Abstract
Progesterone causes vascular smooth muscle cell relaxation through membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ receptor (PAQR) family, and nuclear PRs (nPRs). However, beneficial vascular effects of progesterone in preventing pre-atherosclerosis and the involvement of mPRs and nPRs remain unclear. The results show short- to long-term treatments with 100 nM progesterone (P4) and specific agonists for mPRs, OD 02-0, and nPRs, R5020, inhibited pre-atherosclerotic events in human umbilical vein endothelial cells (HUVECs), decreasing focal adhesion (FA) by monocytes, FA signaling, HUVEC migration and invasion, and vinculin expression. Progesterone and OD 02-0, but not R5020, inhibited phosphorylation of Src and focal adhesion kinase, critical kinases of FA signaling, within 20 min and migration and invasion of HUVECs and monocyte adhesion after 3 h. These inhibitory P4 and 02-0 effects were attenuated with MAP kinase and Pi3k inhibitors, indicating involvement of these kinases in this mPR-mediated action. However, after 16 h, OD 02-0 was no longer effective in inhibiting FA signaling, while both progesterone and R5020 decreased the activity of the two kinases. Knockdown of receptor expression with siRNA confirmed that mPRα mediates short-term and nPR long-term inhibitory effects of progesterone on FA signaling. Thus, progesterone inhibition of FA signaling and pre-atherosclerosis is coordinated through mPRα and nPRs.
Introduction
Atherosclerotic cardiovascular ischemic complications such as myocardial infarction and stroke are the leading causes of morbidity worldwide (Laslett et al. 2012, Fowkes et al. 2013). Atherosclerosis is a complex multistage pathogenic process which arises from vascular endothelial cell dysfunction and shares many characteristics with cancers, such as endothelial cell migration (Gimbrone & García-Cardeña 2016, Theodorou & Boon 2018, Shafi 2020). During the early stages of atherosclerosis, circulating cholesterol-rich lipoproteins accumulate at the site of the focal lesion and in the subendothelial space, triggering an immune and inflammatory response, which in turn promotes the adhesion and permeation of circulating monocytes into the intima (Tabas et al. 2007). The monocytes differentiate into macrophages which promote lipid accumulation (Mestas & Ley 2008, Checkouri et al. 2021), which over time causes the development of atherosclerotic plaques resulting in hardening of the arterial walls and ultimately obstruction of blood flow.
Migration of vascular endothelial cells is a critical angiogenic process in the pathogenesis and progression of atherosclerosis (Bochkov et al. 2006, Zhang et al. 2020). The neovascularization in the intima resulting from abnormal endothelial cell migration promotes the initiation, development, and rupture of atherosclerotic plaques (Fosbrink et al. 2006, Koga et al. 2017). Migration involves the creation of focal adhesions (FAs) at the leading edge of the cell, which interact with the extracellular matrix to generate the force for movement and also breakdown of FA at the trailing edge of the cell (Li et al. 2005). The formation of FA and cell migration by structural proteins including paxillin and vinculin and promotion of angiogenesis and atherosclerosis are controlled by activation (phosphorylation) of two critical protein kinases, cellular Src kinase (Src) and focal adhesion kinase (FAK) (Tarzami et al. 2005, Weis et al. 2008, Jarray et al. 2015, Yamaura et al. 2019). Vinculin is also a vital component in FA during angiogenesis and atherosclerosis (Meyer et al. 1994, Deroanne et al. 1996, Huveneers et al. 2012). However, despite extensive research over the last decade on the factors that regulate these proteins, the involvement of sex steroid hormones in mediating FA and FA signaling remains unresolved due to disparate findings from different studies (Nofer 2012). Angiotensin II enhances the development of atherosclerotic lesions (Daugherty et al. 2000) and monocyte migration (Kintscher et al. 2001) and also stimulates FA in vascular muscle cells (Okuda et al. 1995) and vascular endothelial cells (Montiel et al. 2005, Jiménez et al. 2009). However, information is currently lacking on the potential beneficial effects of sex steroid hormones on angiotensin-enhanced FA in the cardiovascular system and the underlying mechanisms.
The lower risk of developing atherosclerosis and cardiovascular complications in middle-aged premenopausal women than in middle-aged men and postmenopausal women has been attributed to the presence of female sex steroid hormones in the circulation (Man et al. 2020). There is substantial evidence that estrogens exert numerous anti-atherosclerotic effects through regulating endothelial cell functions (Simoncini & Genazzani 2000). In contrast, less information is currently available on anti-atherosclerotic effects of progesterone on vascular endothelial cell functions. However, the relatively few studies on progesterone effects on HUVEC migration have generated contradictory findings. Progesterone was found to activate FAK and enhance HUVEC migration in one study (Zheng et al. 2012), whereas in three other studies progesterone inhibited HUVEC migration and FA or FAK signaling (Otsuki et al. 2001, Bustos et al. 2008, Lee et al. 2015). The finding that the nPR antagonist, RU486, blocked progesterone inhibition of migration suggests it is mediated through the nPR (Lee et al. 2015). However, the involvement of other progesterone receptors (PRs), the membrane progesterone receptors (mPRs) which belong to the progesterone and adipoQ (PAQR) superfamily, in this anti-atherosclerotic action of progesterone has not been investigated. Clearly, additional research is required to confirm the inhibitory effect of progesterone on HUVEC migration and its mechanism of action.
Membrane progesterone alpha (mPRα) is the intermediary in nongenomic actions of progesterone to regulate numerous physiological functions (Thomas 2008, Thomas & Pang 2012, 2013). We have demonstrated that mPRα mediates rapid protective and potentially antihypertensive actions of progesterone in HUVECs through increasing endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) secretion (Pang et al. 2015) and that low concentrations of progesterone and estradiol-17β exert additive effects on NO production (Pang & Thomas 2017). Progesterone also exerts direct beneficial effects on vascular smooth muscle cells (VSMCs) through mPRα to rapidly activate intracellular signaling pathways, decrease cytoplasmic Ca2+ levels, and stimulate Ca2+ uptake by the sarcoplasmic reticulum, resulting in smooth muscle relaxation (Pang & Thomas 2018, 2019). Collectively, these findings suggest that mPRs mediate important beneficial effects of progesterone on vascular tone and blood pressure. However, it is not known whether they have protective role in preventing other cardiovascular complications such as atherosclerotic lesions.
In the present study, we examined the effects of treatments with a physiologically relevant concentration of progesterone (100 nM), in the midrange of circulating progesterone levels in women during the third trimester of pregnancy (Monteiro et al. 2021), on several components of pre-atherosclerosis, monocyte adhesion, migration/invasion, and FA signaling in HUVECs. The receptors mediating these progesterone actions were identified using siRNA and specific agonists for mPRα, Org OD 02-0 (OD 02-0), and nPR, R5020. In addition, the involvement of MAP kinase and PI3K/Akt signaling in mPR-dependent progesterone signaling was examined. Moreover, the influence of progesterone on angiotensin II-enhanced FA signaling was investigated in order to determine whether progesterone is potentially effective in attenuating FA under more severe pre-pathological conditions when FA signaling is elevated.
Materials and methods
Reagents and chemicals
Progesterone was purchased from Sigma-Aldrich, promegestone (R5020) from Perkin Elmer, and Org OD 02-0 (OD 02-0) from Organon (Oss, Netherlands). All other chemical reagents were obtained from Sigma-Aldrich unless otherwise stated. Human mPRα antibody was custom made by Sigma-Genosys (Houston, TX, USA), and its specificity was validated previously (Thomas et al. 2007). Antibodies against phosphorylated FAK (Tyr397, Cat# 8556s), Src (Cat# 2108s) and phosphorylated Src (Tyr416, Cat# 2101s) were purchased from Cell Signaling Technologies. Antibodies for nPR (sc-130071), FAK (Cat# sc-271126), Ki-67 (sc-23900), and vinculin (Cat# sc-73614) were purchased from Santa Cruz Biotechnology. Angiotensin II was obtained from EMD Millipore. The PI3K inhibitor wortmannin and specific MEK1/2 inhibitor AZD6244 were purchased from Selleckchem (Houston, TX, USA).
Cell culture
Ea.hy926 immortalized HUVECs (926 cells, female) and primary HUVECs (male and female mixed) were purchased from American Type Culture Collection (ATCC) and incubated in DMEM medium (Sigma-Aldrich) supplemented with 5% FBS. The medium was changed every 2–3 days, and cells were either immediately used for experiments or subcultured when they were over 90% confluent (passages 2–4). The culture medium was replaced with fresh DMEM without serum for 2–3 h before experimentation. Cells were treated with progestins in the presence or absence of pretreatments (such as inhibitors) for various time periods prior to harvesting for subsequent analysis. Expression levels of mPRα and nPR in the 926 cells were compared with those in the primary HUVECs (passages 2–3) to confirm the physiological relevance of the results obtained with 926 cells.
Immunocytochemistry detection of mPRα and nPR
Immunocytochemical (ICC) analysis on mPRα and nPR expression in 926 cells was performed following procedures published previously using the specific mPRα and nPR antibodies (Pang et al. 2015) used in the Western blot analyses. Briefly, 926 cells were grown on glass coverslips, fixed with 2% paraformaldehyde and 0.5% glutaraldehyde, and then permeabilized with 0.5% Triton X-100. Cells were blocked with 2% bovine serum albumin for 1.5 h and then incubated with mPRα (1:1000), cadherin (1:500, plasma membrane marker, Abcam ab287970), and nPR (1:500) antibodies overnight at 4°C. The cells were washed, followed by incubation with AlexaFluor 488 secondary antibodies for 1 h and washed three times prior to mounting the coverslips on glass slides with ProLong Gold antifade reagent (Invitrogen). Fluorescent-labeled mPR and nPR proteins in 926 cells were visualized using a Nikon inverted microscope with NIS-Elements Imaging System (Nikon).
Western blot analysis
Western blot analyses were performed on 926 cells following procedures published previously (Pang et al. 2015, Pang & Thomas 2017). Cell lysate samples were prepared using 1× RIPA buffer supplemented with protease and phosphatase inhibiters (Thermo Scientific) and incubated in 5× reducing sample buffer (Pierce) for 1 min at room temperature and then boiled for 10 min prior to loading onto a 10% polyacrylamide gel. Proteins were separated by polyacrylamide gel electrophoresis, then transferred from gels onto nitrocellulose membranes (Bio-Rad), washed three times with TBS and blocked with blocking reagent (LI-COR Biosciences, Lincoln, NE, USA). Membranes were then incubated overnight at 4°C in TBS + blocking reagent containing primary antibodies (1:2000). Membranes were subsequently incubated with fluorescent-conjugated secondary antibodies (1:2500) (LI-COR) in PBS for 1 h at room temperature and then washed three times and scanned with the Odyssey® Infrared Imaging System (LI-COR). Relative densities of the protein bands in the Western blot analyses were quantitated using Image J software (https://imagej.nih.gov/ij/), and the phosphorylated Src and FAK bands were all normalized to those of total Src and FAK densitometries, respectively.
Cell invasion assay
926 cells were subcultured in Corning BioCoat Matrigel Invasion Chambers (Corning) in a 24-well plate and incubated for 3 h (mid-term treatment) or 16 h (long-term treatment) with different progestin treatments, and then cells that had penetrated the micropores in the membrane of the chamber inserts were fixed with 10% formalin, stained, and observed following the manufacturer’s instructions. Five fixed microscopic fields of view (upper, lower, left, right, and center) were photographed, and all the cells in the images on the bottom side of the membranes were counted and calculated.
Cell migration wound-healing assay
926 cells were incubated in a 12-well plate overnight in serum-free media prior to making scratches (4–6 scratches/well) in the cell monolayer on the bottom of the plate with a pipette tip. Cells were then incubated with different progestins (3 wells/treatment) for 3 h (mid-term) and 16 h (long term). Plates were photographed at the beginning of the treatments (0 h) and after 3 and 16 h incubation, and distances between the migrating cells from each side of the scratch were measured and gap closure rates were calculated compared to values at 0 h.
Monocyte adhesion assay
926 cells were grown on gelatin-coated coverslips until they were about 85% confluent in a 12-well plate, and then they were treated with progestins for 3 or 16 h in serum-free medium. On the second day, monocytes (ATCC) (~5 x 105/mL) were preincubated with calcein-AM (2 μM) for 30 min, washed with DMEM, and then seeded on top of HUVECs in the wells. After a 2 h incubation, cells were visually checked for adhesion by gently washing them two times with PBS and then fixed with 4% paraformaldehyde for 20 min, followed by mounting the coverslips on glass slides with DAPI-Prolong reagent (Invitrogen). Cells were subsequently observed with FITC filter on Nikon fluorescent inverted microscope, and all adhered cells in five fixed microscopic fields of view (upper, lower, left, right, and center) were photographed, counted and expressed as a percentage of the monocytes adhering in vehicle controls.
Flow cytometry detection of monocytes focal adhesion
At the end of the 16 h coincubation of 926 cells/monocyte mixtures with progestins, cells were washed two times and then detached from the culture wells by coincubation with 0.25% trypsin at 37°C for 5 min. The cell mixtures were centrifuged and washed twice with PBS to remove any traces of trypsin at 4°C and assayed for FA using a flow cytometer (FACSAria™ III, BD Biosciences, Franklin Lakes, NJ, USA) with a FITC detection laser. The monocyte populations were gated, and the ratios of monocytes/HUVECs were calculated.
Knockdown of mPRα and nPR expression with small interfering RNA
Antisense small interfering RNA (siRNA) oligos for mPRα (Silencer Select siRNA) and nPR (h2) were purchased from Thermo Fisher Scientific and Santa Cruz Biotechnology, respectively. 926 endothelial cells were transfected with siRNA (10 nM) or nontargeting oligos (10 nM) using Lipofectamine 3000 (Thermo Scientific). The siRNA mix was replaced with normal medium after 48 h incubation, and cells were cultured for an additional 24 h before being used in experiments.
Statistics
Significances between treatment groups were calculated using GraphPad Prism Software (version 3.02). Results are expressed as means ± s.e.m. of at least six observations, and all experiments were repeated at least three times. Student’s unpaired t-test was used for unpaired comparisons, and one-way or two-way ANOVA with Newman–Keuls’ multiple comparison tests were used to determine differences between multiple experimental treatments.
Results
Characterization of mPRα and nPR expression in 926 cells
Expression levels of mPRα and nPR in incubated 926 cells were examined by Western blot analysis and compared to those in normal primary HUVECs (also from ATCC) to confirm that 926 cells had a similar profile to primary HUVECs for the two receptors. Western blot results confirmed that there were no significant differences in the protein levels of mPRα and nPR (PRa and PRb) between the 926 cells (mixed female and male fetus donors) and primary HUVECs (female fetus donors) after 2–3 passages, suggesting the sex of the fetus donor does not influence the expression of PRs under these experimental conditions (Fig. 1A and B). The expression and localization of mPRα and nPR in the 926 cells were confirmed by ICC (Fig. 1C). The fluorescent images show the presence of mPRα throughout the cytoplasm and on the cell membrane, whereas the nPR is localized in the nucleus.
Characterization of mPRα and nPR expression in immortalized Ea.hy926 (926) HUVECs and in primary HUVECs. (A) Representative Western blot analysis of protein expression of nuclear PR (PRa and PRb) and mPRα in both 926 cells and HUVECs (A). The Western blot analysis was repeated three times, and similar results were obtained on each occasion. (B) Quantitative comparison of the protein bands from the three Western blots of the two cells. Bars in the graph denote means ± s.e.m. (n = 3). Results were analyzed by Student’s t-test, and no significant differences were found in the expression of the same proteins in the two cell lines (B). (C) Immunocytochemical detection of mPRα and nPR in the 926 cells. IgG, control IgG for mPRα antibody (left) and nPR antibody (right). Scale bars in images: 10 μm.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Mid- and long-term effects of progesterone, OD 02-0, and promegestone (R5020) on cell migration/invasion of 926 cells
Treatments with 100 nM progesterone and OD 02-0 for 3 h significantly reduced migration and invasion of the 926 cells compared to vehicle controls in the wound-healing (Fig. 2A and B) and invasion assays (Fig. 2C and D), as shown by greater relative gap widths and fewer cells that invaded, whereas 100 nM R5020 was ineffective in both assays. In contrast, longer term (16 h) treatment with 100 nM R5020 was as effective as progesterone and OD 02-0 in the migration assay (Fig. 2A and B) and cell invasion assay (Fig. 2C and D), significantly reducing migration and the number of cells that passed through the membrane micropores compared to the vehicle controls (Fig. 2A, B, C and D). The Ki-67 protein levels were not significantly altered after 16 h treatments with progesterone, OD 02-0, and R5020, suggesting these treatments did not cause any changes in cell proliferation and/or apoptosis (Supplementary Fig. 1, see section on supplementary materials given at the end of this article). These results suggest that progesterone inhibition of endothelial cell migration/invasion involves both mPR and nPR depending on the treatment duration, the mid-term (3 h) progesterone action being mediated solely through mPRα.
Mid- and long-term effects of progesterone (P4) OD 02-0 (02-0) and R5020 treatments on migration and invasion in Ea.hy926 endothelial cells. (A and B) Representative cell migration images taken at 0, 3, and 16 h of 100 nM progestin treatments in the wound-healing assay (A) and the scratch gap closure ratios of the cells after steroid treatments for 3 and 16 h (B). (C and D) Representative images of cells that had invaded the micropores on the membranes of the incubation chambers at 3 and 16 h (C) and the percentage of invaded cells with 100 nM progestin treatments vs vehicle controls (Veh) (D). Bars in the graphs denote means ± s.e.m. (n = 6 (B), n = 4 (D)). Results were analyzed by one-way ANOVA followed by Newman–Keul’s multiple comparison test. (B and D) * and #, significantly different compared to Veh for the same treatment duration, 3 and 16 h, respectively, in the post hoc test (P < 0.05), ##, P < 0.01.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Mid- and long-term effects of progesterone, OD 02-0, and R5020 on monocyte focal adhesion to 926 cells
Pretreatment of 926 cells with 100 nM progesterone and OD 02-0 for 3 and 16 h significantly reduced the number of monocytes that adhered to them compared to vehicle controls as shown in the fluorescent images, whereas R5020 did not show any effect at 3 h but reduced the numbers of adhered monocytes at 16 h (Fig. 3A and B). These effects at 16 h were confirmed by reductions in the higher energy FITC signals in flow cytometry analyses (Fig. 3C and D). The results showing that all these progestins exert inhibitory effects on focal cell adhesion suggest that both mPRs and nPR are involved in this long-term progesterone action.
Mid- and long-term effects of progesterone (P4), OD 02-0 (02-0), and R5020 treatments on monocyte adhesion to Ea.hy926 cells. (A and B) Representative images of monocytes taken with a fluorescent microscope after 3 and 16 h of 100 nM progestin treatments in the monocyte adhesion assay (A) and the relative percentage of the monocytes adhered to the 926 cells (B). (C) Flow cytometer-gated FITC fluorescent signal (P1) of labeled monocytes from monocytes/926 cell mixtures. (D) Percentage of the total monocytes that had adhered calculated from the flow cytometry results. Veh, vehicle control. Bars in the graphs denote means ± s.e.m. (n = 4–6). Results were analyzed by one-way ANOVA followed by Newman–Keul’s multiple comparison test. (B) * and #, significantly different compared to Veh for the same treatment duration in 3 and 16 h, respectively, in the post hoc test (P < 0.05). (D) * significantly different compared to Veh in the post hoc test (P < 0.05).
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Short-term effects of progesterone, OD 02-0, and R5020 on Src and FAK phosphorylation and role of MAP kinase and PI3/Akt kinases in progesterone and OD 02-0 effects
Concentration-dependent effects of progesterone on phosphorylated Src and FAK (pSrc and pFAK) levels after 20 min treatments showed that 100 nM progesterone significantly decreased both pSrc and pFAK levels, whereas 20 nM progesterone also decreased pFAK significantly but did not significantly lower pSrc (Supplementary Fig. 2A and B). Therefore, for subsequent short-term experiments, 926 cells were incubated for 20 min with 100 nM progesterone and the other progestins. A time-course study showed that the 100 nM progesterone-induced decrease of pSrc and pFAK persisted after incubation for 30, 60, and 90 min (Supplementary Fig. 2C and D). A progestin specificity experiment showed that 100 nM progesterone and OD 02-0, a mPR specific agonist, significantly decreased levels of pSrc and pFAK, whereas the same concentration of R5020, a nPR agonist, was ineffective (Fig. 4A and B). These data suggest that progesterone exerts short-term inhibitory effects on the phosphorylation of Src and FAK through mPR and not through nPR.
Short-term effects of progesterone (P4), OD 02-0 (02-0), and R5020 treatments on phosphorylation of Src and FAK and involvement of MAP and PI3/Akt kinases in the short-term effects of progesterone (P4) and OD 02-0 (02-0) on focal adhesion signaling in Ea.hy926 cells. (A and B) Effects of 100 nM progesterone, 02-0, and R5020 treatments for 20 min on levels of pSrc (A) and pFAK (B). (C, D, E, and F) Cells were pretreated with AZD6244 (AZD, a MAP kinase inhibitor, 100 nM) and wortmannin (WM, an PI3K inhibitor, 500 nM) for 30 min before 20-min treatments with 100 nM P4 and 02-0. The levels of phosphorylated Src (pSrc; C and E) and FAK (pFAK; D and F) were detected using Western blot analysis. Upper panels in figures are representative Western blot images and the lower panels show the quantitative analysis of mean relative densitometry of the protein bands in the images from three assays. Veh, vehicle control; CTL, control groups; M, molecular weight marker. Bars in the graphs denote means± s.e.m. (n = 6). Results were analyzed by one-way (A and B) and two-way (C, D, E, and F) ANOVA followed by Newman–Keul’s multiple comparison test. (A and B) *, significantly different compared to Veh in the post hoc test (P < 0.05). (C, D, E, and F) *, significantly different compared to Veh in in the same treatment group in the post hoc test (P < 0.05).
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Involvement of MAP and PI3/Akt kinases in rapid effects of progesterone and OD 02-0 to decrease phosphorylation of Src and FAK in 926 cells was examined by pretreatment of cells with AZD6244 and wortmannin, potent MAP kinase, and PI3 kinase inhibitors, at concentrations of 100 and 500 nM, respectively, based on the IC50 values provided by the manufacturers. Both AZD6244 (Fig. 4C and D) and wortmannin (Fig. 4E and F) significantly blocked the actions of progesterone and OD 02-0 to decrease pSrc and pFAK levels in 926 cells, suggesting that both kinases play roles in the mPR-mediated rapid action of progesterone to inhibit FA signaling.
Mid-and long-term effects progesterone, OD 02-0, and R5020 on vinculin expression
The effects of progesterone, OD 02-0, and R5020 treatments on protein levels of vinculin, an important component of FA signaling, in 926 cells were examined. A time-course experiment showed that vinculin protein expression was significantly decreased after 2-h treatment with 100 nM progesterone compared to initial (0 h) levels and remained at a significantly lower level after 4-h treatment (Fig. 5A). Treatments with 100 nM OD 02-0 and R5020 mimicked the effects of progesterone at 4 h, significantly downregulating expression levels of vinculin (Fig. 5B). Long-term progestin treatments also decreased the vinculin expression, with R5020 showing the strongest inhibitory effect (Fig. 5C). These data suggest that both mPR and nPR are involved in mid- and long-term progesterone regulation of vinculin expression.
Mid- and long-term effects of progestin treatments on vinculin protein expression in 926 cells. (A and B) (upper panels), Western blot analysis of time course of regulation of vinculin protein expression at 0, 1, 2, and 4 h by 100 nM progesterone (P4) treatments (A), and effects of mid-term (4 h) 100 nM progesterone, OD 02-0 (02-0) and R5020 treatments on vinculin expression (B) and quantitative analysis of relative densitometry of the Western blot bands (lower panels). (C) Effects of long-term (16 h) 100 nM P4, 02-0, and R5020 treatments on the expression levels of vinculin. Veh, vehicle control; vinc, vinculin; act, β-actin; M, molecular weight marker. Upper panels in figures are representative Western blot images and the lower panels show the quantitative analysis of mean relative densitometry of the protein bands in the images from three separate assays. Bars in the graphs denote means± s.e.m. (n = 6). Results were analyzed by one-way ANOVA followed by Newman–Keul’s multiple comparison test. (A, B, and C) *, significantly different compared to Veh in the post hoc test (P<0.05). **, P < 0.01.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Long-term effects of progesterone, OD 02-0, and R5020 on Src and FAK expression
Long-term (16 h) treatments with progesterone, OD 02-0, and R5020 all significantly downregulated Src and FAK protein expression (Fig. 6A and B), suggesting that both mPR and nPR are involved in long-term progesterone regulation of FA-related protein expression. Long-term treatments with 100 nM progesterone and R5020, but not OD 02-0, significantly reduced phosphorylation of Src and FAK (Fig. 6C and D), which suggests that nPR, but not mPR, is responsible for long-term activation and maintenance of FA-related kinase signaling in these cells. An alternative analysis of the Src and FAK phosphorylation Western blot images in which the results are expressed relative to a housekeeping protein, actin, yielded very similar results (Supplementary Figs 3, 6C and D).
Long-term (16 h) effects of progestin treatments on the regulation of focal adhesion-related proteins and their phosphorylation in 926 endothelial cells. (A and B) Effects of 100 nM progesterone (P4), OD 02-0 (02-0), and R5020 treatments on the expression of Src (A) and FAK (B) proteins. (C and D) Effects of 100 nM P4, 02-0, and R5020 treatments on the levels of phosphorylated Src (pSrc; C) and FAK (pFAK; D). Upper panels in figures are representative Western blot images and the lower panels show the quantitative analysis of mean relative densitometry of the protein bands in the images from three separate assays. Veh, vehicle control. Bars in the graphs denote means± s.e.m. (n = 6). Results were analyzed by one-way ANOVA followed by Newman–Keul’s multiple comparison test. (A, B, C and D) *, significantly different compared to Veh in the post hoc test (P < 0.05). **, P < 0.01.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Verification of the roles of mPRα in short-term and nPR in long-term progesterone actions on FA signaling with siRNA
Verification of the involvement of mPR and nPR in FA signaling was performed by knocking down the expression of the two receptors with transfection of siRNA antisense oligos. Successful downregulation of the protein expressions of mPRα and nPR was confirmed by Western blot analysis (Fig. 7A and B). Progesterone-induced short-term decreases of pSrc and pFAK were significantly attenuated by knockdown of mPRα (Fig. 7C and D), whereas long-term progesterone downregulation of pSrc and pFAK was blocked by knockdown of the nPR expression (Fig. 7E and F). An alternative analysis of the Src and FAK phosphorylation Western blot images in which the results are expressed relative to a housekeeping protein, actin, yielded very similar results (Supplementary Figs 3, 7C, D, E, and F). In contrast, knockdown of nPR did not alter the short-term action of progesterone to decrease pSrc and pFAK (Supplementary Fig. 4A, C, and E), and mPRα knockdown did not alter the long-term action of progesterone to decrease phosphorylation of these kinases (Supplementary Fig. 4B, D, and F). These results provide clear evidence that mPRα mediates short-term and nPR mediates long-term progesterone inhibitory actions on FA signaling in endothelial cells.
Identification of the receptors mediating progesterone actions in 926 cells with silence RNAs. (A and B) Western blot verification of siRNA knockdown of the protein expressions of mPRα (A) and nPR (B). (C and D) Effect of knockdown mPRα on the short-term action of progesterone (P4) to decrease the levels of pSrc (C) and pFAK (D). (E and F) Effect of knockdown nPR on the long-term action of progesterone to decrease the levels of pSrc (E) and pFAK (F). NT, nontargeting control oligos; si-mPRα and si-nPR, mPRα and nPR silence RNA oligos. Veh, vehicle controls. Upper panels in figures are representative Western blot images and the lower panels show the quantitative analysis of mean relative densitometry of the protein bands in the images from several assays. Bars in the graphs denote means ± s.e.m. (n = 6). Results were analyzed by one-way ANOVA followed by Newman–Keul’s multiple comparison test. (C, D, E, and F) * , significantly different compared to Veh in the NT group in the post hoc test (P < 0.05), #, significantly different compared to P4 in the NT group in the post hoc test (P < 0.05).
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Effects of progestins on angiotensin II-enhanced FA signaling, migration, and invasion through mPR
Angiotensin II is a potent stimulator of FA in vascular endothelial cells. Experiments were conducted with angiotensin II to determine whether progestin treatments can also attenuate elevated FA signaling and migration and invasion of 926 cells. Cells were pretreated with 0.1 and 1 μM of angiotensin II for 30 min followed by treatments for 20 min with 100 nM of progesterone and OD 02-0. Western blot results showed that both pSrc and pFAK levels were upregulated by 0.1 and 1 μM angiotensin II treatments and that these elevated levels were significantly attenuated by co-treatment of the cells with OD 02-0 (Fig. 8A and B) and progesterone (Supplementary Fig. 5A and B). Angiotensin II treatments (0.1 µM) also significantly increased the invasion and migration in 926 cells, and these effects were also attenuated by co-treatment of the cells with progesterone and OD 02-0 at 3 h (Fig. 8C and E) and with OD 02-0 and R5020 at 16 h (Fig. 8E and F). These results suggest that progesterone also exerts rapid inhibitory effects on elevated FA signaling in 926 cells through mPRs as well as cell migration/invasion at later timepoints through both mPR and nPR.
Short-term effects of OD 02-0 (02-0) treatments on angiotensin II (Ang-II)-enhanced focal adhesion signaling (A and B) and mid- and long-term progestin effects on Ang-II-enhanced invasion and migration (C, D, E, and F) in 926 cells. (A and B) Cells were pretreated with 0, 0.1, and 1 μM Ang-II for 30 min before 02-0 (100 nM, 20 min) treatments. Phosphorylated Src (pSrc; A and B) and FAK (pFAK; C and D) were detected by Western blot analysis. Upper panels in figures are representative Western blot images and the lower panels show the quantitative analysis of mean relative densitometry of the protein bands in the images from several assays. (C, D, E, and F) Effects of P4 and 02-0 on cell invasion and migration at 3 h (Fig. 5C and E) and 02-0 and R5020 at 16 h (Fig. 5D and F) with co-treatment with 0.1 μM Ang-II. M, molecular weight marker (see representative images of Fig. 8C, D, E, and F in Supplementary Fig. 6A, B, C, and D). Bars in the graphs denote means ± s.e.m. (n = 3 (A and B), n = 8). Results were analyzed by one-way ANOVA followed by Newman–Keul’s multiple comparison test; (A, B, C, D, E and F) *, Veh treatments significantly different compared to corresponding Veh treatment alone in the absence of Ang-II treatment in the post hoc test (P < 0.05). (A, B, C, D, E, and F). #, significantly different from Veh in the same Ang-II treatment group in the post hoc test (P < 0.05).
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Discussion
We have previously demonstrated that progesterone exerts beneficial cardiovascular effects on HUVECs through mPRα to upregulate eNOS activity and NO production, which leads to relaxation of vascular smooth muscle cells and a reduction in blood pressure (Pang et al. 2015). The present results show mPRα is also an intermediary in another protective action of progesterone in HUVECs to attenuate early pre-atherosclerotic events. mPRα acts coordinately with nPR to mediate short-term and long-term progesterone inhibitory effects, respectively, on FA signaling through decreasing the phosphorylation of two critical protein kinases, Src and FAK. Interestingly, experiments with AZD6244, a MEK inhibitor, and wortmannin, an Akt inhibitor, provide evidence that rapid progesterone downregulation of Src and FAK phosphorylation involves mPR-dependent MAP kinase and PI3k/Akt signaling. The involvement of MAPkinase in progesterone regulation of FA has not been reported previously. The same signaling pathways have previously been shown to mediate progesterone stimulation of eNOS and NO production in HUVECS through mPRα (Pang et al. 2015), which suggests they are intermediaries in the progesterone regulation of multiple cardiovascular protective functions in endothelial cells. The results show mPRs also participate in longer term progesterone actions with nPR to downregulate the vinculin expression and inhibit monocyte adhesion and HUVEC migration and invasion. These findings demonstrate that progesterone exerts wide-ranging protective effects though both mPRα and nPR in HUVECs to inhibit FA signaling and other early atherosclerotic events. Potential beneficial effects of a combination mPR and nPR agonists on cardiovascular functions need further investigation.
Conflicting results have been reported on the influence of sex hormones on FA in HUVECs. The inhibitory effects of progesterone on FA and HUVEC migration in the present study are consistent with the findings of three research groups (Otsuki et al. 2001, Bustos et al. 2008, Lee et al. 2015) but contradicts those of Zheng and coworkers (Zheng et al. 2012). Progesterone inhibited HUVEC migration in a transwell (invasion) assay through phosphorylation of Src, resulting in inhibition of Rho activity and phosphorylation of FAK and paxillin, an important FA protein (Lee et al. 2015). Progesterone also inhibited monocyte adhesion (Simoncini et al. 2004) and expression of vascular cell adhesion molecule (VCAM-1) (Otsuki et al. 2001), and intracellular adhesion molecule (Bustos et al. 2008), which have important roles in FA and monocyte adhesion via TNF-α. The nPR antagonist RU486 failed to block progesterone inhibition of VCAM-1 expression (Otsuki et al. 2001), which suggests the potential involvement of other receptors such as mPRs. In contrast, Zheng and coworkers found that progesterone treatments increased FAK phosphorylation through nPR and migration of HUVECs, which was blocked by knockdown of FAK expression (Zheng et al. 2012). The reasons for the disparate results obtained in their study are unclear but may be partially due to methodological differences, such as the duration of the migration experiment which was three or more times longer (Zheng et al. 2012). Nonetheless, the preponderance of evidence obtained supports inhibitory migratory and atherosclerotic roles for progesterone in vascular endothelial cells.
A major finding of the present study is that progesterone inhibits FA signaling and the activities of two key FA kinases, Src and FAK. Involvement of potential intermediaries in these progesterone actions, the mPRs and nPR, was investigated primarily using specific agonists for these receptors. Org OD 02-0 (OD 02-0) is a potent mPRα agonist and shows no nPR agonist activity in a nPR-B transactivation assay using a PRE-luciferase reporter system (Kelder et al. 2010), whereas R5020 is a potent nPR agonist (Wilkinson et al. 2008). Importantly, the roles of mPRα and nPR in mediating short-term and long-term inhibitory actions of progesterone, respectively, on FA signaling were confirmed by knocking down their expression with siRNA. Rapid progesterone inhibition of FAK phosphorylation in HUVECs, significantly decreasing it after 5- and 10-min treatments, has been reported previously (Lee et al. 2015). Paxillin, another protein component in FA, was also phosphorylated during this time period (Lee et al. 2015), which suggests this progesterone action also potentially could be mediated through mPRα. Interestingly, long-term progesterone treatment (20 h) was also shown in this study to inhibit the migration of HUVECs through decreasing the level of RhoA protein (Lee et al. 2015), a small GTPase that is prevalent in regulating cell shape, polarity, and locomotion via actin polymerization and cell adhesion.
The present results also provide the first evidence that progesterone also regulates vinculin protein expression in HUVECs, significantly decreasing it after mid-and long-term progesterone treatments through both mPRs and nPR. Previously, an endogenous metabolite of progesterone, 5α-pregnane-3,20-dione, was found to decrease vinculin expression, cell-substrate attachment, and adhesion plaques in MCF-7 breast cancer cells (Wiebe & Muzia 2001). Vinculin is a membrane cytoskeletal protein associated with cell–cell and cell–matrix junctions and functions as one of several interacting proteins involved in the FA that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton (Geiger 1979, Burridge & Feramisco 1980).
A most remarkable discovery in this study is that mPRs and nPR exert similar effects in endothelial vascular cells. Both receptor agonists, OD 02-0 and R5020, exert nongenomic progestin actions to inhibit FAK signaling through decreasing Src and FAK phosphorylation. Furthermore, both of them decrease the expression of vinculin, Src, and FAK proteins, presumably through a genomic mechanism. Finally, both OD 02-0 and R5020 mimic the actions of progesterone to decrease monocyte adhesion and HUVEC migration and invasion. The only clear differences between mPRα- and nPR-mediated responses is that short-term (20 min) progesterone inhibition of Src and FAK activation is regulated through mPRα, whereas long-term (16 h) inhibition of these kinases is mediated through nPR, actions that were subsequently confirmed by siRNA for the receptors. In addition, evidence was obtained that initial inhibition of HUVEC migration and invasion as well as monocyte adhesion at 3 h involves mPRs but not nPR. Thus, progesterone inhibition of FAK signaling is regulated through both mPRα and nPR which exert similar effects but over different time periods to provide a coordinated rapid and sustained anti-atherosclerotic response in vascular endothelial cells. This interesting model of steroid action warrants further study.
The inhibition of pre-atherosclerotic responses in HUVECs demonstrated with progesterone in the present study indicates one plausible mechanism underlying the gender differences in the incidence of atherosclerosis and cardiovascular complications in middle-aged premenopausal women and middle-aged men. The elevated circulating progesterone levels in women during the luteal phase of the menstrual cycle and throughout pregnancy (Muneyyirci-Delale et al. 1999, Man et al. 2020) potentially act via this mechanism to inhibit the development of atherosclerotic lesions, whereas circulating progesterone levels in men are much lower and unlikely to exert this beneficial effect (Muneyyirci-Delale et al. 1999). In view of the present results, the possible use of hormone replacement therapy with formulations containing progesterone or a combination of mPR- and nPR-specific agonists merit consideration as therapeutic treatments for reducing the risk of atherosclerosis in postmenopausal women and also perhaps in aged men, both of whom have very low plasma progesterone levels (Brismaar & Nisson 2009).
HUVECs have been used extensively as a cell model for studying early atherosclerotic events. The atherosclerosis responses of HUVECs to numerous bioactive compounds including hormones, drugs, phytochemicals, and agents that alter gene expression have also frequently been demonstrated in whole animal models or in clinical samples (Choi et al. 2012, Meyer et al. 2014, Pan et al. 2018, Watanabe & Sato 2020, Liu et al. 2022), supporting the use of this cell model for atherosclerosis research. Moreover, cultured HUVECs share many functional characteristics with human arterial endothelial cells (HAECs) where the atherosclerotic plaque develops (Lau et al. 2021). Nonetheless, research shows that endothelial cells from different regions of the vascular system display considerable heterogeneity (Thorin & Shreeve 1998, Chi et al. 2003, Lang et al. 2008), and that the genotype and phenotype of HUVECs differ from those of HAECs (Chi et al. 2003). Consequently, in addition for the need to confirm the present findings with HUVECs in animal models of atherosclerosis, it is beneficial to verify that similar results are also obtained in an arterial endothelial vascular cell model.
In summary, the present results demonstrate that progesterone exerts both short-term and long-term actions through mPRα and nPR, respectively, to inhibit FA signaling in HUVECs. Evidence was obtained for involvement of MAP kinase and PI3k/Akt signaling in short-term FA decreases in Src and FAK phosphorylation mediated by mPRα. Progesterone also decreased the expression of FA proteins vinculin, Src, and FAK through both receptors. This inhibition of FA signaling by progesterone is accompanied by longer term decreases in monocyte adhesion and HUVEC migration and invasion, which could result in inhibition of plaque formation and the progression of atherosclerosis (Fig. 9). Arguably, the most interesting finding is that mPRα and nPR act in a coordinated fashion to inhibit these pre-atherosclerotic processes, with mPRα primarily acting as the intermediary in initial actions of progesterone and nPR in the longer term effects. Thus, progesterone inhibition of FA in HUVECs is a valuable model for exploring the cooperation between membrane and nuclear steroid receptors in integrating cellular responses to steroid hormones and provides an explanation for the presence of both receptors in the same cells. The present results provide clear evidence that both mPR and nPR mediate the initial anti-atherosclerotic actions of progesterone in vascular endothelial cells. The results indicate some of the underlying mechanisms as well as the potential for developing therapeutic treatments for cardiovascular diseases that target both receptors. However, longer term studies in animal models will be required to determine the roles of mPRs and nPRs in preventing the development of subsequent atherosclerotic events.
Schematic description of proposed focal adhesion signaling pathways involved in short-, mid- and long-term progesterone actions mediated by mPRα and nPR in HUVECs. Gi, inhibitory G protein; vinc, vinculin; Src, proto-oncogene tyrosine-protein kinase Src; FAK, focal adhesion kinase; Akt, serine–threonine kinase.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0073
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JME-22-0073.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research project was supported by the Morris L Lichtenstein, Jr, Medical Research Foundation.
Acknowledgement
The authors thank Ms Jing Dong for cell culture and assistance with experiments.
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