulación/genética , Proteínas RGS/genética , Animales , Bovinos/fisiología , Células Cultivadas , Femenino , Fase Folicular/efectos de los fármacos , Fase Folicular/genética , Fase Folicular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Gonadotropinas/farmacología , Células de la Granulosa/efectos de los fármacos , Caballos/fisiología , Folículo Ovárico/efectos de los fármacos , Ovulación/efectos de los fármacos , Ovulación/metabolismo , Proteínas RGS/metabolismo , Activación Transcripcional/efectos de los fármacos

RESUMEN

Vanin-2 (VNN2) is known to be involved in inflammation and leukocyte migration, but its regulation in follicles remains unknown. The objectives of this work were to study the regulation of VNN2 transcripts in bovine follicles prior to ovulation and to characterize the control of its expression in bovine granulosa cells. VNN2 expression was studied using total RNA extracted from granulosa cells of small follicles (2-4 mm in diameter), dominant follicles obtained on Day 5 of the estrous cycle, ovulatory follicles obtained 0-24 h after human chorionic gonadotropin (hCG), and corpora lutea on Day 5 of the cycle. The results from RT-PCR analyses showed that levels of VNN2 mRNA were high in ovulatory follicles 24 h post-hCG but low in the other tissues. In ovulatory follicles, levels of VNN2 mRNA were low at 0 h but significantly up-regulated 12-24 h post-hCG. To determine factors controlling VNN2 gene expression, established primary cultures of granulosa cells isolated from bovine dominant follicles were used. Treatment with forskolin elevated VNN2 mRNA expression as observed in vivo. Mutation studies identified the minimal region conferring basal and forskolin-stimulated VNN2 promoter activities, which were dependent on chicken ovalbumin upstream promoter-transcription factor (COUP-TF), GATA, and Ebox cis-elements. Electrophoretic mobility shift assays identified COUP-TF, GATA4, and upstream stimulating factor proteins as key factors interacting with these elements. Chromatin immunoprecipitation assays confirmed basal and forskolin-induced interactions between these proteins and the VNN2 promoter in bovine granulosa cell cultures. VNN2 promoter activity and mRNA expression were markedly stimulated by forskolin and overexpression of the catalytic subunit of PKA, but inhibited by PKA and ERK1/2 inhibitors. Collectively, the findings from this study describe for the first time the gonadotropin/forskolin-dependent up-regulation of VNN2 transcripts in granulosa cells of preovulatory follicles and provide insights into some of the molecular bases of VNN2 gene expression in follicular cells.

Asunto(s)

Amidohidrolasas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Folículo Ovárico/metabolismo , Proestro , Regiones Promotoras Genéticas , Transcripción Genética , Regulación hacia Arriba , Amidohidrolasas/biosíntesis , Amidohidrolasas/genética , Animales , Bovinos , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Células Cultivadas , Gonadotropina Coriónica/farmacología , Colforsina/farmacología , Cuerpo Lúteo/citología , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/metabolismo , Activadores de Enzimas/farmacología , Femenino , Fármacos para la Fertilidad Femenina/farmacología , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mutación , Folículo Ovárico/citología , Folículo Ovárico/efectos de los fármacos , Proestro/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elementos de Respuesta/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

The ovulatory process involves a complex remodeling of the extracellular matrix during which a desintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1) is thought to play a key role, but its transcriptional regulation in bovine follicles remains largely unknown. The objectives of this study were to characterize the regulation of ADAMTS1 in bovine follicles before ovulation and to determine its transcriptional control in bovine granulosa cells. Regulation of ADAMTS1 was assessed using total RNA isolated from bovine preovulatory follicles obtained at various times after human chorionic gonadotropin treatment. Results from RT-PCR analyses showed that levels of ADAMTS1 mRNA were very low at 0 hours but increased at 6 to 24 hours after human chorionic gonadotropin in granulosa cells. To determine the regulatory mechanisms controlling ADAMTS1 gene expression in vitro, primary cultures of bovine granulosa cells were established, and treatment with forskolin up-regulated ADAMTS1 mRNA levels. Promoter activity assays, 5'-deletion, and site-directed mutagenesis identified a minimal region conferring full-length basal and forskolin-stimulated ADAMTS1 promoter activities, with both being dependent on Ebox cis-acting elements. EMSAs revealed upstream stimulating factor (USF) proteins as key trans-activating factors interacting with Ebox. Chromatin immunoprecipitation assays confirmed such interactions between USF and Ebox in vivo, and USF binding to Ebox elements was increased by forskolin treatment. ADAMTS1 promoter activity and mRNA expression were increased by forskolin and overexpression of the catalytic subunit of protein kinase A, but not by cotreatment with inhibitors of protein kinase A, ERK1/2, and epidermal growth factor receptor signaling pathways. Furthermore, treatment with a soluble epidermal growth factor induced ADAMTS1 mRNA expression in granulosa cells. Collectively, results from this study describe the gonadotropin/forskolin-dependent up-regulation of ADAMTS1 mRNA in granulosa cells of bovine preovulatory follicles in vivo and in vitro and identify for the first time some of the molecular mechanisms responsible for ADAMTS1 promoter activation in follicular cells of a large monoovulatory species.

Asunto(s)

Proteínas ADAM/genética , Regulación de la Expresión Génica , Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas ADAM/metabolismo , Animales , Sitios de Unión/genética , Bovinos , Células Cultivadas , Gonadotropina Coriónica/farmacología , Colforsina/farmacología , Femenino , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Ovulación/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Factores Estimuladores hacia 5'/genética , Factores Estimuladores hacia 5'/metabolismo

RESUMEN

Little is known about the expression and regulation of epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles of large monoovulatory animal species. To characterize the gonadotropin-dependent regulation of EREG and AREG mRNAs in equine follicles prior to ovulation, extracts were prepared from equine follicles collected during estrus between 0 and 39h post-hCG and corpora lutea obtained on day 8 of the estrous cycle (day 0=day of ovulation). Results from RT-PCR/Southern blot analyses showed that levels of EREG and AREG mRNAs were very low in follicles obtained at 0h but increased thereafter (P<0.05), with maximal levels observed 33-39h post-hCG. This significant increase was observed in both granulosa and theca cells. Immunohistochemistry and immunoblot analyses confirmed the hCG-dependent induction of EREG protein in both cell types. RT-PCR/Southern blot analyses of ADAM17, which encodes an enzyme that cleaves and releases soluble bioactive EREG and AREG, showed that levels of its transcript were high and remained constant throughout the period studied. Studies on the hCG-dependent regulation of EREG and AREG in bovine preovulatory follicles in vivo showed that the induction of both transcripts was transient, observed predominantly at 6h post-hCG and localized only in granulosa cells. To characterize the effect of epidermal growth factor receptor (EGFR) activation on the expression of ovulation-related genes in granulosa cells of a large monoovulatory animal species, primary cultures of bovine granulosa cells were established. Results from RT-PCR analyses revealed that EREG and AREG mRNAs were induced by forskolin treatment in vitro; but the EGFR inhibitor PD153035 suppressed the forskolin-dependent induction of several ovulation-related transcripts, including PTGS2, PTGER2, TNFAIP6, PGR, MMP1, VEGFA, and CTSL2 mRNAs. Moreover, these transcripts were induced in granulosa cell cultures by EGF, an analog of EREG and AREG. Collectively, this study identifies differences in the temporal and cellular localization of EREG and AREG expression in equine and bovine preovulatory follicles, and underscores the potential role of follicular EGFR activation in the regulation of ovulation-regulated genes in large monoovulatory species.

Asunto(s)

Gonadotropina Coriónica/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Anfirregulina , Animales , Bovinos , Células Cultivadas , Familia de Proteínas EGF , Epirregulina , Femenino , Caballos , Humanos , Ovulación/efectos de los fármacos , Ovulación/genética

RESUMEN

BACKGROUND: Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. OBJECTIVE: To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. METHODS: Eleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay. RESULTS: Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways. CONCLUSIONS: Pathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies.

Asunto(s)

Asma/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/métodos , Caballos , Animales , Antígenos/inmunología , Enfermedad Crónica , Clonación Molecular , Femenino , Biblioteca de Genes , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Reacción en Cadena de la Polimerasa

RESUMEN

Glanzmann thrombasthenia (GT) is characterized by a defect of platelet aggregation. This autosomal recessive genetic disorder is caused by an abnormality of the platelet glycoprotein receptors alpha IIb or beta III. Recently, we identified a horse with clinical and pathological features of GT. The aim of this study was to describe this case of GT at the molecular level. A point mutation from G to C in exon 2 of ITGA2B causing a substitution of the expected amino acid arginine 72 (Arg(72)) by a proline (Pro(72)) was encountered. This amino acid change may result in abnormal structural conformations that yield an inactive alpha IIb subunit. The genomic DNA analysis showed that this horse was homozygous for the missense mutation.

Asunto(s)

Enfermedades de los Caballos/genética , Integrina beta3/genética , Glicoproteína IIb de Membrana Plaquetaria/genética , Trombastenia/veterinaria , Animales , Secuencia de Bases , ADN Complementario/análisis , ADN Complementario/genética , Femenino , Homocigoto , Caballos , Datos de Secuencia Molecular , Mutación Missense , Mutación Puntual , Análisis de Secuencia de ADN/veterinaria , Trombastenia/genética

RESUMEN

BACKGROUND: Among the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1. RESULTS: 1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in KRAS and BRAF. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced serpinE2 mRNA levels, indicating that expression of serpinE2 is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade. CONCLUSIONS: Our data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

Asunto(s)

Neoplasias Colorrectales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Serpina E2/metabolismo , Animales , Western Blotting , Butadienos/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/genética , Células HCT116 , Humanos , Técnicas In Vitro , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Ratones , Ratones Desnudos , Nitrilos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Interferencia de ARN , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serpina E2/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas ras/genética , Proteínas ras/metabolismo

RESUMEN

The endoplasmic reticulum (ER) is a major site of protein synthesis and facilitates the folding and assembly of newly synthesized proteins. Misfolded proteins are retrotranslocated across the ER membrane and destroyed at the proteasome. DERL1 is an important protein involved in the retrotranslocation and degradation of a subset of misfolded proteins from the ER. We characterized a 2617 bp cDNA from bovine granulosa cells that corresponded to bovine DERL1. Two transcripts of 3 and 2.6 kb were detected by Northern blot analysis, and showed variations in expression among tissues. During follicular development, DERL1 expression was greater in day 5 dominant follicles compared to small follicles, ovulatory follicles, or corpus luteum (CL). Within the CL, DERL1 mRNA expression was intermediate in midcycle, and lowest in late cycle as compared to early in the estrous cycle. Western blot analyses demonstrated the presence of DERL1 in the bovine CL at days 5, 11, and 18 of the estrous cycle. Co-immunoprecipitation using luteal tissues showed that DERL1 interacts with class I MHC but not with VIMP or p97 ATPase. The interaction between DERL1 and MHC I suggests that, in the CL, DERL1 may regulate the integrity of MHC I molecules that are transported to the ER membrane. Furthermore, the greater expression of DERL1 mRNA is associated with the active follicular development and early luteal stages, suggesting a role of DERL1 in tissue remodeling events and maintenance of function in reproductive tissues.

Asunto(s)

Bovinos/genética , Cuerpo Lúteo/metabolismo , Proteínas de la Membrana/genética , Folículo Ovárico/metabolismo , Animales , Bovinos/metabolismo , Clonación Molecular , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Folículo Ovárico/fisiología , ARN Mensajero/metabolismo , Distribución Tisular

RESUMEN

Wound healing in horses is complicated, particularly when wounds are on the limb. The objectives of this study were to clone equine thrombospondin II (THBS2) and secreted protein acidic and cysteine-rich (SPARC) cDNAs and to compare the spatiotemporal expression of mRNAs and proteins during repair of body and limb wounds. These molecules were targeted in view of their potential biological contribution to angiogenesis, which is exacerbated during the repair of limb wounds in horses. Cloning was achieved by screening size-selected cDNA libraries previously derived from 7-day-old wounds. Expression was studied in unwounded skin and in samples from 1, 2, 3, 4, and 6 wk old wounds of the body and limb. Temporal gene expression was determined by semiquantitative RT-PCR, while protein expression was mapped immunohistochemically. The temporal pattern of expression for both genes was similar; wounding caused immediate upregulation of mRNA, which did not return to baseline by the end of the study, and overexpression was noted in body relative to limb wounds. Immunostaining for THBS2 and SPARC was induced by wounding, though no differences in stain location or intensity were detected between body and limb wounds. This study is the first to characterize equine cDNA for THBS2 and SPARC and to document mRNA expression over the different phases of repair. THBS2 and SPARC might modulate angiogenesis during wound healing in the horse, which could protect against the disproportionate fibroplasia commonly afflicting limb wounds and leading to the development of exuberant granulation tissue.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Osteonectina/metabolismo , ARN Mensajero/metabolismo , Trombospondinas/metabolismo , Cicatrización de Heridas/fisiología , Animales , Moléculas de Adhesión Celular/genética , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Caballos , Immunoblotting , Inmunohistoquímica , Modelos Lineales , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Osteonectina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombospondinas/genética

RESUMEN

The development of exuberant granulation tissue, a situation that in some ways resembles the human keloid, compromises both the aesthetic and functional outcomes of wound repair in horses. To help elucidate the underlying molecular mechanisms the spatio-temporal expression of lumican (LUM) mRNA and protein for their potential contributions to tissue remodelling of body and limb wounds, was examined in an established experimental model. Expression was studied in intact skin and in samples of 1-, 2-, 3-, 4- and 6-week-old wounds of the body and forelimb. Temporal gene expression was determined by reverse transcriptase polymerase chain reaction, and protein expression was mapped immunohistochemically. A significant increase in LUM mRNA expression was observed in response to wounding at both anatomical locations, and a significantly higher mRNA level was recorded in thoracic than in limb wounds at weeks 1, 3 and 6 of repair. The immunohistochemical observations partially corroborated the mRNA data. To the authors' knowledge this study is the first to document that the cDNA for LUM is expressed over the different phases of wound repair in horses and suggests that LUM might be involved in both inflammation and remodelling in response to dermal injury. Further studies are now required to verify and quantify the temporal expression of this protein to provide the basis for targeted therapies that might prevent the development of exuberant granulation tissue in horse wound repair.

Asunto(s)

Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Regulación de la Expresión Génica/fisiología , Tejido de Granulación/crecimiento & desarrollo , Caballos/lesiones , Sulfato de Queratano/metabolismo , Cicatrización de Heridas/fisiología , Animales , Proteoglicanos Tipo Condroitín Sulfato/genética , Clonación Molecular , ADN Complementario/genética , Inmunohistoquímica , Sulfato de Queratano/genética , Lumican , ARN Mensajero/genética , ARN Mensajero/metabolismo , Piel/metabolismo , Heridas y Lesiones/metabolismo

RESUMEN

OBJECTIVE: To clone full-length equine pigment epithelium-derived factor (PEDF) complementary DNA (cDNA) and to evaluate its temporal expression during repair of wounds in horses. ANIMALS: 4 clinically normal 2-to 3-year-old Standardbred mares. PROCEDURES: Full-length equine PEDF cDNA was cloned by screening size-selected cDNA libraries derived from biopsy specimens obtained from the wound edge 7 days after experimental creation of a 6.25-cm(2) full-thickness wound in the skin of the lateral thoracic wall. Expression was evaluated in normal skin and in biopsy specimens obtained weekly from experimentally induced wounds on the trunk and limbs of horses. Temporal gene expression was determined by use of reverse transcriptase PCR assay. RESULTS: Equine PEDF shared 87% sequence and 88% peptide homology with human PEDF. Wounding caused upregulation of PEDF mRNA, which did not return to baseline by the end of the study in either anatomic location. Relative overexpression was evident in wounds on the trunk, compared with expression for wounds on the limbs. CONCLUSIONS AND CLINICAL RELEVANCE: This study characterized full-length equine cDNA for PEDF and determined that the gene for PEDF appeared to be upregulated in response to dermal wounding. Although the cause of exuberant granulation tissue is probably multifactorial, these data suggested that PEDF, via its potent antiangiogenic capabilities, may contribute to superior healing in wounds on the trunks of horses by protecting such wounds from excessive formation of vascular granulation tissue that characterizes wounds on the limbs of this species.

Asunto(s)

Proteínas del Ojo/genética , Caballos/genética , Factores de Crecimiento Nervioso/genética , Serpinas/genética , Cicatrización de Heridas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/sangre , Femenino , Expresión Génica , Caballos/sangre , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/sangre , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Serpinas/biosíntesis , Serpinas/sangre

RESUMEN

Healing of wounds located on the distal limbs of horses is often complicated by retarded epithelialization and the development of exuberant granulation tissue (proud flesh). Treatments that definitively resolve this pathological process are still unavailable. Molecular studies of the repair mechanism might contribute to the development of new therapeutic strategies. The study presented herein aimed to clone the full length cDNA and to study the spatio-temporal expression profile of mRNA and protein for LAMR1, previously attributed a role in wound epithelialization, during the repair of body and limb wounds in the horse. Cloning was achieved by screening a cDNA library previously derived from 7-day wound biopsies. Expression was studied in unwounded skin and in samples from 1-, 2-, 3-, 4- and 6-week-old wounds of the body and limb. Temporal gene expression was determined by real time polymerase chain reaction (RT-PCR) while protein expression was mapped immunohistochemically. Full-length cDNA for equine LAMR1 was shown to be highly similar to that of other species. The mRNA expression of LAMR1 was significantly up-regulated only in thoracic wounds, 4 and 6 weeks following wounding (upon epithelialization). Cutaneous wounding induced protein expression at both locations. Our data suggest that up-regulation of LAMR1 protein might favour epithelialization during wound healing. However, its interaction with ligands other than laminin complicates data interpretation. Future studies should quantitatively verify the temporal expression of this protein in order to provide the basis for targeted therapies that might enhance epithelialization.

Asunto(s)

Regulación de la Expresión Génica/fisiología , Caballos/metabolismo , Receptores de Laminina/metabolismo , Cicatrización de Heridas/fisiología , Heridas y Lesiones/veterinaria , Animales , ADN Complementario/genética , Immunoblotting , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Laminina/genética , Piel/metabolismo , Heridas y Lesiones/metabolismo

RESUMEN

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.

Asunto(s)

Estradiol/biosíntesis , Células de la Granulosa/enzimología , Progesterona/biosíntesis , Factor de Crecimiento Transformador beta1/fisiología , 17-Hidroxiesteroide Deshidrogenasas/genética , Andrógenos/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Bovinos , Células Cultivadas , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Estradiol/metabolismo , Estrona/metabolismo , Femenino , Hormona Folículo Estimulante/farmacología , Expresión Génica/efectos de los fármacos , Glutatión Transferasa/genética , Fosfoproteínas/genética , Pregnenolona/metabolismo , Progesterona/metabolismo , ARN Mensajero/análisis , Receptores de HFE/genética , Estimulación Química , Factor de Crecimiento Transformador beta1/metabolismo

RESUMEN

BACKGROUND: Wounds on horse limbs can develop exuberant granulation tissue which resembles the human keloid. Clues gained from the study of over-scarring in horses might help control fibro-proliferative disorders. OBJECTIVES: The aim of the present study was to clone full-length equine ANXA2 cDNA then to study spatio-temporal expression of ANXA2 and MMP1 mRNA and protein, potential contributors to remodeling, during repair of body (normal) and limb (fibro-proliferative) wounds in an established horse wound model. METHODS: Cloning of ANXA2 was achieved by screening size-selected cDNA libraries. Expression was studied in intact skin and in biopsies of 1, 2, 3, 4 and 6-week-old wounds of the body and limb. Temporal gene expression was determined by semi-quantitative RT-PCR while protein expression was mapped immunohistochemically. RESULTS: ANXA2 mRNA was up-regulated only in body wounds, corroborating the superior and prompt tissue turnover at this location. Immunohistochemistry partially substantiated the mRNA data in that increased staining for ANXA2 protein was detected in neo-epidermis which formed more rapidly and completely in body wounds. MMP1 mRNA levels in body wounds significantly surpassed those of limb wounds in week one biopsies. The protein was abundant in migrating epithelium of limb wounds at weeks two and four; conversely, body wounds in which epithelialization was near complete showed diminished staining of MMP1. CONCLUSION: We conclude that ANXA2 and MMP1 might participate in remodeling during wound healing in the horse, and that differences in expression may contribute to the excessive proliferative response seen in the limb.

Asunto(s)

Anexina A2/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Piel/metabolismo , Cicatrización de Heridas/fisiología , Heridas y Lesiones/metabolismo , Animales , Anexina A2/genética , Biopsia , Proliferación Celular , ADN Complementario/genética , Femenino , Regulación de la Expresión Génica , Caballos , Metaloproteinasa 1 de la Matriz/genética , Modelos Animales , ARN Mensajero/metabolismo , Piel/citología , Piel/patología , Heridas y Lesiones/patología

RESUMEN

The protein tyrosine phosphatase (PTP) superfamily of enzymes functions with protein tyrosine kinases to regulate a broad spectrum of fundamental physiological processes. Addition of the PTP inhibitor potassium bisperoxo(1,10-phenanthroline)oxo-vanadate(V) [bpV(phen)] to the culture medium of human ovarian cancer cells (OVCAR-3) resulted in a dose-dependent decrease in the formation of tumors in a 3-D culture system. An evaluation of the potency of bpV(phen) in vivo confirmed the anti-tumor activity. Further study of the mechanism of action revealed a 40% decrease in Cdk2 kinase activity, an elevated level of Cdk2/p27(kip1), and the appearance of Cdk2/SHP-1 complexes. Therefore, a cytostatic dose of a PTP inhibitor increases the intracellular levels of Cdk2/p27(kip) and Cdk2/SHP-1 complexes, which indicate the presence of additional mechanisms underlying the anti-tumor activity.

Asunto(s)

Adenocarcinoma/metabolismo , Proteínas de Unión al Calcio/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Neoplasias Ováricas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Femenino , Humanos , Compuestos Organometálicos/farmacología , Fenantrolinas/farmacología , Células Tumorales Cultivadas