Novel mineralocorticoid receptor mechanisms regulate cardiac tissue inflammation in male mice

in Journal of Endocrinology

Correspondence should be addressed to M J Young: morag.young@baker.edu.au

*(M J Young is now at Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia)

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MR activation in macrophages is critical for the development of cardiac inflammation and fibrosis. We previously showed that MR activation modifies macrophage pro-inflammatory signalling, changing the cardiac tissue response to injury via both direct gene transcription and JNK/AP-1 second messenger pathways. In contrast, MR-mediated renal electrolyte homeostasis is critically determined by DNA-binding-dependent processes. Hence, ascertaining the relative contribution of MR actions via DNA binding or alternative pathways on macrophage behaviour and cardiac inflammation may provide therapeutic opportunities which separate the cardioprotective effects of MR antagonists from their undesirable renal potassium-conserving effects. We developed new macrophage cell lines either lacking MR or harbouring a mutant MR incapable of DNA binding. Western blot analysis demonstrated that MR DNA binding is required for lipopolysaccharide (LPS), but not phorbol 12-myristate-13-acetate (PMA), induction of the MAPK/pJNK pathway in macrophages. Quantitative RTPCR for pro-inflammatory and pro-fibrotic targets revealed subsets of LPS- and PMA-induced genes that were either enhanced or repressed by the MR via actions that do not always require direct MR-DNA binding. Analysis of the MR target gene and profibrotic factor MMP12 identified promoter elements that are regulated by combined MR/MAPK/JNK signalling. Evaluation of cardiac tissue responses to an 8-day DOC/salt challenge in mice selectively lacking MR DNA-binding in macrophages demonstrated levels of inflammatory markers equivalent to WT, indicating non-DNA binding-dependent MR signalling in macrophages is sufficient for DOC/salt-induced tissue inflammation. Our data demonstrate that the MR regulates a macrophage pro-inflammatory phenotype and cardiac tissue inflammation, partially via pathways that do not require DNA binding.

Supplementary Materials

    • Supplementary Methods and Data - Ong 2019
    • Supplementary Figure S1. Regions of interest in the 20kb upstream of the Mmp12 transcription start site. Letters indicate regions with the size of each indicated above in base pairs. Position numbers are relative to the transcription start site. Callout boxes indicate potential binding sites occurring outside of clusters of interest. Abbreviations: bp = base pair, can = canonical, HRE = hormone response element binding site, n/c = non canonical.
    • Supplementary Figure S2. A: Representative imaging of PCR analysis of MR expression in BMDMs prior to immortalisation. A. LEFT IMAGE: From left to right, samples from 3 individual mice with the MRflox/C603S/LysMCre/+ genotype, and 1 WT sample. RIGHT IMAGE: From left to right, 3 samples from individual mice with the MRflox/flox/LysMCre/+ genotype. The MR C603S produces a band the same size as the WT. B. Representative PCR data for iBMDMs derived from WT mice (lanes 1-4), MyMRKO mice (lanes 5-8) and MyMRC603S (lanes 9- 12). Cells were treated with vehicle (-) or LPS (+) for 24hr as indicated. Kidney (lane 13) provided as a control.
    • Supplementary Figure S3. Modification of MR function does not impact LPS induction of ERK or p38MAPK in macrophages. Western blot analysis of LPS induction of ERK1/2 and p38MAPK in iBMDMs with variable MR signaling. Representative western blots are in the upper panels. Average data from 4-6 independent experiments are presented in the graphs in the lower panels as mean arbitrary intensity units (&#x00B1; SEM) corrected for sample loading using total MAPK. Comparison of means by Student&#x2019;s t-test and 2 tailed p-value. *p<0.05, **p<0.01, ***p<0.001 vs vehicle treated. Cell lines used were wild type (WT), myeloid MR knockout (MyMRKO) or mutant MR expressing (MyMRC603S) iBMDM.
    • Supplementary Figure S4. Modification of MR function does not impact PMA induction of JNK, ERK1/2 or p38 in macrophages. Western blot analysis of PMA induction of JNK p54 and p46, ERK1/2 and p38MAPK in iBMDMs with variable MR signaling. Representative Western blots are in the upper panels. Average data from 3 independent experiments are presented in the graphs in the lower panels as log2 fold change in mean arbitrary intensity units (&#x00B1; SEM) induced by PMA over vehicle (VEH), corrected for sample loading using total MAPK. The dashed line indicates a log2 fold change of zero, i.e. no PMA effect beyond VEH. Log transformed data was compared using one-way ANOVA with Bonferroni correction for multiple testing with no statistically different response between macrophages of different genotypes. Cell lines used were wild type (WT), myeloid MR knockout (MyMRKO) or mutant MR expressing C603S in macrophages (MyMRC603S) iBMDM.
    • Supplementary Figure S5. Intact MR-DNA binding allows repression of PMA effect on Mmp12 in HEK293T cells. The response to PMA plus 10nM aldosterone (ALDO) of GREcontaining regions C and D of the Mmp12 promoter (shown in Supplementary Figure S1) was assessed by luciferase reporter assay. Data presented from 3 independent experiments showing mean relative luminescence (on a log2 scale) versus vehicle-treated &#x00B1; SEM. Statistical significance determined by one-way ANOVA with Bonferroni correction for multiple testing. **p<0.01, ****p<0.0001 versus vehicle, NS = not significant. Aldosterone treatment was with 10nM. HEK293T were transfected with 400ng of reporter plasmid with 150ng of WT PRShMR or mutant PRShMRC603S.
    • Supplementary Figure S6. Effect of dexamethasone signalling via GR on TNF-a and PMA induction of Mmp12 promoter regions (A-E) in HEK293T cells. Luciferase reporter assays for the 5 regions of the Mmp12 promoter (illustrated in Supplementary Figure S1) were tested for GR responsiveness in the presence and absence of TNF-a 10ng/mL or PMA 20nM using co-transfected GR and 10nM dexamethasone (10nM, Dex). Data from 3 independent experiments presented as the mean relative luminescence (log2 scale) vs vehicle treated &#x00B1; SEM. One-way ANOVA with Bonferroni post hoc test **p<0.01, ***p<0.001, ****p<0.0001 vs vehicle treated or between groups as indicated; ns = not significant.
    • Supplementary Figure S7. Myofibroblast (a-SMA expressing) cell density in the heart and kidney of uninephrectomised male mice after 8 days of deoxycorticosterone (DOC)/salt. Immunostaining for a-SMA and either point counting interstitial cells or measuring positively stained pixels was used to determine the number of myofibroblasts present. Data presented represent mean cell density (heart) or percentage positive pixels (kidney) &#x00B1; SEM. No statistically significant differences were seen between genotypes and treatment arms. For kidney the percentage of positively stained pixels was used due to lack of discrete cells for counting. Whole kidney tissue results exclude positive staining in vessels and artefact occurring around tissue planes. Statistically significant differences were determined by two-way ANOVA with Bonferroni correction for multiple testing with p<0.05 considered statistically significant, n=7-10. Mouse genotypes were control (CON) MRFlox/Flox, heterozygous (HET) MRC603S/+ and MyMRC603S which has myeloid cells expressing MRC603S/- and MRC603S/Flox in all other cells.
    • Supplementary Figure S8. Collagen staining (picrosirius red) in the heart and kidney of uninephrectomised male mice at 8 days of deoxycorticosterone (DOC)/salt. Staining for collagen by picrosirius red and calculating the percentage of positively stained pixels per field was used to determine the amount of collagen. Data presented represent the mean percentage &#x00B1; SEM of positive pixels per field. In the heart, arteries and arterioles and areas immediately adjacent to the ventricular lumen were excluded. Statistically significant differences were determined by two-way ANOVA with Bonferroni post hoc test, using a cut-off of p<0.05, n=7- 10. No statistically significant difference was found between treatment or genotype groups. Mouse genotypes were control (CON) MRFlox/Flox, heterozygous (HET) MRC603S/+ and MyMRC603S which has myeloid cells expressing MRC603S/- and MRC603S/Flox in all other cells.
    • Supplementary Figure S9. Representative Images for Mac-2 positive staining of heart sections in uninephrectomised male mice at 8 days of deoxycorticosterone (DOC)/salt. Hearts were sectioned at 5uM in the mid coronal plane and tissue resident macrophages identified by Mac-2 positive staining. Images are captured at 20x objective. Quantitative data are presented in Table 2. Presented here are images from control (CON) MRFlox/Flox mice, and MyMRC603S mice which has myeloid cells expressing MRC603S/- and MRC603S/Flox in all other cells.
    • Supplementary Table S1. Primary and secondary antibodies used for Western Blot and immunostaining.
    • Supplementary Table S2. Primers for RT-PCR analysis of MR expression and of MR target genes in iBMDM lines.
    • Supplementary Table S3. Taqman Assay identities for qPCR in gene expression experiments
    • Supplementary Table S4. Aldosterone regulation of selected target genes in macrophages at 2 and 6 h in WT iBMDMs. Aldosterone (10nM) induced gene expression in WT iBMDMs (iMAC) at 2 time points. The data represent 5 independent experiments performed in triplicate, presented as mean expression &#xF0B1; SEM relative to vehicle. Rpl32 was used as the housekeeping gene. The mean of the vehicle-treated group is standardised to 1.00. p-values were calculated using Student&#x2019;s t-test. *p<0.05, **p<0.01, ***p<0.001 vs vehicle.
    • Supplementary Table S5. Augmentation of PMA effects on the expression of selected genes in WT iBMDMs by aldosterone. Aldosterone (ALDO, 10nM) treatment in WT iBMDMs significantly increased the expression of selected inflammatory and profibrotic gene targets induced by PMA in iBMDMs. Responses to ALDO were reversed by spironolactone (MRA, 1uM). Data represent the average of 5 independent experiments, presented as fold change in expression &#xF0B1; SEM relative to vehicle treated cells. Rpl32 was the housekeeping gene. p-value calculated using one-way ANOVA with Bonferroni correction for multiple testing. *p<0.05, **p<0.01, ***p<0.001 for PMA vs Vehicle, ^for PMA + ALDO vs PMA; #for PMA + MRA + ALDO vs PMA + ALDO.
    • Supplementary Table S6. Loss of MR-DNA binding prevents aldosterone augmentation of PMA gene induction at 2 and 6 hr. PMA induced expression of a range of proinflammatory and profibrotic target genes in MyMRKO and MyMRC603S iBMDMs. However, aldosterone (ALDO, 10nM) did not further regulate PMA-induced genes in these iBMDMs. Data represent the average of 3 independent experiments, presented as fold change in expression &#xF0B1; SEM relative to vehicle treated cells. Rpl32 was the housekeeping gene. p-value calculated using one-way ANOVA with Bonferroni correction for multiple testing. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs Vehicle. No statistically significant difference was identified in any variable for PMA + ALDO vs PMA. MyMRKO = myeloid MR-null, MyMRC603S = myeloid cells expressing only non-DNA binding MR (C603S).
    • Supplementary Table S7. Effect of LPS with or without aldosterone (ALDO) and MRA on the expression of selected genes after 2 h and 6 h in WT iBMDMs. LPS induced expression of a range of proinflammatory and profibrotic target genes in WT iBMDMs. However, aldosterone (ALDO, 10nM) did not further regulate LPS-induced genes. Data represent the average of 5 independent experiments presented as fold change in expression &#xF0B1; SEM relative to vehicle treated cells. Rpl32 was the housekeeping gene. p-value calculated using one-way ANOVA with Bonferroni correction for multiple testing. ***p<0.001, ****p<0.0001 vs vehicle; #p<0.05 v LPS.
    • Supplementary Table S8. Expression of LPS target genes in MyMRKO and MyMRC603S iBMDMs in response to aldosterone (ALDO) at 2 h. LPS regulated expression of a range of proinflammatory and profibrotic target genes in iBMDMs derived from MyMRKO and MyMRC603S animals. However, aldosterone (ALDO, 10nM) did not further regulate LPS-induced genes in MyMRKO and MyMRC603S iBMDMs. Data is the average of 3 independent experiments and presented as mean expression relative to vehicle treated cells &#x00B1; SEM normalised against Rpl32 expression as housekeeping gene, with vehicle group mean standardised to a value of 1.00. p-value calculated using one-way ANOVA with Bonferroni correction for multiple testing. *p<0.05, **p<0.01, ***p<0.001 (vs vehicle). In all cases, mean values for LPS versus LPS+ALDO were not significantly different.
    • Supplementary Table S9. Biometric baseline characteristics of uninephrectomised male mice used for the in vivo assessment of a DOC/salt challenge in MyMRC603S mice versus Control. Control (CON), MRC603S/+ (HET) mice heterozygous for C603S mutation; MyMRC603S mice whose myeloid cells are MRC603S/- while other cells are MRC603S/flox. Data is presented as mean &#xF0B1;&#xF020; SEM. Organ wet weights were normalised against tibia length. Two-way ANOVA with p<0.05 was deemed statistically significant. No statistically significant differences were seen for any variable between genotypes and treatment arms. ^Results from two mice were excluded as significant outliers for kidney weight compared to remainder of their group (CON DOC 476mg vs mean 257mg for the remainder, HET Vehicle 172mg vs mean 212mg for the remainder).

 

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