Progesterone triggers Rho kinase-cofilin axis during in vitro and in vivo endometrial decidualization

Abstract

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 progesterone 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, metabolism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualization 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 decidualization 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 = 15) and PCOS patients (N = 9). In parallel, lysates of non-cultured ESCs isolated from proliferative (N = 4) or secretory (N = 4) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N = 3) or secretory endometrium (N = 3), and in proliferative or secretory full tissue samples from controls (N = 8 and N = 9, respectively) or PCOS patients (N = 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 expression 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. MAIN RESULTS AND THE ROLE OF CHANCE During in vitro decidualization, the activities of PKA, protein kinase B (Akt/PKB), and ROCK are increased while the activity of casein kinase 2 (CK2) is decreased; these initial trends are observable after 4-day treatment (P < 0.05) and are further augmented following the 9-day treatment (P < 0.001) with mixtures containing progesterone and 8-Br-cAMP or forskolin. The presence of progesterone is necessary for activation of ROCK, yet it is dispensable in the case of PKA and Akt/PKB; in comparison to controls, PCOS patient-derived ESCs feature dampened response to progesterone. In non-cultured ESCs isolated from secretory vs proliferative phase tissue, only activity of ROCK is increased (P < 0.01). ROCK2 protein levels are slightly elevated in secretory versus proliferative ESCs (relative mean standard deviation < 50%), and ROCK2 mRNA is elevated in mid-secretory versus proliferative full tissue samples (P < 0.05) obtained from controls but not PCOS patients. Activation of ROCK2 downstream signalling results in increase of phospho-S3 CFL1 in secretory endometrium (P < 0.001) as well as in vitro decidualized ESCs (P < 0.01) from controls but not PCOS patients. ROCK2-triggered alterations in the cytoskeleton are reflected by the significantly decreased motility of in vitro decidualized ESCs (P < 0.05). LARGE SCALE DATA Proteomic and phosphoproteomic data are available via ProteomeXchange with identifier PXD026243. LIMITATIONS, REASONS FOR CAUTION The number of biological samples was limited. The duration of protocol for isolation of non-cultured ESCs from tissue can potentially affect phosphorylation pathways in cells, yet the possible artefacts were minimized by the identical treatment of proliferative and secretory samples. WIDER IMPLICATIONS OF THE FINDINGS The study demonstrated the benefits of combining the focussed kinase activity assay with wide-scale phosphoproteomics and showed the need for detailed elaboration of the in vitro decidualization protocols. ROCK was identified as the novel target of interest in decidualization, which requires closer attention in further studies—including the context of decidualization-related subfertility and infertility. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the Estonian Ministry of Education and Research, and the Estonian Research Council (PRG1076, PRG454, PSG230 and PSG608), Enterprise Estonia (EU48695), Horizon 2020 innovation grant (ERIN, Grant no. EU952516) of the European Commission, the COMBIVET ERA Chair, H2020-WIDESPREAD-2018-04 (Grant agreement no. 857418), the Academy of Finland (Project grants 315921 and 321763), the Finnish Medical Foundation and The Sigrid Juselius Foundation. The authors confirm that they have no conflict of interest with respect to the content of this article.

Endocrine disrupting chemicals interfere with decidualization of human primary endometrial stromal cells in vitro

“…Spent culture media were collected and stored at -80°C. At the end of the 9-day treatment, the cells were rinsed with phosphate-buffered saline (PBS; from Sigma-Aldrich, Steinheim, Germany) and lysed as previously reported (34). The lysis buffer components were from the following sources: HEPES and NaCl -Calbiochem (Darmstadt, Germany); EDTA -Scharlau (Barcelona, Spain); Triton X-100 -Ferak (Berlin, Germany); phenylmethylsulfonyl fluoride (PMSF) -AppliChem (Darmstadt, Germany); cOmplete ™ protease inhibitor cocktail -Roche (Basel, Switzerland).…”

“…The total protein concentration in each sample was then normalized to the same level between the samples lysed on the same day using the kinase assay buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.005% Triton X-100, 0.5 mg/mL BSA, 5 mM dithiothreitol). For measuring activity of CK2, the previously described protocol was used (34). Briefly, ARC-1530 probe (final total concentration of 2 nM) was added to the lysate dilutions (total protein concentration of 200-400 mg/mL).…”

“…Decidualization was measured by quantification of PRL in spent culture media samples (from the last 72 h of the in vitro treatment) and assessment of kinase activities in cell lysates following the 9-day in vitro decidualization as described (34). In media samples, the total protein concentration was measured as described above.…”

“…Here, our ambition was to study the impact of a wide selection of EDCs on human endometrial cell decidualization using our recently established in vitro model for decidualization (34). This model is based on primary endometrial stromal cells from hysterectomies (or endometrial biopsies) that are simulated to decidualize with a mixture containing progesterone and estradiol; the process is monitored using a suite of kinases reflecting activation of intracellular signaling, and secretion of PRL as the final outcome (34).…”

“…Here, our ambition was to study the impact of a wide selection of EDCs on human endometrial cell decidualization using our recently established in vitro model for decidualization (34). This model is based on primary endometrial stromal cells from hysterectomies (or endometrial biopsies) that are simulated to decidualize with a mixture containing progesterone and estradiol; the process is monitored using a suite of kinases reflecting activation of intracellular signaling, and secretion of PRL as the final outcome (34). In this way, instead of focusing on one marker, we assessed a characteristic fingerprint involving several previously identified markers of decidualization, which is a complex physiological process that cannot be explained by modulation of a single pathway.…”