Rachel Karry

Mineralocorticoid Receptor Blocker Increases Angiotensin-Converting Enzyme 2 Activity in Congestive Heart Failure Patients

Aldosterone plays an important role in the pathophysiology of congestive heart failure (CHF), and spironolactone improves cardiovascular function and survival rates in patients with CHF. We hypothesized that the mineralocorticoid receptor blockade (MRB) exerted its beneficial effects by reducing oxidative stress and changing the balance between the counter-acting enzymes angiotensin-converting enzyme (ACE) and ACE2. Monocyte-derived macrophages were obtained from 10 patients with CHF before and after 1 month of treatment with spironolactone (25 mg/d). Spironolactone therapy significantly (P<0.005) reduced oxidative stress, as expressed by reduced lipid peroxide content, superoxide ion release, and low-density lipoprotein oxidation by 28%, 53%, and 70%, respectively. Although spironolactone significantly (P<0.01) reduced macrophage ACE activity by 47% and mRNA expression by 53%, ACE2 activity and mRNA expression increased by 300% and 654%, respectively. In mice treated for 2 weeks with eplerenone (200 mg · kg−1 · d−1), cardiac ACE2 activity significantly (P<0.05) increased by 2-fold and was paralleled by increased ACE2 activity in macrophages. The mechanism of aldosterone antagonist action was studied in mouse peritoneal macrophages (MPMs) in vitro. Although ACE activity and mRNA were significantly increased by 250 nmol/L aldosterone, ACE2 was significantly reduced. Cotreatment with eplerenone (2 &mgr;mol/L) attenuated these effects. In MPM obtained from p47 knockout mice, where NADPH oxidase is inactive, as well as in control MPMs treated with NADPH oxidase inhibitor, aldosterone did not increase ACE or decrease ACE2. MRB reduced oxidative stress, decreased ACE activity, and increased ACE2 activity, suggesting a protective role for MRB by possibly increasing generation of angiotensin (1–7) and decreasing formation of angiotensin II. These effects are mediated, at least in part, by NADPH oxidase.

Growth hormone (GH) receptors in prostate cancer: gene expression in human tissues and cell lines and characterization, GH signaling and androgen receptor regulation in LNCaP cells

Various hormones and growth factors have been implicated in progression of prostate cancer, but their role and the underlying molecular mechanism(s) involved remain poorly understood. In this study, we investigated the role of human growth hormone (GH) and its receptor (GHR) in human prostate cancer. We first demonstrated mRNA expression of GHR and of its exon 9-truncated isoform (GHR(tr)) in benign prostate hyperplasia (BPH) and prostate adenocarcinoma patient tissues, as well as in LNCaP, PC3 and DU145 human prostate cancer cell lines. GHR mRNA levels were 80% higher and GHR(tr) only 25% higher, in the carcinoma tissues than in BPH. Both isoforms were also expressed in LNCaP and PC3 cell lines and somewhat less so in DU145 cells. The LNCaP cell GHR protein was further characterized, on the basis of its M(r) of 120kDa, its binding to two different GHR monoclonal antibodies, its high affinity and purely somatogenic binding to (125)I-hGH and its ability to secrete GH binding protein, all characteristic of a functional GHR. Furthermore, GH induced rapid, time- and dose-dependent signaling events in LNCaP cells, including phosphorylation of JAK2 tyrosine kinase, of GHR itself and of STAT5A (JAK2-STAT5A pathway), of p42/p44 MAPK and of Akt/PKB. No effect of GH (72h) could be shown on basal or androgen-induced LNCaP cell proliferation nor on PSA secretion. Interestingly, however, GH caused a rapid (2-12h) though transient striking increase in immunoreactive androgen receptor (AR) levels (< or =5-fold), followed by a slower (24-48h) reduction (< or = 80%), with only modest parallel changes in serine-phosphorylated AR. In conclusion, the GH-induced activation of signaling pathways, its effects on AR protein in LNCaP cells and the isoform-specific regulation of GHR in prostate cancer patient tissues, suggest that GH, most likely in concert with other hormones and growth factors, may play an important role in progression of human prostate cancer.

Urokinase Plasminogen Activator Upregulates Paraoxonase 2 Expression in Macrophages via an NADPH Oxidase-Dependent Mechanism

Objective—Macrophage foam cells are characterized by increased oxidative stress. Macrophage urokinase plasminogen activator (uPA) was shown to contribute to atherosclerosis progression. We hypothesized that uPA atherogenicity is related to its ability to increase macrophage oxidative stress. Increased macrophage oxidative stress in turn was shown to enhance PON2 expression. In the present study we investigated the effect of uPA on macrophage PON2 expression in relation to cellular oxidative stress. Methods and Results—uPA increased PON2 expression in THP-1 macrophages in a dose-dependent manner. This effect required uPA/uPAR interaction and was abolished by cell treatment with antioxidants. uPA increased macrophage oxidative stress, measured by increased lipid peroxides, reactive oxygen species formation, superoxide anion release, and cell-mediated LDL oxidation. These effects were related to uPA-mediated activation of NADPH oxidase, and could not be reproduced in mouse peritoneal macrophages (MPM) harvested from p47phox−/− mice, suggesting a causal relationship between NADPH oxidase activation and the effects of uPA on macrophage oxidative stress and PON2 expression. Finally, MPM from PON2−/− mice were more susceptible to uPA-induced cellular oxidative stress than wild-type MPM, suggesting that PON2 protects against uPA-stimulated macrophage oxidative stress. Conclusions—Upregulation of macrophage PON2 may provide a compensatory protective mechanism against uPA-stimulation of macrophage oxidative stress during atherogenesis.

Growth hormone affects gene expression and proliferation in human prostate cancer cells.

We previously showed that growth hormone (GH) receptors (GHR) are expressed in the most commonly studied human prostate cancer (PCa) cell lines and that GHR isoforms undergo differential, cell-type-specific hormonal regulation. We now report that human GH (hGH) can stimulate/modulate insulin-like growth factor (IGF) and β-oestradiol (E(2) ) receptor (ER(β) ) gene expressions in these cells and interact with IGF-I and E(2) to stimulate androgen-dependent LNCaP cell proliferation. We observed a cell type-dependent, differential regulation of IGF axis gene expression by GH: IGF-I was stimulated in the androgen-dependent LNCaP cells; IGF-II was stimulated in androgen-insensitive (AI) PC3 cells; the IGF-I cognate receptor, IGF-IR, was stimulated in LNCaP cells, but inhibited in PC3 cells; IGF-IIR was stimulated in both LNCaP and PC3 cells. GH also stimulated ER(β) gene expression in LNCaP and PC3 cells, but had little or no effect on any of those genes in AI DU145 cells. The potent androgen analogue, mibolerone, also stimulated IGF-I, IGF-IR and ER(β) , but reduced IGF-IIR mRNAs in LNCaP cells. Furthermore, triiodothyronine (T(3) ) and E(2) also stimulated the expression of those four genes in LNCaP cells, but co-administration of GH had almost no effect. Finally, we also studied the effects of GH, IGF-I and E(2) , alone or in combination, on LNCaP cell proliferation. Importantly, we demonstrated, for the first time, that although GH and IGF-I alone had no effect on LNCaP cell proliferation, concomitant administration for 96 h revealed a permissive role of GH on IGF-I-induced proliferation. GH also appeared to exert a synergistic effect on E(2) -stimulated LNCaP cell proliferation. Taken together, these findings indicate that GH via GHRs, most likely in concert with gonadal steroids, T(3) , IGF system axis and probably other hormones and growth factors, potentially plays an important role in the mechanisms underlying tumour cell growth in PCa.

Paraoxonase activity and expression is modulated by therapeutics in experimental rat nonalcoholic Fatty liver disease.

Objective. The objective of the present study is to investigate the effect of rosiglitazone, metformin, ezetimibe, and valsartan (alone or in combinations) on paraoxonase (PON) activity and PON-mRNA expression in nonalcoholic fatty liver disease (NAFLD). Methods. 54 Male Sprague–Dawley rats were divided to 9 groups: chow diet group (15 weeks); methionine-choline-deficient diet (MCDD) group (15 weeks); MCDD-treated groups for the last 6 weeks with either metformin (M), rosiglitazone (R), metformin plus rosiglitazone (M+R), ezetimibe (E), valsartan (V), or a combination of R+M+V or of R+M+V+E for a total period of 15 weeks. Results. PON activities in serum and liver were decreased in MCDD rats. PON activity in serum increased significantly in all treatment groups. PON activity in liver was also increased significantly, except only in groups R, E, V, R+M+V, and R+M+V+E. Liver PON3 mRNA expression increased significantly in groups R+M, E, V, R+M+V, and R+M+V+E whereas liver PON2 mRNA expression increased significantly in MCDD, R+M, E, V, R+M+V, and R+M+V+E. Conclusions. PON activities in serum and liver were decreased in NAFLD. Treatment with insulin sensitizers, ezetimibe, and valsartan increased PON activity and reduced oxidative stress both in serum and liver.

Dexamethasone in the presence of desipramine enhances MAPK/ERK1/2 signaling possibly via its interference with β-arrestin

Antidepressant medication is the standard treatment for major depression disorder (MDD). However, the response to these treatments is often incomplete and many patients remain refractory. In the present study, we show that the glucocorticoid receptor (GR) agonist dexamethasone (DEX) increased MAPK/ERK1/2 signaling in the presence of the noradrenergic antidepressant, desipramine (DMI), while no such effect was induced by DEX or DMI alone in human neuroblastoma SH-SY5Y cells. This enhancement was dependent on the activation of both α2 adrenergic receptors (AR) and GR. The timing of MAPK/ERK1/2 activation as well as DEX-induced reduction in membranous α2 AR suggests the involvement of a β-arrestin-dependent mechanism. In line with the latter, DEX increased cytosolic and decreased membranous levels of β-arrestin. Concomitantly, DEX induced a time-dependent increase in cytosolic α2 AR-β-arrestin interaction and a decrease in β-arrestin interaction with Mdm2 E3 ubiquitin ligase. All of these effects of DEX were prevented by the GR antagonist RU486. Our data suggest an additional intracellular role for DEX, in which activation of GR interferes with the trafficking and degradation of β-arrestin-α2c-AR complex. We suggest that such an interaction in the presence of DMI can enhance MAPK/ERK1/2 signaling, a key player in neural plasticity and neurogenesis processes, which is impaired in MDD, while stimulated by antidepressants.