Peter J. Mark

Developmental Programming of Renal Glucocorticoid Sensitivity and the Renin-Angiotensin System

Fetal glucocorticoid excess leads to subsequent adult hypertension, but the mechanisms involved in this developmental programming remain largely unknown. In this study we tested the hypothesis that programmed hypertension in rats is linked to altered renal expression of the glucocorticoid receptor, mineralocorticoid receptor, and 11β-hydroxysteroid dehydrogenase type 2 and components of the intrarenal and adipose renin-angiotensin system. The interactive effects of a postnatal diet enriched in omega-3 fatty acids, which prevents emergence of the hypertensive phenotype, were also examined. Maternal dexamethasone (0.75 μg/mL of drinking water from day 13 to term) markedly increased renal expression of the glucocorticoid receptor in 6-month–old offspring, and this was associated with hypomethylation of the glucocorticoid receptor promoter; renal MR was unaffected. In contrast, maternal dexamethasone reduced renal 11β-hydroxysteroid dehydrogenase type 2 in offspring, but this effect was prevented by a high omega-3 diet. Consistent with these effects, renal Na/K-ATPase-α1 was elevated in offspring of dexamethasone-treated mothers, but only in those raised on the standard diet. Maternal dexamethasone also programmed increased expression of renal and adipose angiotensin-converting enzyme and renal renin, but among these changes, only that of renal angiotensin-converting enzyme was prevented by the omega-3 diet. Our data support the hypothesis that programmed hypertension is mediated, in part, by increased renal glucocorticoid sensitivity, with consequent stimulatory effects on Na/K-ATPase-α1 and intrarenal renin-angiotensin system components. Partial prevention of programmed changes in renal gene expression by postnatal dietary omega-3 fatty acids provides insight into how this intervention prevents hypertension induced by fetal glucocorticoid excess.

Changes in the Placental Glucocorticoid Barrier During Rat Pregnancy: Impact on Placental Corticosterone Levels and Regulation by Progesterone

Abstract Glucocorticoid excess in utero inhibits fetal growth and programs adverse outcomes in adult offspring. Access of maternal glucocorticoid to the glucocorticoid receptor (NR3C1) in the placenta and fetus is regulated by metabolism via the 11beta-hydroxysteroid dehydrogenase (HSD11B) enzymes, as well as multidrug resistance P-glycoprotein (ABCB1)-mediated efflux of glucocorticoids from the syncytiotrophoblast. This study determined expression of genes encoding the two HSD11B isoforms (Hsd11b1 and Hsd11b2), the two ABCB1 isoforms (Abcb1a and Abcb1b), and Nr3c1 in the junctional and labyrinth zones of rat placentas at Days 16 and 22 of normal gestation (Day 23 is term). To assess possible regulation of the Hsd11b and Abcb1 isoforms by glucocorticoids and progesterone, their placental expression was also measured at Day 22 after partial progesterone withdrawal from Day 16 (maternal ovariectomy plus full estrogen and partial progesterone replacement) or after treatment with dexamethasone acetate (1 μg/ml of drinking water from Day 13). Expression of Hsd11b1 mRNA increased in the labyrinth zone (the site of maternal-fetal exchange) from Day 16 to Day 22, whereas that of Hsd11b2 fell dramatically. Consistent with these changes, corticosterone levels increased 10-fold in the labyrinth zone over this period. Expression of both Abcb1a and Abcb1b was markedly higher in the labyrinth zone compared with the junctional zone on both days, consistent with the proposed barrier role of ABCB1 in the placenta. Nr3c1 mRNA expression was similar in the two placental zones at Day 16 but increased 3-fold in the labyrinth zone by Day 22. Partial progesterone withdrawal increased Hsd11b1 mRNA and protein expression in the labyrinth zone but decreased Nr3c1 mRNA expression. These data show that the dynamic expression patterns of the placental HSD11Bs in late gestation are associated with dramatic shifts in placental corticosterone. Moreover, the late gestational rise in labyrinthine Hsd11b1 seems to be driven by the normal prepartum fall in progesterone level.

Placental ABCA1 and ABCG1 transporters efflux cholesterol and protect trophoblasts from oxysterol induced toxicity.

ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 mediate the efflux of cholesterol and other sterols. Both transporters are expressed on the fetal capillaries of the placenta and are involved in maternal-to-fetal cholesterol delivery. In this study, we report that ABCA1 and ABCG1 are also present on the syncytiotrophoblast, the maternal facing placental membrane. Syncytial ABCA1 expression is apical, suggesting a role in cholesterol efflux to the mother, while ABCG1 is expressed basolaterally indicating transport to the fetus. Silencing of ABCA1 expression in primary trophoblasts in culture, or pharmacological antagonism by glyburide, decreased cholesterol efflux to apolipoprotein A-I (apoA-I) compared to controls, while ABCG1-silencing decreased cholesterol efflux to high density lipoproteins (HDL). In contrast, treatment with endogenous or synthetic LXR alpha/beta ligands such as T0901317 increased ABCA1 and ABCG1 expression and enhanced cholesterol efflux to apoA-I and HDL, respectively, while treatment with pharmacological PPAR-alpha or -gamma ligands was without effect. Trophoblasts transfected with ABCA1 or ABCG1 siRNA were more sensitive to toxic oxysterols substrates (25-hydroxycholesterol and 7-ketocholesterol) compared to mock-transfected cells, while prior treatment with T0901317 reduced oxysterol-mediated toxicity. These results identify syncytial ABCA1 and ABCG1 as important, inducible cholesterol transporters which also prevent placental accumulation of cytotoxic oxysterols.

A structure-based mutational analysis of cyclophilin 40 identifies key residues in the core tetratricopeptide repeat domain that mediate binding to Hsp90.

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90β construct and qualitatively assessed for binding using a β-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90β using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding.

Expression of the estrogen receptor-associated immunophilins, cyclophilin 40 and FKBP52, in breast cancer

The estrogen receptor α (ERα) is implicated in the development of breast cancer. The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with ERα and other steroid receptors in mutually exclusive heterocomplexes and may differentially modulate receptor activity. Since previous studies have not assessed the levels of these immunophilins in breast cancer, we examined 10 breast cancer cell lines for mRNA and protein expression of CyP40 and FKBP52 and for amplification of the CyP40 gene. In addition, 26 breast carcinomas, including seven with matched normal breast tissue, were examined for mRNA expression of both immunophilins. CyP40 and FKBP52 were ubiquitously expressed in breast cancer cell lines, but there were significant differences in their pattern of expression. FKBP52 protein levels were generally an order of magnitude greater than those for CyP40. FKBP52 mRNA expression correlated strongly with protein expression and was significantly higher in ERα-positive compared with ERα-negative cell lines. However, CyP40 mRNA expression did not correlate with protein expression, nor did expression of this immunophilin correlate with ERα status. Relatively high expression of CyP40 in one cell line (BT-20) could be attributed to amplification of the CyP40 gene. Both immunophilins were also ubiquitously expressed in breast carcinomas, and we demonstrate for the first time that both CyP40 and FKBP52 mRNA are overexpressed in breast tumors compared to matched normal breast controls. The overexpression of CyP40 and FKBP52, coupled with relative differences in their expression in tumors, may have important functional implications for ERα and other steroid receptors in breast cancer.

Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock

Abstract The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42°C for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37°C, those exposed to heat stress (42°C for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate, hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Maternal dietary omega-3 fatty acids and placental function

The developing fetus requires substantial amounts of fatty acids to support rapid cellular growth and activity. Although the fatty acid composition delivered to the fetus is largely determined by maternal circulating levels, the placenta preferentially transfers physiologically important long-chain polyunsaturated fatty acids (LC-PUFAs), particularly omega-3 (n-3) PUFAs. Maternal dietary supplementation with n-3 PUFAs during pregnancy has been shown to increase gestation length, enhance fetal growth, and reduce the risk of pregnancy complications, although the precise mechanisms governing these effects remain uncertain. Omega-3 PUFAs are involved in several physiological pathways which could account for these effects, including anti-inflammatory, pro-resolving, and anti-oxidative pathways. Recent studies have shown that maternal dietary n-3 PUFA supplementation during rat pregnancy can reduce placental oxidative damage and increase placental levels of pro-resolving mediators, effects associated with enhanced fetal and placental growth. Because several placental disorders, such as intrauterine growth restriction, preeclampsia, and gestational diabetes mellitus, are associated with heightened placental inflammation and oxidative stress, there is considerable interest in the potential for dietary n-3 PUFAs as a therapeutic intervention for these disorders. In this study, we review the impact of dietary n-3 PUFAs on placental function, with particular focus on placental inflammation, inflammatory resolution, and oxidative stress.

Oxysterols exert proinflammatory effects in placental trophoblasts via TLR4-dependent, cholesterol-sensitive activation of NF-κB.

Oxidized cholesterol metabolites (oxysterols) promote inflammation in a variety of cell types and are thought to be involved in a number of disease pathologies. Oxysterol concentrations are increased in pregnancy, together with systemic oxidative stress and inflammation. We tested the hypothesis that oxysterols 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-ketoC) promote placental trophoblast inflammation, and determined the mechanisms involved. Treatment of primary trophoblasts in culture with 25-OHC and 7-ketoC increased the production of proinflammatory cytokines (interleukin-6, macrophage inflammatory protein-1β and tumour necrosis factor-α) in a concentration-dependent fashion. Inhibition of TLR4 activation using selective inhibitors of TLR4 complex formation (OxPAPC) or signalling transmission (CLI095) prevented lipopolysaccharide (LPS)- and oxysterol-induced inflammatory cytokine production. Pretreatment of trophoblasts with selective inhibitors of I-kB kinase activity (parthenolide and TPCA-1) reduced oxysterol- and LPS-stimulated inflammatory responses, consistent with the involvement of the nuclear factor kappa B (NF-κB) pathway downstream of TLR4 signalling. Both oxysterols also increased the phosphorylation and nuclear localization of NF-κB subunit p65/RelA. Oxysterols are also known to activate liver X receptors (LXRs) which can inhibit inflammatory signalling, either directly or indirectly via membrane cholesterol reduction. Treatment with the LXR agonist, T0901317, exerted significant anti-inflammatory effects, reducing LPS- and oxysterol-driven cytokine production. Treatment with methyl-β-cyclodextrin to deplete membrane microdomain cholesterol and thereby disrupt TLR4 signalling, similarly abrogated their effects. Together, these findings indicate that although oxysterols likely activate both pro- and anti-inflammatory pathways in the placenta, the predominant effect is the promotion of placental inflammation via TLR4-dependent activation of NF-κB.

Maternal Dietary Omega-3 Fatty Acid Supplementation Reduces Placental Oxidative Stress and Increases Fetal and Placental Growth in the Rat

ABSTRACT Placental oxidative stress plays a key role in the pathophysiology of several placenta-related disorders including intrauterine growth restriction. Oxidative stress occurs when accumulation of reactive oxygen species damages DNA, proteins, and lipids, an outcome normally limited by antioxidant defenses. Dietary supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) may limit oxidative stress by increasing antioxidant capacity, but n-3 PUFAs are also highly susceptible to lipid peroxidation; so n-3 PUFA supplementation is potentially harmful. Here we examined the effect of n-3 PUFAs on placental oxidative stress and on placental and fetal growth in the rat. We also investigated whether diet-induced changes in maternal plasma fatty acid profiles are associated with comparable changes in placental and fetal tissues. Rats were fed either standard or high n-3 PUFA diets from Day 1 of pregnancy, and tissues were collected on Day 17 or 22 (term = Day 23). Dietary supplementation with n-3 PUFAs increased fetal (6%) and placental (12%) weights at Day 22, the latter attributable primarily to growth of the labyrinth zone (LZ). Increased LZ weight was accompanied by reduced LZ F2-isoprostanes (by 31% and 11% at Days 17 and 22, respectively), a marker of oxidative damage. Maternal plasma PUFA profiles were altered by dietary fatty acid intake and were strongly predictive of corresponding profiles in placental and fetal tissues. Our data indicate that n-3 PUFA supplementation reduces placental oxidative stress and enhances placental and fetal growth. Moreover, fatty acid profiles in the mother, placenta, and fetus are highly dependent on dietary fatty acid intake.

Antioxidant Defenses in the Rat Placenta in Late Gestation: Increased Labyrinthine Expression of Superoxide Dismutases, Glutathione Peroxidase 3, and Uncoupling Protein 2

Placental oxidative stress plays a key role in the pathophysiology of placenta-related disorders, most notably preeclampsia (PE) and intrauterine growth restriction (IUGR). Oxidative stress occurs when accumulation of reactive oxygen species (ROS) damages DNA, proteins and lipids, an outcome that is limited by antioxidant enzymes; mitochondrial uncoupling protein 2 (UCP2) may also limit oxidative stress by reducing ROS production. Here we characterized placental antioxidant defenses during normal gestation and following glucocorticoid-induced IUGR. Placentas were collected on Days 16 and 22 of normal rat pregnancy (term = Day 23) and at Day 22 after dexamethasone treatment from Day 13. Expression of several genes encoding antioxidant enzymes (Sod1, Sod2, Sod3, Cat, Gpx3, Txn1, Txnrd1, Txnrd2, and Txnrd3) and Ucp2 was measured by quantitative RT-PCR in the labyrinth (LZ) and junctional zones (JZ) of the placenta. Expression of Sod1 and Ucp2 mRNAs and the activity of xanthine oxidase, a source of ROS, all increased from Days 16 to 22 in both placental zones, whereas Sod2 and Gpx3 increased only in the rapidly growing LZ. In contrast, Sod3 and Txnrd1 expression fell in the LZ over this period, whereas total superoxide dismutase activity remained stable. Dexamethasone treatment reduced fetal-placental growth and LZ expression of Ucp2 but increased JZ expression of Txn1. Indices of placental oxidative damage (TBARS, F2-isoprostanes, and 8-OHdG) did not change with gestational age or dexamethasone, indicative of adequate antioxidant protection. Overall, our data suggest that the rat placenta is protected from oxidative stress by the dynamic zone- and stage-dependent expression of antioxidant defense genes.