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Syndecan-4 regulates extravillous trophoblast migration by coordinating protein kinase C activation.

ABSTRACT: Extravillous trophoblast (EVT) invasion is an essential component of human placentation. Poor EVT invasion is associated with obstetrical complications including preeclampsia. Integration of cues from the extracellular environment is required for directional EVT invasion, but how EVTs coordinate responses to these cues is not well understood. Syndecan-4 (SDC4) is a transmembrane heparan sulfate proteoglycan that binds to, and modulates the activity of, many extracellular proteins implicated in placental development. Therefore, we determined the functional importance of SDC4 for EVT invasion. We found that SDC4 is expressed by a first trimester EVT line (HTR8), and in EVTs in placenta throughout pregnancy, with higher expression during early pregnancy than at term. Higher expression was also observed in placentas from preeclampsia compared to normotensive pregnancies. SDC4-deficient HTR8 EVTs exhibited reduced migration and Matrigel-based invasion, both under basal conditions and following exposure to basic fibroblast growth factor and heparin-binding epidermal growth factor. SDC4-deficient HTR8 EVTs also showed reduced protein kinase C-alpha (PKC?) and AKT phosphorylation. SDC4 directly bound to activated PKC? in EVTs, and inhibition of PKC? decreased EVT invasion and migration. Our findings reveal an essential role of SDC4 as a regulator of EVT motility, in part through coordination of PKC? activation.

SUBMITTER: Jeyarajah MJ

PROVIDER: S-EPMC6629623 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Syndecan-4 regulates extravillous trophoblast migration by coordinating protein kinase C activation.

Jeyarajah Mariyan J MJ Jaju Bhattad Gargi G Kops Brianna F BF Renaud Stephen J SJ

Scientific reports 20190715 1

Extravillous trophoblast (EVT) invasion is an essential component of human placentation. Poor EVT invasion is associated with obstetrical complications including preeclampsia. Integration of cues from the extracellular environment is required for directional EVT invasion, but how EVTs coordinate responses to these cues is not well understood. Syndecan-4 (SDC4) is a transmembrane heparan sulfate proteoglycan that binds to, and modulates the activity of, many extracellular proteins implicated in p ...[more]

PMID: 31308409

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Project description:Mutations in the LINC-HELLP non-coding RNA (HELLPAR) have been associated with familial forms of the pregnancy-specific HELLP syndrome. These mutations negatively affect extravillous trophoblast (EVT) differentiation from a proliferative to an invasive state and disturb the binding of RNA splicing complex proteins PCBP1, PCBP2, and YBX1 to LINC-HELLP. In this study, by using both in vitro and ex vivo experiments, we investigate if these proteins are involved in the regulation of EVT invasion during placentation. Additionally, we study if this regulation is due to alternative mRNA splicing. HTR-8/SVneo extravillous trophoblasts and human first trimester placental explants were used to investigate the effect of siRNA-mediated downregulation of PCBP1, PCBP2, and YBX1 genes on the differentiation of EVTs. Transwell invasion assays and proliferation assays indicated that upon knockdown of PCBP2 and, to a lesser extent, YBX1 and PCBP1, EVTs fail to differentiate toward an invasive phenotype. The same pattern was observed in placental explants where PCBP2 knockdown led to approximately 80% reduction in the number of explants showing any EVT outgrowth. Of the ones that still did show EVT outgrowth, the percentage of proliferating EVTs was significantly higher compared to explants transfected with non-targeting control siRNAs. To further investigate this effect of PCBP2 silencing on EVTs, we performed whole transcriptome sequencing (RNA-seq) on HTR-8/SVneo cells after PCBP2 knockdown. PCBP2 knockdown was found to have minimal effect on mRNA expression levels. In contrast, PCBP2 silencing led to a switch in splicing for a large number of genes with predominant functions in cellular assembly and organization, cellular function and maintenance, and cellular growth and proliferation and the cell cycle. EVTs, upon differentiation, alter their function to be able to invade the decidua of the mother by changing their cellular assembly and their proliferative activity by exiting the cell cycle. PCBP2 appears to be a paramount regulator of these differentiation mechanisms, where its disturbed binding to LINC-HELLP could contribute to dysfunctional placental development as seen in the HELLP syndrome.

Project description:BackgroundSERPINE2, one of the potent serpins belonging to the plasminogen activator (PA) system, is involved in the tissue remodeling. We previously demonstrated the expression patterns of Serpine2 in the mouse placenta and uterus, indicating that Serpine2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. In this study, we further investigated the expression pattern of SERPINE2 in the human placenta and explored possible functional roles of SERPINE2 in regulating trophoblast activity.MethodsPlacental tissues from various trimesters were collected for real-time reverse-transcription polymerase chain reaction quantification. Immunohistochemical staining was performed in placental tissues to assure localization of SERPINE2. SERPINE2 small interfering (si) RNA was applied to suppress its expression in villous explants and extravillous trophoblast-like 3A cells. Subsequent experiments to evaluate SERPINE2 levels, villous outgrowth, trophoblast invasion, and tube formation were performed.ResultsSERPINE2 messenger RNA was detected in the human placenta during pregnancy with the highest levels in the third trimester. The SERPINE2 protein was present in villous syncytiotrophoblasts and trophoblasts of chorionic villi for anti-SERPINE2 immunostaining. Extravillous trophoblasts in the chorionic plate and basal plate confronting the invasive face of anchoring villi were also positive. In most decidual cells, SERPINE2 was observed in the cytoplasm. In addition, fibrinoid deposit was weakly immunoreactive. Introduction of SERPINE2 siRNA into villous explants and trophoblast cells led to significantly reduced villous outgrowth, and trophoblastic migration and invasion. Moreover, capillary-like network formation of 3A cells in Matrigel was greatly attenuated by SERPINE2 siRNA and SERPINE2 antiserum.ConclusionsThese data identify the temporal and spatial SERPINE2 distribution in the human placenta and suggest its possible role in modulating tissue remodeling of extravillous trophoblasts in the placenta during pregnancy.

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Syndecan-4 regulates extravillous trophoblast migration by coordinating protein kinase C activation.

Publications

Syndecan-4 regulates extravillous trophoblast migration by coordinating protein kinase C activation.

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