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1.
Cells ; 13(10)2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38786034

RESUMEN

Lysophosphatidic acid (LPA) species, prevalent in the tumor microenvironment (TME), adversely impact various cancers. In ovarian cancer, the 18:0 and 20:4 LPA species are selectively associated with shorter relapse-free survival, indicating distinct effects on cellular signaling networks. Macrophages represent a cell type of high relevance in the TME, but the impact of LPA on these cells remains obscure. Here, we uncovered distinct LPA-species-specific responses in human monocyte-derived macrophages through unbiased phosphoproteomics, with 87 and 161 phosphosites upregulated by 20:4 and 18:0 LPA, respectively, and only 24 shared sites. Specificity was even more pronounced for downregulated phosphosites (163 versus 5 sites). Considering the high levels 20:4 LPA in the TME and its selective association with poor survival, this finding may hold significant implications. Pathway analysis pinpointed RHO/RAC1 GTPase signaling as the predominantly impacted target, including AHRGEF and DOCK guanine exchange factors, ARHGAP GTPase activating proteins, and regulatory protein kinases. Consistent with these findings, exposure to 20:4 resulted in strong alterations to the actin filament network and a consequent enhancement of macrophage migration. Moreover, 20:4 LPA induced p38 phosphorylation, a response not mirrored by 18:0 LPA, whereas the pattern for AKT was reversed. Furthermore, RNA profiling identified genes involved in cholesterol/lipid metabolism as selective targets of 20:4 LPA. These findings imply that the two LPA species cooperatively regulate different pathways to support functions essential for pro-tumorigenic macrophages within the TME. These include cellular survival via AKT activation and migration through RHO/RAC1 and p38 signaling.


Asunto(s)
Lisofosfolípidos , Macrófagos , Proteómica , Transducción de Señal , Humanos , Lisofosfolípidos/metabolismo , Macrófagos/metabolismo , Proteómica/métodos , Fosforilación/efectos de los fármacos , Fosfoproteínas/metabolismo
2.
Mol Oncol ; 16(17): 3146-3166, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35451191

RESUMEN

Survival of ovarian carcinoma is associated with the abundance of immunosuppressed CD163high CD206high tumor-associated macrophages (TAMs) and high levels of arachidonic acid (AA) in the tumor microenvironment. Here, we show that both associations are functionally linked. Transcriptional profiling revealed that high CD163 and CD206/MRC1 expression in TAMs is strongly associated with an inhibition of cytokine-triggered signaling, mirrored by an impaired transcriptional response to interferons and IL-6 in monocyte-derived macrophages by AA. This inhibition of pro-inflammatory signaling is caused by dysfunctions of the cognate receptors, indicated by the inhibition of JAK1, JAK2, STAT1, and STAT3 phosphorylation, and by the displacement of the interferon receptor IFNAR1, STAT1 and other immune-regulatory proteins from lipid rafts. AA exposure led to a dramatic accumulation of free AA in lipid rafts, which appears to be mechanistically crucial, as the inhibition of its incorporation into phospholipids did not affect the AA-mediated interference with STAT1 phosphorylation. Inhibition of interferon-triggered STAT1 phosphorylation by AA was reversed by water-soluble cholesterol, known to prevent the perturbation of lipid raft structure by AA. These findings suggest that the pharmacologic restoration of lipid raft functions in TAMs may contribute to the development new therapeutic approaches.


Asunto(s)
Neoplasias , Microambiente Tumoral , Ácido Araquidónico/metabolismo , Humanos , Macrófagos/metabolismo , Microdominios de Membrana/metabolismo , Neoplasias/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal
4.
Theranostics ; 11(3): 1377-1395, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33391540

RESUMEN

Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions. Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release. Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca2+-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options. Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.


Asunto(s)
Ácido Araquidónico/farmacología , Macrófagos/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Calcio/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Femenino , Fosfolipasas A2 Grupo II/metabolismo , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/metabolismo , Neoplasias Ováricas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos
5.
J Clin Med ; 9(6)2020 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-32570986

RESUMEN

Epithelial-mesenchymal transition (EMT) is an important process of cell remodeling characterized by the gradual loss of the epithelial phenotype and progressive gain of a mesenchymal phenotype. EMT is not an all-or-nothing process, but instead a transition of epithelial to mesenchymal cells with intermediate cell states. Recently, EMT was described in endometriosis, and many EMT-specific pathways like Twist, Snail, Slug, Zinc finger E-box-binding homeobox 1/2 (ZEB1/2), E/N-cadherin, keratins, and claudins are involved. However, as pointed out in this review, a comparison of the eutopic endometrium of women with and without endometriosis yielded only subtle changes of these EMT markers. Furthermore, only very few alterations in cell-cell contacts could be found but without changes in the epithelial phenotype. This suggests only a partial EMT which is not a prerequisite for the detachment of endometrial cells and, thus, not critical for the first step(s) in the pathogenesis of endometriosis. In contrast, the majority of changes in the EMT-related marker expression were found in the ectopic endometrium, especially in the three endometriotic entities, ovarian, peritoneal, and deep infiltrating endometriosis (DIE), compared with the eutopic endometrium. In this review, we examine the most important EMT pathways described in endometriosis and propose that partial EMT might result from the interaction of endometrial implants with their surrounding microenvironment.

6.
Reprod Biomed Online ; 40(6): 769-778, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32362572

RESUMEN

RESEARCH QUESTION: How closely related are adenomyotic and endometrial glands? DESIGN: In this study, the mRNA and protein database www.proteinatlas.org was searched for proteins expressed predominantly in the endometrial glands. Specificity was tested with tissue microarrays. Biopsy specimens of endometrial, adenomyotic tissue, or both, were collected after surgery from 21 women without endometriosis, 20 women with endometriosis, 18 women with adenomyosis together with endometriosis and 12 women with adenomyosis alone. Tissue expression was analysed by immunohistochemistry. RESULTS: Two proteins were identified: calcyphosine (CAPS), and msh homeobox 1 (MSX1). A high abundance and good specificity in endometrial glands were found. Both proteins, CAPS and MSX1, showed a high specificity for endometrium and are both localized in the luminal cells and epithelial cells of the glandular and adenomyotic glands. No significant differences were found between CAPS- and MSX1-positive endometrial glands between cases with and without endometriosis. Also, no cycle-specific different expression was found. Furthermore, a close relationship between the adenomyotic glands and the endometrial glands for CAPS (range 63.0-98.3%) and for MSX1 (range 87.1-99.3%) could be demonstrated. Only 11.2% and 6.8% negative glands for CAPS and MSX1 were identified in all tissues from all patients, respectively; none were negative for both proteins. CONCLUSIONS: Taken together, our results show that the protein expression pattern of adenomyosis is nearly identical to those of the endometrium with and without endometriosis, thus suggesting endometrial glands as the main source for adenomyotic glands.


Asunto(s)
Adenomiosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Endometriosis/metabolismo , Endometrio/metabolismo , Factor de Transcripción MSX1/metabolismo , Adenomiosis/patología , Adenomiosis/cirugía , Adulto , Endometriosis/patología , Endometriosis/cirugía , Endometrio/patología , Femenino , Humanos , Persona de Mediana Edad
7.
Arch Gynecol Obstet ; 301(4): 1003-1011, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32140805

RESUMEN

PURPOSE: Claudins as the major components of tight junctions are important in maintaining cell-cell integrity and thus function as a barrier. Dysregulation of the claudins is often associated with loss of the epithelial phenotype, a process called epithelial-mesenchymal transition (EMT), which most often results in gain of migrative and invasive properties. However, the role of claudins in the endometrium or endometriosis has only rarely been examined. METHODS: In this study, we investigated localization of claudin-2 and claudin-3 in the eutopic and ectopic endometrium with immunohistochemistry. A detailed quantification with HSCORE was performed for claudin-2 and claudin-3 in endometrium without endometriosis and in cases with endometriosis compared to the three endometriotic entities: peritoneal, ovarian, and deep-infiltrating endometriosis. RESULTS: We found a preferential localization of both claudins in the glandular and the luminal epithelial cells in the endometrium with and without endometriosis. Quantification of localization of both claudins showed no differences in eutopic endometrium of control cases compared to cases with endometriosis. Furthermore, both claudins are localized highly similar in the ectopic compared to the eutopic endometrium, which is in clear contrast to previously published data for claudin-3. CONCLUSION: From our results, we conclude that localization of claudin-2 and claudin-3 is highly stable in eutopic and ectopic endometrium without any loss of the epithelial phenotype and thus do not contribute to the pathogenesis of endometriosis.


Asunto(s)
Claudina-2/metabolismo , Claudina-3/metabolismo , Endometriosis/genética , Endometrio/patología , Estudios de Casos y Controles , Endometriosis/patología , Femenino , Humanos
8.
Int J Mol Sci ; 20(24)2019 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-31835434

RESUMEN

Transforming growth factor-ßs (TGF-ßs) signal after binding to the TGF-ß receptors TßRI and TßRII. Recently, however, betaglycan (BG) was identified as an important co-receptor, especially for TGF-ß2. Both proteins are involved in several testicular functions. Thus, we analyzed the importance of BG for TGF-ß1/2 signaling in Sertoli cells with ELISAs, qRT-PCR, siRNA silencing and BrdU assays. TGF-ß1 as well as TGF-ß2 reduced shedding of membrane-bound BG (mBG), thus reducing the amount of soluble BG (sBG), which is often an antagonist to TGF-ß signaling. Treatment of Sertoli cells with GM6001, a matrix metalloproteinases (MMP) inhibitor, also counteracted BG shedding, thus suggesting MMPs to be mainly involved in shedding. Interestingly, TGF-ß2 but not TGF-ß1 enhanced secretion of tissue inhibitor of metalloproteinases 3 (TIMP3), a potent inhibitor of MMPs. Furthermore, recombinant TIMP3 attenuated BG shedding. Co-stimulation with TIMP3 and TGF-ß1 reduced phosphorylation of Smad3, while a combination of TIMP3/TGF-ß2 increased it. Silencing of BG as well as TIMP3 reduced TGF-ß2-induced phosphorylation of Smad2 and Smad3 significantly, once more highlighting the importance of BG for TGF-ß2 signaling. In contrast, this effect was not observed with TIMP3/TGF-ß1. Silencing of BG and TIMP3 decreased significantly Sertoli cell proliferation. Taken together, BG shedding serves a major role in TGF-ß2 signaling in Sertoli cells.


Asunto(s)
Proliferación Celular , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Células de Sertoli/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta2/metabolismo , Animales , Línea Celular , Dipéptidos/farmacología , Masculino , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratas , Células de Sertoli/citología , Proteínas Smad/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo
9.
J Steroid Biochem Mol Biol ; 191: 105372, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31042565

RESUMEN

Cardiotonic steroids such as ouabain are potent inhibitors of the sodium pump and have been widely used for centuries in the treatment of congestive heart failure. In recent decades, however, they have also been identified as hormone-like molecules that trigger signaling cascades of physiological relevance by using the various sodium pump α subunit isoforms as receptors. The murine Leydig cell line MLTC-1 expresses both the ubiquitous, relatively ouabain-insensitive α1 isoform of the sodium pump and the ouabain-sensitive α3 isoform that is normally found in neuronal cells. The physiological relevance of the simultaneous presence of the two isoforms in Leydig cells has not been previously addressed. MLTC-1 Leydig cells contain lipid droplets (LDs) and are capable of progesterone biosynthesis when stimulated by luteinizing hormone (LH). When exposed to low nanomolar concentrations of ouabain, they respond with stimulation of Erk1/2, CREB, and ATF-1 phosphorylation, LD enlargement, and perilipin2 mobilization to the LDs. As a result, progesterone biosynthesis is augmented. Abrogation of α3 isoform expression by siRNA prevents all of the above responses, indicating that it is the hormone/receptor-like interaction of ouabain exclusively with this isoform that triggers the signaling events that normally occur when LH binds to its receptor. Considering that ouabain is produced endogenously and is found in seminal fluid, one can speculate that effects of this substance on germ and somatic cells of the testis might play a role in male reproductive physiology.


Asunto(s)
Cardiotónicos/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Esteroides/metabolismo , Animales , Vías Biosintéticas/efectos de los fármacos , Línea Celular , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Progesterona/metabolismo , Isoformas de Proteínas/metabolismo
10.
Reproduction ; 158(2): R41-R47, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30978694

RESUMEN

A diagnosis of endometriosis is based upon the histological identification of endometrial tissue at ectopic sites which are commonly located on the pelvic organs, the peritoneum and ovary. In rare cases, ectopic lesions can be found in other organs, such as kidney, bladder, lung or brain. Diagnosis is achieved by laparoscopic intervention followed by histological confirmation of endometriotic tissue. Prevalence is estimated at approximately 10% in the general female population with many patients experiencing pain and/or infertility. Currently, the implantation hypothesis by Sampson is the most accepted hypothesis about the pathogenesis of endometriosis. However, the occurrence of endometriosis in patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome who sometimes lack a uterus or endometrium seems to suggest metaplasia as a cause of endometriosis. A critical reevaluation of the literature about MRKH does not reveal conclusive evidence of an association of uterus/endometrium agenesis and endometriosis. Most often only MRI diagnoses of uterus/endometrium agenesis and only very rarely conclusive histological evidence of the endometriotic lesions are presented. In contrast, whenever biopsies were performed endometriosis always appeared together with uterus/endometrium remnants. Taken together, we suggest that MRKH patients only develop endometriosis if a uterus/endometrium is present which underscores and not contradicts the implantation hypothesis of Sampson.


Asunto(s)
Trastornos del Desarrollo Sexual 46, XX/complicaciones , Endometriosis/etiología , Conductos Paramesonéfricos/anomalías , Anomalías Congénitas , Femenino , Humanos , Metaplasia , Útero/anomalías
11.
Reprod Sci ; 26(9): 1181-1192, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-30514158

RESUMEN

Claudins are the major components of tight junctions and are often deregulated in human cancer, permitting escape of cancer cells along with the acquisition of invasive properties. Similarly, endometrial cells also show invasive capabilities; however, the role of tight junctions in endometriosis has only rarely been examined. In this study, we analyzed the protein expression and localization of claudin-7 and claudin-11 in human eutopic and ectopic endometrium and endometrial cell lines. We identified claudin-7 primarily at the basolateral junctions of the glandular epithelial cells in eutopic endometrium as well as in the ectopic lesions in nearly all glands and cysts. Quantification of claudin-7 localization by HSCORE showed a slight increase in peritoneal and deep infiltrating endometriosis (DIE) compared to eutopic endometrium. In contrast, claudin-11 was localized mainly in the apicolateral junctions in nearly all glandular epithelial cells of the eutopic endometrium. Interestingly, we observed a deregulation of claudin-11 localization to a basal or basolateral localization in ovarian (P < .001), peritoneal (P < .01), and DIE (P < .05) and a moderately decreased abundance in ovarian endometriosis. In endometrial cell lines, claudin-7 was only present in epithelial Ishikawa cells, and silencing by small-interfering RNA increased cell invasiveness. In contrast, claudin-11 could be demonstrated in Ishikawa and endometriotic 12Z and 49Z cells. Silencing of claudin-11 decreased invasiveness of 12Z slightly but significantly in 49Z. We suggest that although claudin-7 and claudin-11 can be found in nearly all eutopic and ectopic epithelial cells, the impaired localization of claudin-11 in ectopic endometrium might contribute to the pathogenesis of endometriosis.


Asunto(s)
Claudinas/metabolismo , Endometriosis/metabolismo , Endometrio/metabolismo , Células Epiteliales/metabolismo , Ciclo Menstrual/metabolismo , Enfermedades Peritoneales/metabolismo , Línea Celular , Femenino , Humanos , Uniones Estrechas/metabolismo
12.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(11): 1369-1377, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30591146

RESUMEN

Extracellular lysophosphatidic acid (LPA) and the G-protein-coupled LPA receptors (LPAR) are involved in cell migration and invasion and found in the human endometrium. However, underlying mechanisms resulting in cellular invasion have been rarely investigated. We used stromal endometrial T-HESC, epithelial endometriotic 12Z, 49Z and Ishikawa cells. Interestingly, proliferation of T-HESC cells was strongly increased after LPA treatment, whereas the epithelial cell lines only showed a moderate increase. LPA increased invasion of 12Z and 49Z strongly and significantly. The LPAR inhibitor Ki16425 (LPAR1/3) attenuated significantly LPA-induced invasiveness of 12Z, which was confirmed by LPAR1 and LPAR3 siRNAs, showing that both LPA receptors contribute to invasiveness of 12Z cells. Investigation of cell invasion with an antibody-based protease array revealed mainly differences in cathepsins and especially cathepsin B between 12Z compared to the less invasive Ishikawa. Stimulation with LPA showed a time- and dose-dependent increased secretion of cathepsin B which was inhibited by the Gq inhibitor YM-254890 and Gi/o inhibitor pertussis toxin in the 12Z cells, again highlighting the importance of LPAR1/3. The activity of intracellular and secreted cathepsin B was significantly upregulated in LPA-treated samples. Inhibition of cathepsin B with the specific inhibitor CA074 significantly reduced LPA-increased invasion of 12Z. Our results reveal a novel role of LPA-mediated secretion of cathepsin B which stimulated invasion of endometriotic epithelial cells mainly via LPAR1 and LPAR3. These findings may deepen our understanding how endometriotic cells invade into ectopic sites, and provide new insights into the role of LPA and cathepsin B in cellular invasion.


Asunto(s)
Catepsina B/metabolismo , Endometriosis/metabolismo , Lisofosfolípidos/efectos adversos , Regulación hacia Arriba , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Isoxazoles/farmacología , Propionatos/farmacología , Receptores del Ácido Lisofosfatídico/metabolismo , Factores de Tiempo
13.
J Steroid Biochem Mol Biol ; 182: 50-61, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29684479

RESUMEN

Although dehydroepiandrosterone sulfate (DHEAS) constitutes the most abundant steroid in humans, in-depth investigations of its effects are rather scarce. We address here DHEAS effects on the estrogen receptor-positive metastatic human breast cancer cell line MCF-7. We focus on DHEAS-mediated signaling that might influence expression of claudin-1 and matrix metalloproteinase-9 (MMP-9), both known to be critical factors for migration and invasiveness of various cancers, including breast cancer cells. Physiological concentrations of DHEAS trigger persistent phosphorylation of Erk1/2 in MCF-7 cells. Exposure of these cells for 24 h to 1 µM DHEAS also leads to a significant reduction of claudin-1 expression that cannot be prevented by high concentrations of the steroid sulfatase inhibitor STX64, indicating that desulfation and further conversion of DHEAS to some other steroid hormone is not required for this action. In addition, exposure of MCF-7 cells to the same concentration of DHEAS completely abolishes MMP-9 expression and considerably impairs cell migratory behavior. Abrogation of Gnα11 expression by siRNA prevents the stimulatory effect of DHEAS on Erk1/2 phosphorylation, consistent with a G-protein-coupled receptor being involved in the DHEAS-induced signaling. Nevertheless, Gnα11 also has direct effects that do not depend on DHEAS; thus, when Gnα11 expression is suppressed, expression of claudin-1 and MMP-9 as well as cell migration are significantly reduced. This is the first report demonstrating direct involvement of DHEAS and Gnα11 in the regulation of claudin-1 and MMP-9 expression and migration of MCF-7 cells.


Asunto(s)
Neoplasias de la Mama/patología , Movimiento Celular/efectos de los fármacos , Claudina-1/metabolismo , Sulfato de Deshidroepiandrosterona/farmacología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Claudina-1/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP/genética , Humanos , Células MCF-7 , Metaloproteinasa 9 de la Matriz/genética , Fosforilación , ARN Interferente Pequeño/genética , Transducción de Señal
14.
Reprod Sci ; 25(7): 1106-1115, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-28992748

RESUMEN

To analyze whether the endometrial and endometriotic microenvironment is involved in the pathogenesis of endometriosis, we characterized the stromal composition. We used CD90 for fibroblasts, α-smooth muscle actin for myofibroblasts as well as CD10 and CD140b for mesenchymal stromal cells. Quantification of eutopic endometrial stroma of cases without endometriosis showed a high percentage of stromal cells positive for CD140b (80.7%) and CD10 (67.4%), a moderate number of CD90-positive cells (57.9%), and very few α-smooth muscle actin-positive cells (8.5%). These values are highly similar to cases with endometriosis showing only minor changes: CD140b (76.7%), CD10 (63%), CD90 (53.9%), and α-smooth muscle actin (6.9%). There are no significant differences in the composition of CD140b- and CD10-positive stromal cells between the eutopic endometrial stroma and the 3 different endometriotic entities (ovarian, peritoneal, and deep infiltrating endometriosis), except for a significant difference between CD10-positive stromal cells in peritoneal lesions compared to ovarian lesions. However, the percentage of CD90-positive stromal cells was reduced in the 3 different endometriotic entities compared to the endometrium, especially significant in the ovarian lesions. In contrast, the percentage of α-smooth muscle actin-positive cells in the ovary was moderately increased. Taken together, the marker signature of eutopic endometrial and endometriotic stromal cells resembles mostly mesenchymal stromal cells. Our results show clearly that the proportion of the different stromal cell types in the endometrium with or without endometriosis does not differ significantly, thus suggesting that the stromal eutopic endometrial microenvironment does not contribute to the pathogenesis of endometriosis.


Asunto(s)
Endometriosis/patología , Endometrio/citología , Endometrio/patología , Células del Estroma/citología , Células del Estroma/patología , Adulto , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Neprilisina/análisis , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/análisis , Células del Estroma/metabolismo , Antígenos Thy-1/análisis
15.
Biochim Biophys Acta ; 1863(11): 2809-2819, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27599714

RESUMEN

In addition to the ubiquitous α1 isoform of the sodium pump, sperm cells also express a male-specific α4 isoform whose function has been associated with sperm motility, fertility, and capacitation. Here we investigate in the murine spermatogenic cell line GC-2 interactions of the α4 isoform with the cardiotonic steroid ouabain in signaling cascades involved in the non-classical action of steroid hormones. Exposure of GC-2 cells to low concentrations of ouabain stimulates the phosphorylation of Erk1/2 and of the transcription factors CREB and ATF-1. As a consequence of this signaling cascade, ouabain stimulates on the mRNA level the expression of integrins αv, ß3 and α5, whose expression is also modulated by the cAMP response element. Increased expression of integrins αv and ß3 is also seen in cultures of seminiferous tubules exposed to 10nM ouabain. At the protein level we observed a significant stimulation of ß3 integrin expression by ouabain. Abrogation of α4 isoform expression by siRNA leads to the complete suppression of all ouabain-induced signaling mentioned above, including its stimulatory effect on the expression of ß3 integrin. The results presented here demonstrate for the first time the induction of signaling cascades through the interaction of ouabain with the α4 isoform in a germ-cell derived cell line. The novel finding that these interactions lead to increased expression of integrins in GC-2 cells and the confirmation of these results in the ex vivo experiments indicate that hormone/receptor-like interactions of ouabain with the α4 isoform might be of significance for male physiology.


Asunto(s)
Hormonas Esteroides Gonadales/metabolismo , Integrinas/metabolismo , Ouabaína/farmacología , Túbulos Seminíferos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espermatozoides/efectos de los fármacos , Factor de Transcripción Activador 1/metabolismo , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Integrinas/genética , Masculino , Ratones , Fosforilación , Interferencia de ARN , Ratas Wistar , Túbulos Seminíferos/enzimología , ATPasa Intercambiadora de Sodio-Potasio/genética , Espermatozoides/enzimología , Técnicas de Cultivo de Tejidos , Transfección
16.
Cell Signal ; 28(8): 1075-85, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27164415

RESUMEN

In the classical signaling pathway, testosterone regulates gene expression by activating the cytosolic/nuclear androgen receptor. In the non-classical pathway, testosterone activates cytosolic signaling cascades that are normally triggered by growth factors. The nature of the receptor involved in this signaling pathway is a source of controversy. In the Sertoli cell line 93RS2, which lacks the classical AR, we determined that testosterone stimulates the non-classical signaling pathway, characterized by the phosphorylation of Erk1/2 and transcription factors CREB and ATF-1. We also demonstrated that testosterone increases the expression of the tight junction (TJ) proteins claudin-1 and claudin-5. Both of these proteins are known to be essential constituents of TJs between Sertoli cells, and as a consequence of their increased expression transepithelial resistance across Sertoli cell monolayers is increased. ZIP9 is a Zn(2+)transporter that was recently shown to be a membrane-bound testosterone receptor. Silencing its expression in 93RS2 Sertoli cells by siRNA completely prevents Erk1/2, CREB, and ATF-1 phosphorylation as well the stimulation of claudin-1 and -5 expression and TJ formation between neighboring cells. The study presented here demonstrates for the first time that in Sertoli cells testosterone acts through the receptor ZIP9 to trigger the non-classical signaling cascade, resulting in increased claudin expression and TJ formation. Since TJ formation is a prerequisite for the maintenance of the blood-testis barrier, the testosterone/ZIP9 effects might be significant for male physiology. Further assessment of these interactions will help to supplement our knowledge concerning the mechanism by which testosterone plays a role in male fertility.


Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Claudina-1/metabolismo , Claudina-5/metabolismo , Células de Sertoli/metabolismo , Transducción de Señal , Testosterona/metabolismo , Uniones Estrechas/metabolismo , Factor de Transcripción Activador 1/metabolismo , Animales , Western Blotting , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Impedancia Eléctrica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Silenciador del Gen/efectos de los fármacos , Masculino , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ratas , Células de Sertoli/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Testosterona/farmacología , Uniones Estrechas/efectos de los fármacos
17.
PLoS One ; 11(3): e0150143, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26938869

RESUMEN

Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 µM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the regulation and maintenance of male fertility.


Asunto(s)
Barrera Hematotesticular/efectos de los fármacos , Claudina-3/metabolismo , Claudina-5/metabolismo , Sulfato de Deshidroepiandrosterona/química , Regulación de la Expresión Génica , Células de Sertoli/metabolismo , Animales , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Silenciador del Gen , Masculino , Ratones , Microscopía Fluorescente , Fosforilación , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Piel/patología , Esteril-Sulfatasa/metabolismo , Ácidos Sulfónicos/metabolismo , Uniones Estrechas
18.
Mol Cell Endocrinol ; 405: 1-13, 2015 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-25666991

RESUMEN

The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the α4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well.


Asunto(s)
Cardiotónicos/farmacología , Claudina-1/metabolismo , Claudinas/metabolismo , Expresión Génica/efectos de los fármacos , Ouabaína/farmacología , Células de Sertoli/metabolismo , Animales , Barrera Hematotesticular/citología , Línea Celular , Claudina-1/genética , Claudinas/genética , Masculino , Ratas , Células de Sertoli/efectos de los fármacos , Uniones Estrechas/metabolismo , Activación Transcripcional
19.
Biochim Biophys Acta ; 1833(3): 511-9, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23220124

RESUMEN

Sertoli cells express α1 and α4 isoforms of the catalytic subunit of Na(+),K(+)-ATPase (sodium pump). Our recent findings demonstrated that interactions of the α4 isoform with cardiotonic steroids (CTS) like ouabain induce signaling cascades that resemble the so-called non-classical testosterone pathway characterized by activation of the c-Src/c-Raf/Erk1/2/CREB signaling cascade. Here we investigate a possible physiological significance of the activated cascade. The results obtained in the current investigation show that the ouabain-induced signaling cascade also leads to the activation of the CREB-related activating transcription factor 1 (ATF-1) in the Sertoli cell line 93RS2 in a concentration- and time-dependent manner, as demonstrated by detection of ATF-1 phosphorylated on Ser63 in western blots. The ouabain-activated ATF-1 protein was found to localize to the cell nuclei. The sodium pump α4 isoform mediates this activation, as it is ablated when cells are incubated with siRNA to the α4 isoform. Ouabain also leads to increased expression of steroidogenic acute regulator (StAR) protein, which has been shown to be a downstream consequence of CREB/ATF-1 activation. Taking into consideration that CTS are most likely produced endogenously, the demonstrated induction of StAR expression by ouabain establishes a link between CTS, the α4 isoform of the sodium pump, and steroidogenesis crucial for male fertility and reproduction.


Asunto(s)
Factor de Transcripción Activador 1/metabolismo , Cardiotónicos/farmacología , Ouabaína/farmacología , Fosfoproteínas/metabolismo , Células de Sertoli/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Factor de Transcripción Activador 1/genética , Animales , Western Blotting , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Técnicas para Inmunoenzimas , Masculino , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Isoformas de Proteínas , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genética
20.
Biochim Biophys Acta ; 1813(12): 2118-24, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21820472

RESUMEN

The α4 isoform of the Na(+),K(+)-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.


Asunto(s)
Glicósidos Cardíacos/farmacología , Ouabaína/farmacología , Células de Sertoli/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Proteína Tirosina Quinasa CSK , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Transporte Iónico/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Fosforilación/efectos de los fármacos , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/citología , Células de Sertoli/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genética , Testosterona/farmacología , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo
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