Rachel V. Richardson

Glucocorticoids and foetal heart maturation; implications for prematurity and foetal programming

Glucocorticoids are steroid hormones, essential in mammals to prepare for life after birth. Blood levels of glucocorticoids (cortisol in most mammals including humans; corticosterone in rats and mice) rise dramatically shortly before birth. This is mimicked clinically in the routine administration of synthetic glucocorticoids to pregnant women threatened by a preterm birth or to preterm infants to improve neonatal survival. Whilst effects on lung are well documented and essential for postnatal survival, those on heart are less well known. In this study, we review recent evidence for a crucial role of glucocorticoids in late gestational heart maturation. Either insufficient or excessive glucocorticoid exposure before birth may alter the normal glucocorticoid-regulated trajectory of heart maturation with potential life-long consequences.

Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy

Significance We have discovered a role for the glucocorticoid receptor (GR) in coordinating cell division. We find enrichment of GR to mitotic spindles and demonstrate that GR knockdown causes accumulation of mitotic defects, including delayed anaphase, ternary chromosome segregation, and death in mitosis. Mitotic GR function requires the ligand-binding domain but not ligand binding, revealing a nontranscriptional and ligand-independent mechanism of action. Analysis of GR haploinsufficient cells and tissues reveals increased aneuploidy and DNA damage, and mice show an increased incidence of tumors in vivo, with further GR loss within those incident tumors. We also identify reduced GR expression in several common human cancers, thereby implicating GR as a novel tumor suppressor gene. The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.

Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

Insufficient alveolar gas exchange capacity is a major contributor to lung disease. During lung development, a population of distal epithelial progenitors first produce bronchiolar-fated and subsequently alveolar-fated progeny. The mechanisms controlling this bronchiolar-to-alveolar developmental transition remain largely unknown. We developed a novel grafting assay to test if lung epithelial progenitors are intrinsically programmed or if alveolar cell identity is determined by environmental factors. These experiments revealed that embryonic lung epithelial identity is extrinsically determined. We show that both glucocorticoid and STAT3 signalling can control the timing of alveolar initiation, but that neither pathway is absolutely required for alveolar fate specification; rather, glucocorticoid receptor and STAT3 work in parallel to promote alveolar differentiation. Thus, developmental acquisition of lung alveolar fate is a robust process controlled by at least two independent extrinsic signalling inputs. Further elucidation of these pathways might provide therapeutic opportunities for restoring alveolar capacity. Highlighted Article: Glucocorticoid receptor and STAT3 signalling act in parallel, but cross-regulated, pathways to control epithelial differentiation during lung alveolar development in mice.

Cardiac GR and MR: From Development to Pathology

The efficacy of mineralocorticoid receptor (MR) antagonism in the treatment of certain patients with heart failure has highlighted the pivotal role of aldosterone and MR in heart disease. The glucocorticoid (GC) receptor (GR) is also expressed in heart, but the role of cardiac GR had received much less attention until recently. GR and MR are highly homologous in both structure and function, although not in cellular readout. Recent evidence in animal models has uncovered a tonic role for GC action via GR in cardiomyocytes in prevention of heart disease. Here, we review this evidence and the implications for a balance between GR and MR activation in the early life maturation of the heart and its subsequent health and disease.

Glucocorticoid receptor alters isovolumetric contraction and restrains cardiac fibrosis

Corticosteroids directly affect the heart and vasculature and are implicated in the pathogenesis of heart failure. Attention is focussed upon the role of the mineralocorticoid receptor (MR) in mediating pro-fibrotic and other adverse effects of corticosteroids upon the heart. In contrast, the role of the glucocorticoid receptor (GR) in the heart and vasculature is less well understood. We addressed this in mice with cardiomyocyte and vascular smooth muscle deletion of GR (SMGRKO mice). Survival of SMGRKO mice to weaning was reduced compared with that of littermate controls. Doppler measurements of blood flow across the mitral valve showed an elongated isovolumetric contraction time in surviving adult SMGRKO mice, indicating impairment of the initial left ventricular contractile phase. Although heart weight was elevated in both genders, only male SMGRKO mice showed evidence of pathological cardiomyocyte hypertrophy, associated with increased myosin heavy chain-β expression. Left ventricular fibrosis, evident in both genders, was associated with elevated levels of mRNA encoding MR as well as proteins involved in cardiac remodelling and fibrosis. However, MR antagonism with spironolactone from birth only modestly attenuated the increase in pro-fibrotic gene expression in SMGRKO mice, suggesting that elevated MR signalling is not the primary driver of cardiac fibrosis in SMGRKO mice, and cardiac fibrosis can be dissociated from MR activation. Thus, GR contributes to systolic function and restrains normal cardiac growth, the latter through gender-specific mechanisms. Our findings suggest the GR:MR balance is critical in corticosteroid signalling in specific cardiac cell types.

RENAL AND BLOOD PRESSURE RESPONSE TO A HIGH-SALT DIET IN MICE WITH REDUCED GLOBAL EXPRESSION OF THE GLUCOCORTICOID RECEPTOR

Salt-sensitive hypertension is common in glucocorticoid excess. Glucocorticoid resistance also presents with hypercortisolemia and hypertension but the relationship between salt intake and blood pressure (BP) is not well defined. GRβgeo/+ mice have global glucocorticoid receptor (GR) haploinsufficiency and increased BP. Here we examined the effect of high salt diet on BP, salt excretion and renal blood flow in GRβgeo/+mice. Basal BP was ∼10 mmHg higher in male GRβgeo/+ mice than in GR+/+ littermates. This modest increase was amplified by ∼10 mmHg following a high-salt diet in GRβgeo/+ mice. High salt reduced urinary aldosterone excretion but increased renal mineralocorticoid receptor expression in both genotypes. Corticosterone, and to a lesser extent deoxycorticosterone, excretion was increased in GRβgeo/+ mice following a high-salt challenge, consistent with enhanced 24 h production. GR+/+ mice increased fractional sodium excretion and reduced renal vascular resistance during the high salt challenge, retaining neutral sodium balance. In contrast, sodium excretion and renal vascular resistance did not adapt to high salt in GRβgeo/+ mice, resulting in transient sodium retention and sustained hypertension. With high-salt diet, Slc12a3 and Scnn1a mRNAs were higher in GRβgeo/+ than controls, and this was reflected in an exaggerated natriuretic response to thiazide and benzamil, inhibitors of NCC and ENaC, respectively. Reduction in GR expression causes salt-sensitivity and an adaptive failure of the renal vasculature and tubule, most likely reflecting sustained mineralocorticoid receptor activation. This provides a mechanistic basis to understand the hypertension associated with loss-of-function polymorphisms in GR in the context of habitually high salt intake.

26 Advancement of fetal heart maturation with precocious glucocorticoid treatment is dependent upon maternal genotype

Glucocorticoids are routinely administered to pregnant women at risk of pre-term delivery to mature fetal organs and improve neonatal survival. Here, we tested the hypotheses that (i) antenatal glucocorticoid exposure, prior to the normal increase in glucocorticoid levels, will advance fetal heart maturation and (ii) this would depend on maternal GR genotype. Female GR+/- and GR+/+ mice were crossed with GR+/- males to generate GR+/+ and GR+/- fetuses. GR+/- intercrosses also generated GR-/- (glucocorticoid-resistant controls). Dexamethasone (100 µg/kg/day) or vehicle was administered in the drinking water of pregnant dams from E12.5 (n = 3–6/group), 2d prior to the initiation of fetal glucocorticoid production. In utero high frequency ultrasound was performed at E15.5. Dexamethasone treatment of GR+/+ dams did not affect myocardial performance index (MPI), a measure of systolic and diastolic function, in either GR+/- or GR+/+ fetuses. However, compared to vehicle, dexamethasone treatment of GR+/- dams decreased MPI (indicating improved cardiac function) in their GR+/- fetuses (mean ± SEM: vehicle = 0.746 ± 0.020, dex = 0.620 ± 0.028; P < 0.01, n = 13–16) with no effect on MPI in their GR+/+ fetuses (mean ± SEM: vehicle = 0.713 ± 0.044, dex = 0.687 ± 0.082, n = 4–7). Examination of the influence of maternal genotype showed elevated MPI in GR+/- fetuses in GR+/- dams compared to GR+/+ dams (mean ± SEM: GR+/- dams = 0.769 ± 0.027, GR+/+ dams = 0.585 ± 0.075, n = 4), which was reversed by dexamethasone treatment (2-way ANOVA interaction P ≤ 0.01). Precocious GR activation therefore improves fetal heart function, but only in GR+/- fetuses from GR+/- dams, suggesting fetal heart maturation is dependent on both fetal and maternal factors. Fetal factors could include reduced GR density in GR+/- mice and maternal factors may include the higher circulating plasma levels of glucocorticoid in GR+/- mice.

19 Sex Differences in Pathological Remodelling Caused by Cardiomyocyte/Vascular Smooth Muscle Glucocorticoid Receptor Defficiency

Variation in the human glucocorticoid receptor (GR) gene associates with relative glucocorticoid resistance, hypertension and increased cardiovascular disease risk. Here, the contribution of cardiac GR to disease risk was assessed in male and female “SMGRKO” mice with cardiomyocyte and vascular smooth muscle deletion of GR. SMGRKO mice, generated from GR “floxed” (congenic on C57BL/6J) x SM22α-Cre crosses, have reduced cardiac GR protein and mRNA levels (by 52% and 57%, respectively), compared to Cre-negative littermate controls. Interestingly, fewer female SMGKRO mice survive to weaning [SMGRKO: Control; females 23:68(25%); males 42:52(45%)]. Doppler measurements (Visual Sonics Vevo770 ultrasound) of blood flow within the left ventricle show a detrimental increase in the myocardial performance index, which represents combined systolic and diastolic function, in both sexes of adult SMGRKO mice, primarily due to greater isovolumetric contraction time (p < 0.05) indicating impairment of the initial left ventricular contractile phase. In males only, cardiomyocyte size and heart weight (% body weight) are increased (Control: 0.5 ± 0.02%; SMGRKO: 0.55 ± 0.01%, p < 0.05), as are levels of mRNA encoding myosin heavy chain-β, a marker of pathological cardiac hypertrophy (p < 0.05). Both sexes showed left ventricular fibrosis (histopathology) and elevated levels of mRNA encoding pro-fibrotic factors. Thus, cardiomyocyte/smooth muscle GR-deficiency causes the same functional impairment of isovolumetric contraction but differential pathological changes in the left ventricle of male and surviving female mice. Whether pathology is worse in females that died before weaning is under investigation. Nevertheless, these findings support a role for cardiomyocyte GR in determination of cardiovascular disease risk, though without overt signs of cardiac hypertrophy in females.

A ROLE FOR THE GLUCOCORTICOID RECEPTOR IN CARDIAC REMODELLING

Variation in the glucocorticoid receptor (GR) associates with relative glucocorticoid resistance, hypertension and increased cardiovascular disease risk in humans. Mice heterozygous for the glucocorticoid receptor (GR+/−) are similarly glucocorticoid resistant with raised circulating glucocorticoid levels and elevated blood pressure; susceptibility to heart disease is uncharacterised. Here we describe the cardiac phenotype of adult male GR+/− mice and show evidence of impaired cardiac remodelling in response to pharmacological challenge. Heart weight (% body weight) is unchanged in 12 week old GR+/− mice (WT:0.57±0.03%, GR+/−:0.61±0.03%), but cardiomyocyte cross-sectional area is reduced (WT:240±21 µm2, GR+/−:193±9.0 µm2, p<0.05), and cardiac nuclei density is increased (nuclei/field; WT:67±1, GR+/−:74±2, p<0.05), suggesting GR+/− mice have more but smaller cardiomyocytes than WT. Whilst histological analysis does not reveal differences in fibrosis, cardiac levels of mRNA encoding connective tissue growth factor are reduced in GR+/− mice (WT:100±12%, GR+/−:65±4%, p<0.05) implying subtle alterations in pro-fibrotic signalling. Echocardiography demonstrates comparable cardiac function in 10 week old GR+/− and WT mice. Intriguingly, preliminary data show cardiac hypertrophy in response to angiotensin II infusion (100 ngkg-1 min-1 by osmotic mini-pump) is attenuated in GR+/− mice (heart weight/tibia length Vehicle: WT 7.47±0.326 mg/mm, GR+/−7.29 mg/mm; AngII: WT 8.66±0.249 mg/mm, GR+/−8.07±0.217 mg/mm, p<0.05 (AngII treatment)). These data show that GR deficiency alters the size and number of cardiomyocytes and that, whilst adult GR+/− mice match WT cardiac function under basal conditions, cardiac remodelling following pathological challenge is attenuated. Further characterisation of the GR+/− cardiac phenotype may provide critical insights into how GR variation in humans increases risk of heart disease.