TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population.

The environmental toxin 2,3,7,8 tetrachlorodibenzo p-dioxin (TCDD) plays an important role in the development of chloracne. Chloracne is characterized by hyperkeratosis of the interfollicular squamous epithelium and metaplasia of sebaceous glands. Dysregulation of keratinocyte terminal differentiation leading to accelerated formation of the cornified envelope as a result of TCDD-mediated aryl hydrocarbon receptor (AHR) activation has been implicated as one of the molecular pathogenic mechanisms contributing to the development of chloracne. In addition, chloracne is characterized by altered skin stem cell characteristics, and it has been speculated that the phenotype of chloracne closely matches that of c-Myc overexpressing transgenic mice. Therefore, we sought to determine whether TCDD plays a role in regulation of the skin stem cell population. We have proposed in this report that TCDD may directly or indirectly (via AHR receptor cross-talk) upregulate c-Myc via epidermal growth factor receptor-extracellular signal regulated kinase (EGFR-ERK) axis stimulation, which may correspond with an increase in human epidermal stem cell activation and differentiation of EPSCs into keratinocytes, with eventual depletion of the epidermal stem cell compartment of the skin. Thus, TCDD may cause increased epidermal stem cell turnover during chloracne.

Angiotensin type 1 receptor resistance to blockade in the opossum proximal tubule cell due to variations in the binding pocket.

Blockade of the angiotensin (ANG) II receptor type 1 (AT(1)R) with angiotensin receptor blockers (ARBs) is widely used in the treatment of hypertension. However, ARBs are variably effective in reducing blood pressure, likely due, in part, to polymorphisms in the ARB binding pocket of the AT(1)R. Therefore, we need a better understanding of variations/polymorphisms that alter binding of ARBs in heterogeneous patient populations. The opossum proximal tubule cell (OKP) line is commonly used in research to evaluate renal sodium handling and therefore blood pressure. Investigating this issue, we found natural sequence variations in the opossum AT(1)R paralleling those observed in the human AT(1)R. Therefore, we posited that these sequence variations may explain ARB resistance. We demonstrate that OKP cells express AT(1)R mRNA, bind (125)I-labeled ANG II, and exhibit ANG II-induced phosphorylation of Jak2. However, Jak2 phosphorylation is not inhibited by five different ARBs commonly used to treat hypertension. Additionally, nonradioactive ANG II competes (125)I-ANG II efficiently, whereas a 10-fold molar excess of olmesartan and the ANG II receptor type 2 blocker PD-123319 is unable to block (125)I-ANG II binding. In contrast, ANG II binding to OKP cells stably expressing rat AT(1A)Rs, which have a conserved AT(1)R-binding pocket with human AT(1)R, is efficiently inhibited by olmesartan. A novel observation was that resistance to ARB binding to opossum AT(1)Rs correlates with variations from the human receptor at positions 108, 163, 192, and 198 within the ARB-binding pocket. These observations highlight the potential utility of evaluating AT(1)R polymorphisms within the ARB-binding pocket in various hypertensive populations.

Molecular and metabolic mechanisms of cardiac dysfunction in diabetes.

Diabetes mellitus type 2 (T2DM) is a widespread chronic medical condition with prevalence bordering on the verge of an epidemic. It is of great concern that cardiovascular disease is more common in patients with diabetes than the non-diabetic population. While hypertensive and ischemic heart disease is more common in diabetic patients, there is another type of heart disease in diabetes that is not associated with hypertension or coronary artery disease. This muscle functional disorder is termed "diabetic cardiomyopathy". Diastolic dysfunction characterized by impaired diastolic relaxation time and reduced contractility precedes systolic dysfunction and is the main pathogenic hallmark of this condition. Even though the pathogenesis of "diabetic cardiomyopathy" is still controversial, impaired cardiac insulin sensitivity and metabolic overload are emerging as major molecular and metabolic mechanisms for cardiac dysfunction. Systemic insulin resistance, hyperinsulinemia, dysregulation of adipokine secretion, increases in circulating levels of inflammatory mediators, aberrant activation of renin angiotensin aldosterone system (RAAS), and increased oxidative stress contribute dysregulated insulin and metabolic signaling in the heart and development of diastolic dysfunction. In addition, maladaptive calcium homeostasis and endothelial cell dysregulation endoplasmic reticular stress play a potential role in cardiomyocyte fibrosis/diastolic dysfunction. In this review, we will focus on emerging molecular and metabolic pathways underlying cardiac dysfunction in diabetes. Elucidation of these mechanisms should provide a better understanding of the various cardiac abnormalities associated with diastolic dysfunction and its progression to systolic dysfunction and heart failure.

Insulin resistance and heart failure: molecular mechanisms.

This article addresses the issue of insulin resistance and associated reductions in cardiac insulin metabolic signaling, which is emerging as a major factor in the development of heart failure, and assumes more importance because of an epidemic increase in obesity and the cardiorenal metabolic syndrome in our aging population. The effects of cardiac insulin resistance are exacerbated by metabolic, endocrine, and cytokine alterations associated with systemic insulin resistance. Understanding the molecular mechanisms linking insulin resistance and heart failure may help to design new and more effective mechanism-based drugs to improve myocardial and systemic insulin resistance.

Mitochondria and Oxidative Stress in the Cardiorenal Metabolic Syndrome.

Mitochondria play a fundamental role in the maintenance of normal structure, function, and survival of tissues. There is considerable evidence for mitochondrial dysfunction in association with metabolic diseases including insulin resistance, obesity, diabetes, and the cardiorenal metabolic syndrome. The phenomenon of reactive oxygen species (ROS)-induced ROS release through interactions between cytosolic and mitochondrial oxidative stress contributes to a vicious cycle of enhanced oxidative stress and mitochondrial dysfunction. Activation of the cytosolic and mitochondrial NADPH oxidase system, impairment of the mitochondrial electron transport, activation of p66shc pathway-targeting mitochondria, endoplasmic reticular stress, and activation of the mammalian target of the rapamycin-S6 kinase pathway underlie dysregulation of mitochondrial dynamics and promote mitochondrial oxidative stress. These processes are further modulated by acetyltransferases including sirtuin 1 and sirtuin 3, the former regulating nuclear acetylation and the latter regulating mitochondrial acetylation. The regulation of mitochondrial functions by microRNAs forms an additional layer of molecular control of mitochondrial oxidative stress. Alcohol further exacerbates mitochondrial oxidative stress induced by overnutrition and promotes the development of metabolic diseases.

Over-nutrition and metabolic cardiomyopathy.

Cardiovascular disease, which accounts for the highest morbidity and mortality in the United States, has several major risk factors, including aging and diabetes. Overweight and obesity, especially abdominal obesity, have been increasingly implicated as independent risk factors in the development of cardiovascular disease. Metabolic and/or diabetic cardiomyopathy has been especially associated with excess body weight caused by chronic over-nutrition and high-fat feeding. In the initial stages, obesity is now understood to cause significant dysregulation of cardiac fatty acid and glucose metabolism. These abnormalities are due, in part, to increased oxidative stress, which in turn can cause deleterious effects on intracellular signaling pathways that control cellular growth and proliferation. This increase in oxidative stress is coupled with reduced anti-oxidant species and dysregulation of metabolic signaling pathways. The cardiomyopathy seen with obesity is associated with increased interstitial fibrosis and diastolic dysfunction. Over time, evolving abnormalities include hypertrophy and systolic dysfunction, eventually leading to heart failure.

Phosphoprotein Phosphatase PP2A Regulation of Insulin Receptor Substrate 1 and Insulin Metabolic Signaling.

Insulin (INS) metabolic signaling is important for normal cardiovascular and renal function as well as for exerting the classic actions of INS, such as glucose uptake in skeletal muscle tissue. There is emerging evidence that tyrosine phosphatases as well as protein kinases have important modulating roles in INS metabolic signaling in both cardiovascular and classically INS- sensitive tissues. For example, increases in phosphatase activity may partially explain how angiotensin II and aldosterone attenuate activation of the INS receptor substrate protein 1 (IRS-1)-phosphatidylinositol 3 kinase-protein kinase B pathway, thereby promoting INS resistance. On the other hand, phosphatase activation may also exert beneficial and cardiovascular protective effects in conditions such as overnutrition by blocking serine phosphorylation of IRS-1, thereby improving downstream INS metabolic signaling. Both the beneficial and the detrimental effects exerted by the activation of phosphatases will be covered in this report.

RAS-Mediated Adaptive Mechanisms in Cardiovascular Tissues: Confounding Factors of RAS Blockade Therapy and Alternative Approaches.

Since the classic experiments by Tigerstedt and Bergman that established the role of renin in hypertension a century ago, aggressive efforts have been launched to effectively block the renin-angiotensin system (RAS). Blockade of RAS is advocated at multiple levels by direct renin inhibitor, angiotensin-converting enzyme inhibitor and/or angiotensin II type 1 receptor blocker, or aldosterone inhibitor (spironolactone), and has now become part of the standard of care to control hypertension and related metabolic diseases including diabetes. However, recent lessons learned from randomized clinical trials question the wisdom of blocking RAS at multiple levels. In this context, it is highly pertinent that components of RAS are evolutionarily conserved, and novel physiological/adaptive/protective roles for renin and angiotensin-converting enzyme are currently emerging. Angiotensin II, the classical RAS effector peptide responsible for hypertension, hypertrophy, fluid retention and fibrosis, manifests its cardiovascular protective effect when it activates the angiotensin II type 2 receptor. Additionally, angiotensin-converting enzyme 2 and the angiotensin II metabolite Ang-(1-7) that acts through the Mas proto-oncogene constitute the cardiovascular and renal protective branch of RAS. It is conceivable that modulating this vasodilative/anti-inflammatory branch of RAS by activation of the RAS components that constitute this branch may offer a safer long-term treatment strategy to balance RAS activity and achieve homeostasis compared to chronic multilevel RAS inhibition.

Ligand-dependent complex formation between the Angiotensin II receptor subtype AT2 and Na+/H+ exchanger NHE6 in mammalian cells.

Involvement of Angiotensin II (Ang II) in the regulation of sodium levels by modulating the Na+/H+ exchangers is demonstrated in many tissues. Screening of a mouse 17-day fetus cDNA library with the Angiotensin II receptor AT2 as the bait in yeast two-hybrid assay led us to identify an AT2-interacting mouse fetus peptide that shared 98% amino acid identity with the corresponding region of the human NHE6. NCBI Blast search showed that the clone 6430520C02 (GenBank Accession # AK032326) of the mouse genome project carried the complete sequence of this new mouse NHE6 isoform. The human and mouse NHE6 peptides share 97% overall homology. Further analysis showed that the region spanning the third intracellular loop and C-terminal cytoplasmic tail of the AT2 directly interacted with a 182 amino acid region that spans the predicted 5th intracellular loop and the initial part of the C-terminus of the mouse NHE6 in yeast two-hybrid assay. This 182-amino acid region that interacted with the AT2 also shares 98% homology with the corresponding region of rat NHE6 and therefore is highly conserved across species. We detected widespread expression of this NHE6 isoform in several rat tissues including 10-day fetus, 17-day fetus, and 30-day post-natal tissues of heart, brain, kidney and muscle. Moreover, the AT2 co-immunoiprecipitated with a hemagglutinin tagged NHE6 when expressed in human cell line MCF-7, and activated by AngII. This ligand-dependent complex formation between the AT2 and NHE6 suggests that the hormone Ang II may act as a regulator of NHE6, and Ang II-mediated direct protein-protein interaction between AT2 and NHE6 could be a mechanism for modulating the functions of the ubiquitously expressed NHE6 in different tissues.

Role of Phe308 in the seventh transmembrane domain of the AT2 receptor in ligand binding and signaling.

Studies on Angiotensin II (Ang II) receptor type AT1 have suggested that interaction between the two highly conserved residues, Tyr292 in the 7th transmembrane domain (TMD) and the Asp74 in the 2nd TMD, is critical for linking the Ang II binding and AT1 receptor-Gq protein coupling. In the Ang II receptor type AT2, the Asp is conserved (Asp90 in 2nd TMD), however, there is no Tyr residue in the 7th TMD and Phe308 occupies the analogous position to Tyr292 of the AT1. Replacing this Phe308 with Ala reduced receptor affinity to peptidic ligands (125)I-Ang II (K(d) = 0.37 nM) and (125)I-CGP42112A (K(d) = 0.56 nM), but retained the ability of the AT2 to reduce cGMP levels in Xenopus oocytes. Thus, the Phe308 of the AT2 does not mimic the role of Tyr292 of the AT1 in the receptor activation upon Ang II binding. We have also shown that the M8 mutant of the AT2 with the 7th TMD similar to that of wild type AT2 can couple to PLC like the AT1 and bind the AT2-specific ligands with high affinity. Since the Ang II is shown to bind to both the AT1 and the AT2 in an identical manner, we propose that the absence of Tyr in the 7th TMD of the AT2 does not prevent the receptor from coupling to Gq-protein, rather may contribute to the freedom of the AT2 to couple to trimeric G-proteins in both G- betagamma dependent and independent manners upon Ang II binding.