Vitamin D metabolites influence the expression of various genes involved in calcium homeostasis, cell differentiation, and regulation of the immune system. Expression of these genes is mediated by the activation of the nuclear vitamin D receptor (VDR). Previous studies have shown that a hormonally active form of vitamin D, 1␣,25-dihydroxyvitamin D3, exerts anticoagulant effects in cultured monocytic cells. To clarify whether activation of VDR plays any antithrombotic actions in vivo, hemostatic/thrombogenic systems were examined in normocalcemic VDR knock-out (KO) mice on a high calcium diet and compared with wild type and hypocalcemic VDRKO mice that were fed a regular diet. Platelet aggregation was enhanced significantly in normocalcemic VDRKO mice compared with wild type and hypocalcemic VDRKO mice. Aortic endothelial nitric-oxide (NO) synthase expression and urinary NOx excretions were reduced in hypocalcemic VDRKO mice, but not in normocalcemic VDRKO mice. Northern blot and RT-PCR analyses revealed that the gene expression of antithrombin in the liver as well as that of thrombomodulin in the aorta, liver and kidney was down-regulated in hypo-and normocalcemic VDRKO mice. Whereas tissue factor mRNA expression in the liver and kidney was up-regulated in VDRKO mice regardless of plasma calcium level. Furthermore, VDRKO mice manifested an exacerbated multi-organ thrombus formation after exogenous lipopolysaccharide injection regardless of the calcemic conditions. These results demonstrate that activation of nuclear VDR elicits antithrombotic effects in vivo, and suggest that the VDR system may play a physiological role in the maintenance of antithrombotic homeostasis.

LKB1 encodes a serine/threonine kinase, which functions upstream of the AMP-activated protein kinase (AMPK) superfamily. To clarify the role of LKB1 in heart, we generated and characterized cardiac myocyte-specific LKB1 knock-out (KO) mice using ␣-myosin heavy chain-Cre deletor strain. LKB1-KO mice displayed biatrial enlargement with atrial fibrillation and cardiac dysfunction at 4 weeks of age. Left ventricular hypertrophy was observed in LKB1-KO mice at 12 weeks but not 4 weeks of age. Collagen I and III mRNA expression was elevated in atria at 4 weeks, and atrial fibrosis was seen at 12 weeks. LKB1-KO mice displayed cardiac dysfunction and atrial fibrillation and died within 6 months of age. Indicative of a prohypertrophic environment, the phosphorylation of AMPK and eEF2 was reduced, whereas mammalian target of rapamycin (mTOR) phosphorylation and p70S6 kinase phosphorylation were increased in both the atria and ventricles of LKB1-deficient mice. Consistent with vascular endothelial growth factor mRNA and protein levels being significantly reduced in LKB1-KO mice, these mice also exhibited a reduction in capillary density of both atria and ventricles. In cultured cardiac myocytes, LKB1 silencing induced hypertrophy, which was ameliorated by the expression of a constitutively active form AMPK or by treatment with the inhibitor of mTOR, rapamycin. These findings indicate that LKB1 signaling in cardiac myocytes is essential for normal development of the atria and ventricles. Cardiac hypertrophy and dysfunction in LKB1-deficient hearts are associated with alterations in AMPK and mTOR/ p70S6 kinase/eEF2 signaling and with a reduction in vascular endothelial growth factor expression and vessel rarefaction.LKB1 is a ubiquitously expressed serine/threonine kinase that functions upstream of the 13 members of the AMP-activated protein kinase (AMPK) 5 superfamily (1). Analyses of LKB1-knock-out (KO) mouse models reveal that this kinase controls the physiological functions of many tissues. For instance, whole body LKB1-KO mice display embryonic lethality due to defects in the development of the neural tube, mesenchymal cell death, and abnormal vascular development (2). In addition, tissue-specific ablation of LKB1 has revealed that LKB1 also plays a major role in the regulation of cellular metabolism, likely via its role as an upstream AMPK-kinase (3-5). Although the role of LKB1 in the regulation of metabolic processes is under intense investigation, LKB1 also functions as a tumor suppressor, and its loss leads to the development of Peutz-Jeghers syndrome (6, 7). Importantly, at a cellular level LKB1 controls proliferation and cell polarity (8), which contributes to the tumor-inhibitory actions of LKB1.Consistent with the role of LKB1 in cell growth control, our recent work has shown that increased cardiac myocyte LKB1 activity contributes to the inhibition of protein synthesis involved in cardiac hypertrophy (9). Conversely, reduced LKB1 expression and/or activity has been shown in the hypertrophied hearts of various ...

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E2) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E2-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E2 and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E2-induced hepcidin expression. BMP6 expression induced by E2 was abolished by GPR30 silencing. Finally, both E2 and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E2 and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.

Adiponectin (APN) is an adipocyte-derived factor that exists at high concentrations in serum and has anti-inflammatory and systemic vascular-protective properties. In this study, we investigated the role of APN in pulmonary vascular homeostasis. We found that APN localizes to the luminal side of blood vessels in lung and acts in vitro to block TNF-alpha-induced E-selectin upregulation in pulmonary artery endothelial cells. Targeted deletion of the APN gene in mice leads to a vascular phenotype in lung characterized by E-selectin upregulation and age-dependent increases in perivascular inflammatory cell infiltration and pulmonary arterial pressures. Taken together, these findings demonstrate an important role for APN in lung vascular homeostasis and suggest that APN-deficient states may contribute to the pathogenesis of inflammatory pulmonary vascular disease and to the development of pulmonary hypertension.

Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-␤1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.Androgen exerts a variety of biological effects in many target organs, including male genitalia, brain, and skeletal tissues (1, 2). Most of these actions are mediated through the transcriptional control of particular sets of target genes by the nuclear androgen receptor (AR).1 AR is a ligand-inducible transcription factor that belongs to the nuclear receptor superfamily (3, 4). Upon hormone binding, AR is translocated from the cytosol into the nucleus where it binds specific promoter elements. A number of coregulators and/or coregulator complexes are then recruited to AR, which then activates or represses the transcription of various target genes (2, 3, 5-7). To clarify the physiological function of androgen via AR transcriptional regulation, we generated AR knock-out (ARKO) mice by means of the Cre-loxP system and have reported that ARKO male mice manifest late-onset obesity (8), high turnover osteopenia (9), and impaired brain masculinization (10).In addition to the classic target tissues of androgens, the AR gene is also expressed in mammalian cardiomyocytes, suggesting that androgens may play a role in the heart (11). In fact, Marsh and colleagues (11) have shown that androgens produce cardiac hypertrophy by a direct, receptor-specific mechanism. They also revealed that androgens regulate functional expression of an L-type calcium channel in isolated rat ventricular myocytes, leading to a modulation of cardiac performance in males (12). Li et a...