Effect of GRP75 inhibition on cell stress ın trophoblasts modeled with Preeclampsia

Preeclampsia (PE) is a disease characterized by uteroplacental vascular dysfunction resulting from the insufficient invasion of extravillous trophoblasts (EVTs) into the uterine spiral arteries. This condition leads to intermittent arterial blood flow and repeated ischemia/reperfusion episodes, ultimately resulting in hypoxia-induced oxidative stress. It is known that such oxidative stress increases mitochondrial reactive oxygen species (mtROS) in placental tissue and trophoblast cell lines, leading to mitochondrial dysfunction.Calcium (Ca²⁺) is transferred from the endoplasmic reticulum (ER) to the mitochondria via mitochondria-associated ER membranes (MAMs). Although Ca²⁺ is normally required for mitochondrial oxidative phosphorylation, its overload disrupts this process, enhances ROS production, and causes dysfunction in both the mitochondria and ER, triggering cell death.In this thesis study, we aimed to develop a potential therapeutic approach for PE by improving cell viability and trophoblast invasion through the inhibition of Ca²⁺ transfer in a hypoxia-induced oxidative stress environment designed to mimic the PE model. In this context, it was targeted to prevent excessive Ca²⁺ transfer by reducing the expression of Glucose-Regulated Protein 75 (GRP75), which functions as a 'molecular bridge' in MAMs.The JAR trophoblast cell line was used in our project. Hydrogen peroxide (H₂O₂) was applied to induce an in vitro hypoxia-related oxidative stress condition mimicking PE, and the chemical agent MKT077 was used to reduce GRP75 expression. Accordingly, four groups were included in our study: (1) sham, (2) H₂O₂, (3) H₂O₂ + MKT077, and (4) MKT077.An EC50 analysis was performed to determine the most appropriate doses of H₂O₂ and MKT077 to be applied in each group. To evaluate the suppressive effect of MKT077 on JAR trophoblast cells, GRP75 protein expression levels, the ER stress marker Glucose-Regulated Protein 78 (GRP78), and the PE biomarker soluble fmslike tyrosine kinase-1 (sFLT1) were analyzed immunocytochemically. In addition, the effects of this chemical on cell viability, cell migration, and cell death in JAR trophoblasts were also evaluated.Our project is the first study to investigate the effects of GRP75 on trophoblasts. The findings from this study are expected to contribute to elucidating the underlying mechanisms of PE and to the development of potential therapeutic strategies.