Pastuschek Jana, Poetzsch Jenny, Morales-Prieto Diana M, Schleußner Ekkehard, Markert Udo R, Georgiev Georgi
Placenta-Lab, Department of Obstetrics, University Hospital Jena, Germany.
Placenta-Lab, Department of Obstetrics, University Hospital Jena, Germany; Institute of Immunology, Hannover Medical School, Hannover, Germany.
J Reprod Immunol. 2015 Apr;108:48-55. doi: 10.1016/j.jri.2015.03.002. Epub 2015 Mar 16.
The development of the follicle and competent oocyte is highly coordinated, requiring interplay among several systems. These implicate endocrine, immune, and metabolic signals, intrafollicular paracrine factors from theca, mural, and cumulus granulosa cells, and the oocyte itself. Granulosa cells play a key role in their interaction. COV434 is one of the few human granulosa cell lines that can be used as an in vitro model for ovarian research. We aimed to evaluate the possible activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway by IL-6-type cytokines leukemia inhibitory factor (LIF) and oncostatin M (OSM) in COV434 cells. Expression of GP130 (glycoprotein 130), STAT3 (signal transducer and activators of transcription 3), PIAS3 (protein inhibitor of activated STAT 3), and SOCS3 (suppressor of cytokine signaling 3) genes after stimulation with LIF or OSM was assessed using RT-qPCR (real-time PCR). GP130 transcripts were significantly upregulated after incubation with LIF or OSM for 24h. Expression of the STAT3 gene was stimulated only after incubation with LIF, but not OSM. SOCS3 showed significant upregulation for all time periods and the levels of PIAS3 were initially down- and after 24h upregulated. Furthermore, the major signaling components of the JAK/STAT pathway, GP130 and STAT3, and the kinase activation patterns of STAT3, were examined at protein level. We found constitutive protein expression for GP130, STAT3, pSTAT3(ser727) and upregulation of pSTAT3(tyr705) by LIF and OSM. Our results demonstrate the activation of the JAK/STAT pathway by LIF and OSM in human granulosa cells.
卵泡和有功能的卵母细胞的发育高度协调，需要多个系统之间的相互作用。这些系统涉及内分泌、免疫和代谢信号，卵泡膜、壁层和卵丘颗粒细胞的卵泡内旁分泌因子，以及卵母细胞本身。颗粒细胞在它们的相互作用中起关键作用。COV434是少数可作为卵巢研究体外模型的人颗粒细胞系之一。我们旨在评估白细胞介素-6型细胞因子白血病抑制因子（LIF）和抑瘤素M（OSM）在COV434细胞中对Janus激酶/信号转导和转录激活因子（JAK/STAT）信号通路的可能激活作用。使用实时定量聚合酶链反应（RT-qPCR）评估LIF或OSM刺激后糖蛋白130（GP130）、信号转导和转录激活因子3（STAT3）、活化STAT3的蛋白抑制剂（PIAS3）和细胞因子信号转导抑制因子3（SOCS3）基因的表达。与LIF或OSM孵育24小时后，GP130转录本显著上调。仅在与LIF孵育后，而不是与OSM孵育后，STAT3基因的表达受到刺激。SOCS3在所有时间段均显著上调，PIAS3水平最初下降，24小时后上调。此外，在蛋白水平检测了JAK/STAT通路的主要信号成分GP130和STAT3，以及STAT3的激酶激活模式。我们发现GP130、STAT3、磷酸化STAT3（ser727）的组成型蛋白表达以及LIF和OSM对磷酸化STAT3（tyr705）的上调。我们的结果证明了LIF和OSM在人颗粒细胞中激活了JAK/STAT通路。
