Progesterone triggers Rho kinase-cofilin axis during in vitro and in vivo endometrial decidualization Darja Lavogina 1,2, *, Artjom Stepanjuk 1,3 , Maire Peters 1,4 , Ku ¨ lli Samuel 1 , Sergo Kasvandik 5 , Masuma Khatun 6 , Riikka K. Arffman 6 , Erki Enkvist 2 , Kaido Viht 2 , Sergei Kopanchuk 2 , Freddy La ¨ttekivi 7,8 , Agne Velthut-Meikas 9 , Asko Uri 2 , Terhi T. Piltonen 6 , Ago Rinken 2 , and Andres Salumets 1,4,10,11, * 1 Competence Centre on Health Technologies, Tartu, Estonia 2 Department of Bioorganic Chemistry, Institute of Chemistry, University of Tartu, Tartu, Estonia 3 Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia 4 Department of Obstetrics and Gynaecology, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia 5 Proteomics Core Facility, Institute of Technology, University of Tartu, Tartu, Estonia 6 Department of Obstetrics and Gynecology, PEDEGO Research Unit, Medical Research Center, Oulu University Hospital, University of Oulu, Oulu, Finland 7 Department of Pathophysiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia 8 COMBIVET ERA Chair, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Estonia 9 Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia 10 Estonian Genome Centre, Institute of Genomics, University of Tartu, Tartu, Estonia 11 Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden *Correspondence address. Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu, Estonia. Tel: þ372 737 5296; E-mail: darja.lavogina@ut.ee https://orcid.org/0000-0001-5167-2817 (D.L.); Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, 14186 Stockholm, Sweden. Tel: þ46 70 245 7834; E-mail: andres.salumets@ki.se http://orcid.org/0000-0002-1251-8160 (A.S.) Submitted on November 30, 2020; resubmitted on May 28, 2021; editorial decision on June 07, 2021 STUDY QUESTION: Can a combination of the focussed protein kinase assays and a wide-scale proteomic screen pinpoint novel, clinically relevant players in decidualization in vitro and in vivo? SUMMARY ANSWER: Rho-dependent protein kinase (ROCK) activity is elevated in response to the combined treatment with proges- terone and 8-Br-cAMP during in vitro decidualization, mirrored by increase of ROCK2 mRNA and protein levels and the phosphorylation levels of its downstream target Cofilin-1 (CFL1) in secretory versus proliferative endometrium. WHAT IS KNOWN ALREADY: Decidualization is associated with extensive changes in gene expression profile, proliferation, metabo- lism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualiza- tion of endometrial stromal cells (ESCs) can be reportedly induced using multiple protocols with variable physiological relevance. In our previous studies, cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA)/prolactin axis that is classically upregulated during deciduali- zation showed dampened activation in ESCs isolated from polycystic ovary syndrome (PCOS) patients as compared to controls. STUDY DESIGN, SIZE, DURATION: In vitro decidualization studies were carried out in passage 2 ESCs isolated from controls (N ¼ 15) and PCOS patients (N ¼ 9). In parallel, lysates of non-cultured ESCs isolated from proliferative (N ¼ 4) or secretory (N ¼ 4) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N ¼ 3) or secretory endo- metrium (N ¼ 3), and in proliferative or secretory full tissue samples from controls (N ¼ 8 and N ¼ 9, respectively) or PCOS patients (N ¼ 10 for both phases). PARTICIPANTS/MATERIALS, SETTING, METHODS: The activities of four target kinases were explored using kinase-responsive probes and selective inhibitors in lysates of in vitro decidualized ESCs and non-cultured ESCs isolated from tissue at different phases of the menstrual cycle. In the latter lysates, wide-scale proteomic and phosphoproteomic studies were further carried out. ROCK2 mRNA ex- pression was explored in full tissue samples from controls or PCOS patients. The immunofluorescent staining of phosphorylated CFL1 was performed in full endometrial tissue samples, and in the in vitro decidualized fixed ESCs from controls or PCOS patients. Finally, the cellular migration properties were explored in live in vitro decidualized ESCs. V C The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com Human Reproduction, Vol.36, No.8, pp. 2230–2248, 2021 Advance Access Publication on July 16, 2021 doi:10.1093/humrep/deab161 ORIGINAL ARTICLE Reproductive biology Downloaded from https://academic.oup.com/humrep/article/36/8/2230/6322743 by guest on 16 June 2023