Abstract
The mineralocorticoid receptor (MR) is a ligand-activated transcription factor that regulates cardiorenal physiology and disease. Ligand-dependent MR transactivation involves a conformational change in the MR and recruitment of coregulatory proteins to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate the circadian protein TIMELESS as a selective regulator of MR transactivation. TIMELESS has an established role in cell cycle regulation and DNA repair. TIMELESS may not be central to mammalian clock function and does not bind DNA; however, RNA and protein levels oscillate over 24 h. Co-expression of TIMELESS down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. TIMELESS markedly inhibited MR transactivation of synthetic and native gene promoters and expression of MR target genes in H9c2 cardiac myoblasts. Immunofluorescence showed aldosterone induces colocalisation of TIMELESS and MR, although a direct interaction was not confirmed by coimmunoprecipitation. Potential regulation of circadian clock targets cryptochrome 1 and 2 by TIMELESS was not detected. However, our data suggest that these effects may involve TIMELESS coactivation of oestrogen receptor alpha (ERα). Taken together, these data suggest that TIMELESS may contribute to MR transcriptional outputs via enhancing ERα inhibitory actions on MR transactivation. Given the variable expression of TIMELESS in different cell types, these data offer new opportunities for the development of MR modulators with selective actions.
Introduction
The mineralocorticoid receptor (MR) has a fundamental physiological role in renal epithelial tissues regulating blood pressure and electrolytes. The MR is also expressed throughout the cardiovascular system where the pathophysiology of inappropriate MR activation is established (Young & Clyne 2021). While studies investigating the contribution of MR activation to cardiovascular inflammation and cardiac fibrosis have largely driven our understanding of the role of the MR in non-epithelial cell types, the molecular mechanisms that define tissue-selective MR transcriptional activity in physiology are poorly understood and may offer new insights into the pathophysiological sequelae of inappropriate MR activation (Yang & Young 2009).
The MR is a member of the nuclear receptor superfamily of transcription factors and is most closely related to the steroid hormone receptors – glucocorticoid receptor (GR), progesterone receptor, androgen receptor (AR), oestrogen receptor (ER) – all of which demonstrate ligand- and cell-selective actions (Arriza et al. 1987). The MR is unique amongst this family in that it binds several classes of steroid hormone – aldosterone, cortisol (corticosterone in rodents) and progesterone (Fuller & Young 2005). Pre-receptor metabolism by 11βHSD2 activity in renal principal cells enables aldosterone to bind the MR in the presence of higher cortisol levels (Funder et al. 1988); in non-epithelial tissues, 11βHSD2 is absent, and the MR is preferentially bound by cortisol. In addition to pre-receptor metabolism of hormones, our data show that cortisol and aldosterone binding to the MR induces subtly different conformational changes in the receptor. This reveals unique surfaces for coregulatory protein interactions and suggests a mechanism for ligand-specific receptor function for the MR, as has been shown for other nuclear receptors (Yang et al. 2011).
While nuclear receptors are structurally conserved, coregulators are structurally and functionally diverse and are recruited in a ligand- and cell-type-specific manner (Norris et al. 2002). Many coregulatory proteins perform essential roles required for gene transcription, histone acetylation and deacetylation, nuclear transport, RNA polymerase activity and many others (Thakur & Paramanik 2009), and may impart tissue and ligand specificity to receptor activity. There are now >500 known coregulatory proteins for the steroid hormone receptor family; however, to date, only ~15 have been described for the MR (Yang & Young 2016). We previously demonstrated a key role for circadian transcription factors circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1) for modulating MR transactivation (Fletcher et al. 2019). We also showed that the human homologue of TIMELESS, a core component of the molecular clock in Drosophila, can bind and activate the oestrogen receptor (ERα) (Magne Nde et al. 2018). These links between circadian systems and nuclear receptor function raise the possibility that TIMELESS may also regulate MR transactivation.
TIMELESS was originally described in Drosophila as an essential element of the Drosophila circadian clock (Hardin 2005). TIMELESS encodes a protein product (dTIM) that interacts with the circadian protein Period (PER). dTIM acts to control the subcellular localisation and stability of PER and thus determines the period of the circadian cycle (Gekakis et al. 1995, Urtishak et al. 2009). A mammalian homologue of dTIM has been described, which shares sequence homology to dTIM2, or dTIMEOUT, rather than dTIM. Whether mammalian TIMELESS oscillates across the circadian day in line with other members of the circadian clock is equivocal. However, it has been shown to oscillate and regulate circadian clock time in suprachiasmatic nucleus (SCN) via the negative-feedback arm of the molecular clock (Engelen et al. 2013) and in the heart (Fletcher et al. 2019). Other functions of TIMELESS include regulation of the cell cycle, development, response to cell stress and cancer progression (Koike et al. 1998, Barnes et al. 2003, Unsal-Kaçmaz et al. 2005).
The molecular circadian clock is a cell-autonomous transcription/translation negative feedback loop found in virtually all cells and regulates up to 20–30% of all genes (Top & Young 2018). In mammals, the positive regulators CLOCK and BMAL dimerise and transactivate negative regulators including PER and cryptochrome (Cry) homologues, which in turn repress CLOCK/BMAL actions (Patke et al. 2020). Cell-autonomous oscillation of circadian clock proteins in peripheral tissues is entrained in a hierarchical manner by light detected by the SCN regulating neurohormonal signals to align peripheral clocks with environmental cues such as light (Patke et al. 2020). Cortisol acting via the GR is an accepted ‘zeitgeber’ in peripheral cells, and the MR may also modulate the cellular clock via cooperative signalling with core clock proteins CLOCK and BMAL (Fletcher et al. 2019, Kanki & Young 2021).
We, therefore, hypothesise that TIMELESS is a coregulatory protein of the MR that can modify the regulation of MR target genes and offer new insights into MR–circadian clock interactions and temporal MR transcription. Given that the cellular environment also plays a critical role in defining nuclear receptor function via regulation of the expression and subcellular localisation of coactivators and corepressors, we investigated the role of TIMELESS in the control of MR transactivation using H9c2 rat cardiomyoblasts and HEK293 embryonic kidney cells.
Methods
Cell culture and transfection
HEK293 and H9c2 cell lines were maintained at 37°C with a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1 mM l-glutamine, 1 mM non-essential amino acids, 1% penicillin/streptomycin and 10% (v/v) fetal bovine serum. Cell lines were routinely passaged when 90% confluent. For the transfection assay, 105 cells were seeded into 24-well plates for H9c2 cells and 105 HEK293 cells. Cells were stably transfected with plasmids using H9c2 cells, Fugene® 6 Transfection Reagent (Promega) and following the manufacturer’s protocol.
Plasmids for transfection were pRShMR (200 ng), MMTV-luc (200 ng), CMV-TIM (0–200 ng), pUC19 (0–200 ng), pGL4.23-CNKSR3-Luc (200 ng) and pGL4.23-GILZ-Luc (200 ng) (Ziera et al. 2009), pCMV-ERα (200 ng) and β-galactosidase (Bgal). Twenty-four hours after transfection, the media was replaced with 500 μL DMEM containing 10% charcoal-stripped FCS and the cells were incubated for a further 4 h before the addition of either 10 nM aldosterone, cortisol or oestrogen in the relevant experiments.
The following day the plates were washed with 500 μL PBS, and 150 μL of Passive Lysis Buffer (Sigma) were added to each well and cells were incubated on a rocking platform for 30 min at room temperature. One hundred microlitres from each well was subsequently transferred to a 96-well microplate, 100 μL of luciferase reagent was added and luciferase activity was measured immediately at 405 nm using an EnVision multilabel plate reader (PerkinElmer). Beta-galactosidase expression was assayed in 50 μL of lysate following incubation with chlorophenol red-β-D-galactopyranoside substrate. Experiments were performed three times in triplicate.
Western blot
Fifteen microlitres of each sample was loaded onto 10% acrylamide gels alongside 5 μL of Precision Plus Protein Dual Colour Molecular Weight Marker (Bio-Rad) and separated by electrophoresis (1 h, at 100 V). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane overnight at 12 V at 4°C. Membranes were blocked in TBST containing 5% skim milk (fat-free) for 1 h at room temperature and probed with antibodies for either TIMELESS, MR, SSRP1, XRCC6/Ku70, CRY2 or beta-actin in TBST supplemented with 3% BSA (1:5000 or 1:1000, respectively) overnight at 4°C. Membranes were washed 3× with TBST and incubated with secondary antibodies (goat anti-rabbit IgG HRP for TIMELESS, goat anti-mouse IgG for the MR, Abcam; Supplementary Table 1, see section on supplementary materials given at the end of this article) in TBST, at 4°C for 1 h in the dark. Membranes were washed 3× and visualised using a G:Box Chemi XX9 (Syngene, Cambridge, UK).
Coimmunoprecipitation
A total of 4 × 105 HEK293 cells were seeded into 6-well plates in 2 mL DMEM supplemented with FCS. Twenty-four hours later, cells were transfected with 1 μg of pRShMR, CMV-TIM, CMV-SSRP1 or CMV-XRCC6/Ku70 and incubated for 24 h. DMEM was replaced with DMEM containing 10% charcoal-stripped FCS and 10 nM aldosterone after 4 h. At 1 h incubation with aldosterone, cells were washed with ice-cold PBS and 250 μL of lysis buffer was added (Tris-HCL pH 8.0, 25 mM, NaCl 150 mM, EDTA 2 mM, NP-40 1% supplemented with Pierce™ protease and phosphatase inhibitor cocktail). Lysates were centrifuged for 15 min at 14,000 g at 4°C and the supernatant was transferred to new tubes. Fifity microlitres of Protein A Sepharose Beads (Sigma) were used for the coimmunoprecipitation, following the manufacturer’s protocol. Five microlitres of primary antibodies for MR, TIMELESS, SSRP1, XXRC6/Ku70 or negative IgG (Supplementary Table 1) were used as capture antibody, or control, and incubated for 1 h at 4°C. The beads were washed 3× with lysis buffer containing 0.1% NP-40. Lysates and the beads were incubated overnight at 4°C and washed 3×. All steps were performed in the presence of 1 μM aldosterone to stabilise the MR. Samples were denatured at 100°C for 5 min and eluted in the sample buffer (1× Laemmli buffer and 5% β-mercaptoethanol). Control for input consisted of 15 μL of the HEK293 cell lysate.
Regulation of endogenous MR target genes
H9c2 and HEK293 cells were seeded in six-well plates in DMEM plus FCS as described earlier. Cells were transfected with CMV-TIM (100 ng) or siRNA directed at TIM, plus appropriate controls. After 24 h, media were replaced with DMEM plus charcoal-stripped FCS, and 4 h later, cells were treated with either 10 nM aldosterone alone or in combination with spironolactone (1 μM). Cells were washed with PBS and lysed after 5 h and RNA was isolated with TRI-Reagent (Sigma-Aldrich) and samples were incubated with DNAse I (TURBO DNA-free™ Kit, Life Technologies), following the manufacturer’s protocols.
Quantitative RT-PCR
cDNA was synthesised using 250 ng total RNA using the SuperScript® III First-Strand Synthesis System for RT-PCR (Life Technologies). Quantitative RT-PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) in 384-well plates using Power SYBR® Green PCR Master Mix (Life Technologies) and gene-specific primers (Supplementary Table 1). Regulation of target genes was expressed relative to GAPDH, and experiments were performed three times in triplicate.
Immunocytochemistry
H9c2 cells were grown on sterile coverslips in 6-well plates seeded with 5 × 104 cells and cells were transfected with PRshMR (100 ng) and CMV-TIM (100 ng) as described earlier. After 24 h, the media were replaced with DMEM and charcoal-stripped FCS. Four hours later, 10 nM aldosterone or vehicle was added for 30 min to the appropriate wells.
The plate was then washed twice with PBS, 2 mL of ice-cold methanol was added to each well for 20 min at −20°C, washed 3× 10 min in PBS and 2 mL of immunofluroescence buffer was applied for 10 min. After a further 3× 10 min washes, 2 drops of Image-iT® FX Signal Enhancer (Life Technologies) were applied to each slide for 1 h and washed 3× 10 min. Non-specific staining was blocked by incubation with 200 μL goat normal serum (30 min) and the cells were washed 2× 5 min in PBS before incubation overnight at 4°C with TIMELESS (1:200) and/or MR (1:100) antibodies in 1% BSA (Sigma). The coverslips were washed with PBS 3× 10 min and incubated with the appropriate secondary antibody (1:200) in 1% BSA in the dark, at room temperature for 1 h. Following another 3x 10 min wash, the coverslips were air dried, mounted with 0.06 μg of DAPI, diluted into 100 μL of fluorescent mounting medium (Dako) and dried overnight. The absence of primary antibody served as a negative control. Images were visualised with an Olympus BX60 microscope, connected to an Olympus U-RFL-T camera. The primary antibody for the MR was detected by a green, fluorescent secondary antibody (at 466 nm). The TIMELESS primary antibody was detected by a red fluorescent secondary antibody (at 546 nm).
RNA interference
HEK293 cells were seeded in six-well plates, grown to 60% confluence and transfected with scrambled siRNA or siRNA specific to TIMELESS (Trilencer-27 Origene, MD, USA and sc-270603 Santa Cruz, TX, USA), as per the manufacturers’ protocol and over a range of concentrations 100–400 nM, 1 μM and 2 μM. Subsequent hormone treatment, RNA extraction and Western blot proceeded as described in previous sections.
Statistical analysis
Data were analysed using the GraphPad PRISM software package (GraphPad). Data are presented as mean ± s.e.m. One-way ANOVA is conducted to distinguish any statistically significant results. Unpaired t-tests are used to distinguish the statistical significance between individual conditions. P < 0.05 was considered statistically significant.
Results
Endogenous expression of TIMELESS in H9c2 and HEK203 cells
Western blot for TIMELESS in H9c2 and HEK293 cells showed that TIMELESS was expressed at markedly higher levels in HEK293 cells vs H9c2 cells (Fig. 1).
Representative Western blot for TIMELESS in HEK293 cells and H9c2 cells. Actin served as loading control. TIMELESS protein levels are higher in HEK293 cells. Representative blot of two independent experiments.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-21-0279
TIMELESS has unique coregulatory actions for steroid hormone receptors
TIMELESS was previously shown to stimulate ER activity in HEK293 cells (Magne Nde et al. 2018). Therefore, we investigated whether TIMELESS could modulate other steroid hormone receptors in these cells. TIMELESS enhanced basal and ligand-induced activity of the AR and GR on an MMTV-luc reporter but inhibited MR transactivation of this reporter (Fig. 2). We next examined the dose- and ligand-dependency of this effect on MR. TIMELESS dose-dependently inhibited MMTV-luc transactivation by both 10 nM aldosterone and 10 nM cortisol in HEK293 cells (Fig. 3A).
Ligand-dependent transactivation activity of the (A) androgen receptor (AR) and (B) glucocorticoid receptor (GR) by 10 nM ligand was increased by cotransfection of 100 ng TIMELESS in Hek293 cells. In contrast, transactivation of the MR with 10 nM aldosterone was inhibited by TIMELESS. N = 3 experiments, *P < 0.01, **P < 0.001.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-21-0279
Cotransfection of TIMELESS (50 ng or 200 ng) inhibited MR transactivation of the MMTV-luc reporter in (A) HEK293 and (B) H9c2 cardiac cells in a dose-dependent manner. Aldosterone (10 nM, grey bars) or cortisol (10 nM, black bars) was used. N = 3 experiments, *P < 0.05, ***P < 0.0001 vs empty vector control.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-21-0279
TIMELESS regulation of MR transactivation in H9c9 cardiac cells
Given our previously published studies for circadian regulation of MR in the heart (Fletcher et al. 2019), we investigated whether TIMELESS also coregulates the MR in H9c2 cardiac cells. Cells transfected with full-length hMR and 50 ng or 200 ng CMV-TIM or vector control and treated with 10 nM aldosterone, cortisol or vehicle showed that increasing levels of TIMELESS inhibited MR transactivation of the MMTV-luc reporter indicating that TIMELESS was more efficacious in these cells (Fig. 3B) and TIMELESS corepression of MR in response to 10 nM aldosterone and 10 nM cortisol was equivalent (Fig. 3B). TIMELESS did not coregulate GR transactivation of MMTV.luc in H9c2 cells (Supplementary Fig. 1). We next investigated if the effect of TIMELESS in H9C2 cells was specific to the MMTV reporter or could also occur at native promoters for MR target genes Connector Enhancer of Kinase Suppressor of Ras 3 (CNKSR3) and glucocorticoid-induced leucine-zipper protein (GILZ). Our data showed that TIMELESS corepressed MR transactivation of these promoters activated by either aldosterone or cortisol (Fig. 4). In contrast, TIMELESS had little or no effect on the CNKSR3.luc and GILZ.luc reporters in HEK293 cells (Supplementary Fig. 2).
CMV-TIM 50 ng and 200 ng corepressed MR transactivation of the GILZ-luc and CNKSR3-luc reporters in H9c2 cardiac cells induced by either 10 nM aldosterone or 10 nM cortisol. N = 3 experiments, ***P < 0.0001.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-21-0279
TIMELESS inhibits endogenous MR target gene expression
TIMELESS inhibited expression of selected endogenous MR target genes; serum- and glucocorticoid-regulated kinase 1 (Sgk1), Gilz and Per1 genes were induced 2–3-fold by 10 nM aldosterone in H9c2 rat cardiac cells at 5 h and blocked by spironolactone cotreatment or by cotransfection of 100 ng CMV-TIM (Fig. 5). MR mRNA levels were not altered in these cells. Expression of CNKSR3 was neither strongly induced by aldosterone nor regulated by TIMELESS in H9c2 cells suggesting promoter-specific regulation by aldosterone and TIMELESS. Moreover, induction of Sgk1, Gilz, Per1 and Cnksr3 by aldosterone was not down-regulated by TIMELESS in HEK293 cells, and only FK506 binding protein 5 (FKBP5) was modestly reduced in the presence of TIMELESS in these cells (Supplementary Fig. 3).
GiLZ, SGK-1 and Per1 gene expression in H9c2 cells in response to 10 nM aldosterone is inhibited by cotransfection of 100 ng CMV-TIM (black bars). No change in MR levels was detected. N = 3 experiments, *P < 0.01. Veh, vehicle; al, aldosterone; al.sp., aldosterone plus spironolactone.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-21-0279
MR and TIMELESS colocalise in the nucleus but do not interact directly
Nuclear receptors frequently interact with coregulatory proteins via conserved sequences conforming to one or more leucine-x-x-leucine-leucine (LxxLL; where x is any amino acid) motifs that bind to the activator function 2 region of the LBD. TIMELESS contains three LxxLL motifs, at least one of which is necessary for the binding of TIMELESS to the ERα (Magne Nde et al. 2018). Coimmunoprecipitation assays using the HEK293 cell line transfected with CMV-TIM or pRShMR showed that primary antibodies for TIMELESS and MR successfully pulled down the relevant proteins from HEK293 cell lysate using the corresponding antibody (Fig. 6A and B). However, the Western blot for MR did not detect a band at ~107 kDa in the TIMELESS antibody pull-down sample (Fig. 6B). Western blot for TIMELESS also showed no difference in band intensity between the negative control and MR pull-down lanes indicating that a complex containing TIMELESS was not immunoprecipitated with the MR antibody and that the MR and TIMELESS do not physically interact. In silico investigation of three protein–protein interacting databases (https://www.ebi.ac.uk/intact/home; http://gpsprot.org/; https://string-db.org), to identify potential MR-TIMELESS interactions, identified potential interactions between TIMELESS and MR coregulators SSRP1 and XRCC6/Ku70 (Yang et al. 2011, Young et al. 2015). However, immunoprecipitation of MR, TIMELESS, SSRP1 or XRCC6/Ku70 did not detect an interaction at 1 h of aldosterone treatment (Supplementary Figs 4 and 5). Given that CRY2 can repress transcription and is regulated by TIMELESS via protein interaction, we performed Western blot for CRY2 in the MR and TIMELESS coimmunoprecipitation assays but did not find evidence of CRY2 interaction (data not shown). We also evaluated mRNA levels of Cry1 and Cry2 in cells transfected with TIMELESS and did not detect an induction of Cry1/2 with TIMELESS or aldosterone 10 nM for 5 h. In contrast, Per1 was induced by aldosterone and neither Per1 or Per2 transcripts were regulated by TIMELESS (Supplementary Fig. 6).
CoIP for transfected TIMELESS and hMR. (A) Western blot probed with MR antibody shows 110 kDa band in input and MR pull-down lanes (MR Ab). (B) Western blot probed with TIMELESS antibody. HEK293 input detected two bands (130, 110 kDa in the lysate and TIMELESS pull-down lanes (TIM Ab). No bands are observed in No Ab, IgG Ab and MR Ab pull-down lanes. Representative blots of two independent experiments.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-21-0279
TIMELESS is located predominantly within the nucleus (Gotter et al. 2007, Yoshizawa-Sugata & Masai 2007), whereas the unliganded MR can reside in multiple subcellular localisations depending upon the cell type (Fejes-Tóth et al. 1998, Gomez-Sanchez et al. 2006). Immunofluorescent staining of MR and TIMELESS in the H9c2 cell line showed the two proteins to be predominantly located in the nucleus in both the untreated and aldosterone-treated conditions. Some faint MR staining was detected in the cytosol. However, the intensity of MR staining increased in the presence of aldosterone. When the MR and TIMELESS channels were merged, a bright yellow colour was detected in the nuclei of cells treated with aldosterone, indicating colocalisation of the MR and TIMELESS within the nucleus in the presence of ligand (Fig. 7).
Co-localisation of the MR and TIM in H9c2 cells. H9c2 cells were transfected with MR and TIMELESS and were either untreated or treated with 10 nM aldosterone. Immunostaining is as follows; DAPI (Blue), MR (Green), TIMELESS (Red) and MR/TIMELESS merged (Yellow).
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-21-0279
ERα and TIMELESS repress MR transactivation
Given previous reports that ERα inhibits MR transcription and TIMELESS coactivates ERα, we tested whether there is an additive or synergistic effect of TIMELESS and ERα on MR transactivation in both HEK293 and H9c2 cells. Repression of MR transactivation was significantly greater in the presence of cotransfected ERα in both cell lines. The addition of oestrogen slightly increased the repressive effect in H9C2 cells but not HEK293 cells. Coexpression of ERα repressed MR transactivation of MMTV.luc in both cell lines, whereas the addition of TIMELESS and or treatment with oestrogen further enhanced MR repression in H9c2 cells. In HEK293 cells, cotransfection of TIMELESS plus ERα repressed MR transactivation regardless of the presence of oestrogen (Fig. 8). Thus, there is potential that TIMELESS and ERα may act together to repress the MR in selected cell types.
Coexpression of ERα (100 ng) inhibited MR transactivation in both cell lines. The addition of TIMELESS (black bars) enhanced MR inhibition in H9c2 cells only. Treatment with oestrogen (E2) repressed MR transactivation of MMTV.luc in the presence of endogenous ERα expression in H9c2 cells and in the presence of cotransfected ERα in HEK293 cells. *P < 0.001 vs no treatment, **P < 0.01 vs empty vector control.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-21-0279
RNA interference
TIMELESS is ubiquitously expressed but at variable levels in different cell types. Transfection of 2 independent siRNA targeting TIMELESS expression in HEK293 was not unable to inhibit TIMELESS at 100, 200, 400 ng siRNA and assessing TIMELESS protein at different time points after transfection (1–3 days following transfection) (Supplementary Fig. 7). Higher concentrations of siRNA were also tested but were without effect. This outcome is most likely due to the critical role of TIMELESS for cell cycle regulation, DNA repair processes and thus cell survival in HEK293 embryonic fibroblasts (Mao et al. 2013, Elgohary et al. 2015).
Discussion
The current study supports the hypothesis that TIMELESS serves as a corepressor of the MR and suppresses ligand-dependent MR activity in cardiac cells and embryonic kidney fibroblasts. This contrasts with other members of the steroid hormone family, which are coactivated by TIMELESS. We further demonstrated that TIMELESS corepression of the MR was not ligand-specific and occurred at two native target gene promoters as well as the MMTV-luc reporter. While our data do not support direct TIMELESS–MR interactions, TIMELESS is an established binding partner of core circadian proteins that are also known to regulate MR transcription. Given that TIMELESS contributes to the fine-tuning of circadian timekeeping within cells and fluctuates across the circadian cycle, the actions of TIMELESS may contribute to temporal modulation of MR transactivation in various cell types as part of a transcriptional complex. Evidence also supports TIMELESS as a potent coactivator of GR function in some cells; thus, variable TIMELESS serve to regulate the balance of GR vs MR signalling. This has relevance for cortisol action in those cell types where the MR is predominantly a cortisol receptor, as is the case in many non-epithelial cell types.
TIMELESS regulation of PER via protein–protein interaction and thus the period of the circadian cycle contributes a new layer to the recently described link between MR and the circadian molecular clock (Gekakis et al. 1995, Gotter et al. 2000, Urtishak et al. 2009). The MR is an established transcriptional regulator of Per1 and Per2, but not TIMELESS, in response to both aldosterone and cortisol and can thus potentially modify the pace of the circadian clock. Our findings suggest that increased TIMELESS can mitigate MR activation, which may counter or enhance downstream effects on circadian timekeeping mediated by the MR in response to variation in corticosteroid levels. However, we have also demonstrated circadian transcription factors CLOCK/BMAL in (mammalian) cardiac cells as both positive and negative regulators of MR transactivation, depending upon the target gene promoter (Fletcher et al. 2019). It is thus likely that net MR transcriptional activity is determined by a complex set of transcriptional regulators, of which some oscillate across the day. Disruption of the circadian clock has well recognised wide-reaching effects on cell function and significant implications for health and disease (Ruan et al. 2021). The impact of a disordered circadian clock on MR and other steroid hormone receptors may therefore play an important role in promoting cellular dysfunction by modulating the cellular response to hormonal ligands for the MR.
The role of TIMELESS in the control of the mammalian circadian clock is a moot point. The circadian clock is organised into a series of positive and negative feedback loops that are controlled by core clock proteins CLOCK and BMAL1 (Kondratov & Antoch 2007). TIMELESS is expressed at high levels within the SCN, and it shows a low amplitude of oscillation relative to other circadian genes (Barnes et al. 2003). Whereas some studies show that when TIMELESS function is inhibited via blocking phosphorylation, the circadian clock still functions (Mao et al. 2013). Some studies have identified direct interactions between TIMELESS and circadian clock proteins including CLOCK, BMAL, PER and CRY (Cai & Chiu 2021, Cai et al. 2021). PER1/2 are inhibited by elevated TIMELESS, which inhibits CLOCK–BMAL transactivation via a protein–protein interaction (Sangoram et al. 1998, Barnes et al. 2003) and TIMELESS can directly control PER1 (Takumi et al. 1999) and CRY1 and CRY2 interaction via phosphorylation of CRY1/2 (Griffin et al. 1999, Unsal-Kaçmaz et al. 2005). Thus, MR and TIMELESS have the potential to act independently and in concert to modify multiple components of the cellular clock, which in turn will have a net effect on the regulation of cell function. In the present study, we demonstrated that the MR and TIMELESS colocalise in the nucleus of cardiac myoblast cells treated with aldosterone, suggesting an interaction; however, coimmunoprecipitation of the two proteins from a cell treated with aldosterone was not clear, suggesting that the MR and TIMELESS do not form a direct interaction. Nor did CRY2 form part of this complex. Similarly, we evaluated TIMELESS interactions with MR coregulators SSRP1 and XRCC6/Ku70 which we previously showed can act as MR coregulators (Yang & Young 2009). However, SSRP1 and XRCC6/Ku70 did not coprecipitate with TIMELESS after 1 hour of incubation with aldosterone. However, it is possible that they may form part of a time-sensitive common transcriptional complex, or the coimmunoprecipitation may have been impacted by the inherent instability of the MR and limitations of the available antibodies despite our best efforts.
TIMELESS levels fluctuate across the cell cycle with low levels present in both the G0 and G1 phases and high levels being present in the G2, S and M phases (Unsal-Kaçmaz et al. 2005) and interact with several cell cycle proteins via direct protein–protein interactions. Checkpoint kinase 1 (Chk1), ataxia telangiectasia related (ATR), ATR Interacting Protein (ATRIP), claspin and minichromosome Maintenance Complex Component 2 (2MCM2) are key interactions with TIMELESS that are modulated by ‘replicative stress’ or DNA damage. Down-regulation of TIMELESS results in reduced cell survival supporting the important role of TIMELESS in coupling the cell cycle to the circadian clock (Unsal-Kaçmaz et al. 2005, Gotter et al. 2007, Yoshizawa-Sugata & Masai 2007, Leman et al. 2010, Smith-Roe et al. 2011). Depletion of TIMELESS results in chromosome fragmentation as well as defects in the damage repair processes and mitotic progression compromises replication and intra-S checkpoints of the cell cycle compromising cell viability (Unsal-Kaçmaz et al. 2005, Leman et al. 2010). Conversely, higher levels of TIMELESS are linked to stemness or dedifferentiated cells, which is consistent with the high levels of TIMELESS in the HEK293 embryonic cells vs the relatively differentiated H9c2 cardiac myoblast cells in the present study. TIMELESS is thus also associated with many forms of cancer. In breast cancer, hypomethylation of the TIMELESS promoter results in overactivation of TIMELESS and activation of many critical genes in later stages of the disease (Fu et al. 2012, Magne Nde et al. 2018). In contrast, lower expression of TIMELESS is closely associated with survival in breast cancer, and in renal cell carcinoma, lower levels of TIMELESS are associated with tumour-free tissue (Mazzoccoli et al. 2012). The role of TIMELESS in tumourigenesis relates to dysregulation of DNA repair and cell stress, but also involves dysregulation of steroid receptors in the tumour environment (Yang et al. 2010, Relles et al. 2013). Thus, the effect of TIMELESS on cell function can be significantly regulated by multiple cellular events, which will also have an impact on the activation of the MR and the other steroid hormone receptors.
Regulation of classic MR target genes by increased TIMELESS expression in cardiac but not HEK293 suggests that the actions of TIMELESS on MR transactivation can vary between cells. Aldosterone induction of Gilz, Sgk1 and Per1 was inhibited by TIMELESS equivalent to spironolactone treatment in cardiac cells indicating that TIMELESS is a strong regulator of MR transactivation activity in these cells. In contrast, MR target gene expression of CNKSR3 was not regulated by TIMELESS indicating promoter-specific effects of TIMELESS on MR transactivation. This is consistent with previous demonstrations of the importance of DNA binding of transcriptional complexes at specific promoter sequences, as well as ligand, for defining nuclear receptor confirmation and thus its activity (Mohideen-Abdul et al. 2017). Interestingly, previous studies show that both Gilz and Sgk1 undergo alternative splicing, creating multiple isoforms (Fiol et al. 2007, Simon et al. 2007, Robinson et al. 2012). It is therefore proposed that regulation of Gilz and Sgk1 may also occur by a pre-mRNA splicing complex and TIMELESS may form a part of this; however, this will require independent testing.
The localisation of both MR and TIMELESS separately within the cell has been previously documented separately and is consistent with our findings. The subcellular location of the MR is generally dependent on the presence of ligand where the MR is located in both the nucleus and the cytosol in the absence of ligand and is almost exclusively nuclear upon ligand binding (Gomez-Sanchez et al. 2006). Cardiac cells are one exception in that the nuclear localisation signal is constitutively active in these cells and the MR appears more nuclear vs other cell types (Fejes-Tóth et al. 1998, Gomez-Sanchez et al. 2006). In contrast, TIMELESS is a predominately nuclear protein in all cells and is specifically located within the nucleoli (Gotter et al. 2007, Yoshizawa-Sugata & Masai 2007). Regardless of the nuclear localisation of both MR and TIMELESS in untreated cells, co-immunofluorescent staining clearly shows that in the presence of aldosterone the proteins colocalise within the nucleus and can thus coordinate to regulate target gene sets in the presence of aldosterone.
Given that TIMELESS is a nuclear protein, it is thus well placed to interact with and regulate many transcription factors and pathways. There are many examples of TIMELESS forming protein–protein interactions with circadian proteins, nuclear receptors and transcription factors (Gekakis et al. 1995). Thus, TIMELESS interaction with the MR as part of a transcriptional, via control of cell cycle more broadly, or via regulation of MR transcriptional partners, is likely to be important for the MR at one or more times of the day. We have shown that circadian proteins CLOCK and BMAL directly modulate MR signalling. Given that TIMELESS has established interactions with CLOCK, BMAL and other key circadian proteins, TIMELESS modulation of the MR, GR, and other steroid hormone receptors, may occur via control of CLOCK and BMAL actions. In the present study, we showed that coexpression of ERα repressed MR transactivation separately in both cell lines, but the addition of TIMELESS and or treatment with oestrogen further enhanced MR repression in H9c2 cells but not HEK203 cells. Thus, there is potential that TIMELESS and ERα may act together to repress the MR in selected cell types via previously reported actions of ERα on MR transcription.
TIMELESS levels are linked to both ‘stemness’ in cancer cells and falling levels are linked to ageing thus there is considerable scope for variable MR activation outcomes across the life cycle of many cell types (Terzibasi-Tozzini et al. 2017, Shen et al. 2018, Zou et al. 2020). This has relevance for the choice of cell lines for the present study given that HEK293 cells are embryonic and therefore considerably higher endogenous expression of TIMELESS compared to H9c2 cardiac myoblasts. It may thus be easier to detect a change in MR transactivation in H9c2 cardiac with transfected TIMELESS, compared to HEK293 cells due to TIMELESS being present at limiting amounts in one cell type, not in the other. However, given that the MR can readily and robustly transactivate reporter genes in HEK293 cells (Yang & Eliott 2017), it is likely that other limiting coregulatory proteins expressed in the cardiac cells contribute to the corepressor complex.
Significance
The impact of cardiac failure on global health is immense. In Australia alone, 45,000 people were diagnosed, as well as 17,900 deaths were recorded in 2009 (AIHW 2022). MR antagonists, such as spironolactone and eplerenone, are known to vastly improve the probability of survival in those who have experienced episodes of cardiac failure but are limited by side effects arising due to global MR blockade (Pitt et al. 1999, 2003, Zannad et al. 2011). Coregulatory proteins of nuclear receptors are often expressed in a tissue-specific manner and at variable levels and thus provide a mechanism for achieving and modulating steroid hormone receptor function.
The current study aimed to determine a coregulatory protein for the MR that has the potential to modulate the MR in a cell-specific manner and thus mitigate the side effects of global MR blockade. Understanding the importance of cellular systems that modify MR transactivation including interactions with coregulatory proteins such as TIMELESS is an important step towards understanding variable cellular actions of the MR as well as informing the rational design of MR modulators for the treatment of cardiac and possibly other diseases where corticosteroid signalling plays a key role.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JME-21-0279.
Declaration of interest
Research funds have been received by M J Y from AstraZeneca for research activities that are independent of the current work.
Funding
M J Y: Baker Trust Alice Baker and Eleanor Shaw Gender Equity Fellowship. NHMRC GNT494811. The Baker Heart and Diabetes Institute and Hudson Institute of Medical Research are supported by the Victorian Government’s Operational Infrastructure Scheme.
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