Laboratorio de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República, Instituto de Higiene, Montevideo, Uruguay.
Cell Biology Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay.
Am J Reprod Immunol. 2022 Jun;87(6):e13534. doi: 10.1111/aji.13534. Epub 2022 Mar 14.
Persistent hypoxia and inflammation beyond early pregnancy are involved in a bad outcome because of defective trophoblast invasiveness. Tissue transglutaminase (TG2) coregulates several cell functions. An aberrant expression and/or transamidation activity could contribute to placental dysfunction.
The first-trimester trophoblast cell line (Swan-71) was used to study TG2 expression and cell functions in the absence or presence of inflammatory cytokines (TNF-α, IL-1β) or chemical hypoxia (CoCl ). We analyzed The concentration of cytokines in the supernatant by ELISA; Cell migration by scratch assay; NF-κB activation by detection of nuclear p65 by immunofluorescence or flow cytometry using a Swan-71 NF-κB-hrGFP reporter cell line. Tissue transglutaminase expression was analyzed by immunoblot and confocal microscopy. Expression of spliced mRNA variants of tissue transglutaminase was analyzed by RT-PCR. Transamidation activity was assessed by flow cytometry using 5-(biotinamido)-pentylamine substrate.
Chemical hypoxia and TGase inhibition, but not inflammatory stimuli, decreased Swan-71 migration. IL-6 production was also decreased by chemical hypoxia, but increased by inflammation. Intracellular TGase activity was increased by all stimuli, but NF-κB activation was observed only in the presence of proinflammatory cytokines. TG2 expression was decreased by CoCl and TNF-α. Translocation of TG2 and p65 to nuclei was observed only with TNF-α, without colocalization. Differential relative expression of spliced variants of mRNA was observed between CoCl and inflammatory stimuli.
The observed decrease in total TG2 expression and relative increase in short variants under hypoxia conditions could contribute to impaired trophoblast invasion and impact on pregnancy outcome.
由于滋养细胞浸润缺陷，妊娠早期后持续缺氧和炎症与不良结局有关。组织转谷氨酰胺酶（TG2）共同调节多种细胞功能。异常表达和/或转酰胺酶活性可能导致胎盘功能障碍。
使用早孕期滋养细胞株（Swan-71），在不存在或存在炎症细胞因子（TNF-α、IL-1β）或化学缺氧（CoCl2）的情况下，研究 TG2 的表达和细胞功能。我们通过 ELISA 分析上清液中细胞因子的浓度；通过划痕试验分析细胞迁移；通过免疫荧光或使用 Swan-71 NF-κB-hrGFP 报告细胞系通过流式细胞术检测核 p65 来分析 NF-κB 激活。通过免疫印迹和共聚焦显微镜分析组织转谷氨酰胺酶的表达。通过 RT-PCR 分析组织转谷氨酰胺酶剪接 mRNA 变体的表达。通过使用 5-（生物素氨基）戊基胺底物的流式细胞术评估转酰胺酶活性。
化学缺氧和 TGase 抑制，但不是炎症刺激，降低了 Swan-71 的迁移。化学缺氧也降低了 IL-6 的产生，但炎症增加了。所有刺激均增加了细胞内 TGase 活性，但仅在存在促炎细胞因子的情况下才观察到 NF-κB 激活。CoCl2 和 TNF-α 降低了 TG2 的表达。仅在 TNF-α存在下观察到 TG2 和 p65 向核易位，但没有共定位。CoCl2 和炎症刺激之间观察到 mRNA 剪接变体的差异相对表达。
在缺氧条件下观察到总 TG2 表达减少和短变体相对增加，可能导致滋养细胞侵袭受损，并影响妊娠结局。
