Shobha Ghosh

The mammalian amiloride-insensitive non-specific salt taste receptor is a vanilloid receptor-1 variant

The amiloride‐insensitive salt taste receptor is the predominant transducer of salt taste in some mammalian species, including humans. The physiological, pharmacological and biochemical properties of the amiloride‐insensitive salt taste receptor were investigated by RT‐PCR, by the measurement of unilateral apical Na+ fluxes in polarized rat fungiform taste receptor cells and by chorda tympani taste nerve recordings. The chorda tympani responses to NaCl, KCl, NH4Cl and CaCl2 were recorded in Sprague‐Dawley rats, and in wild‐type and vanilloid receptor‐1 (VR‐1) knockout mice. The chorda tympani responses to mineral salts were monitored in the presence of vanilloids (resiniferatoxin and capsaicin), VR‐1 antagonists (capsazepine and SB‐366791), and at elevated temperatures. The results indicate that the amiloride‐insensitive salt taste receptor is a constitutively active non‐selective cation channel derived from the VR‐1 gene. It accounts for all of the amiloride‐insensitive chorda tympani taste nerve response to Na+ salts and part of the response to K+, NH4+ and Ca2+ salts. It is activated by vanilloids and temperature (> 38°C), and is inhibited by VR‐1 antagonists. In the presence of vanilloids, external pH and ATP lower the temperature threshold of the channel. This allows for increased salt taste sensitivity without an increase in temperature. VR‐1 knockout mice demonstrate no functional amiloride‐insensitive salt taste receptor and no salt taste sensitivity to vanilloids and temperature. We conclude that the mammalian non‐specific salt taste receptor is a VR‐1 variant.

Curcumin ameliorates renal failure in 5/6 nephrectomized rats: role of inflammation

TNF-alpha and NF-kappaB play important roles in the development of inflammation in chronic renal failure (CRF). In hepatic cells, curcumin is shown to antagonize TNF-alpha-elicited NF-kappaB activation. In this study, we hypothesized that if inflammation plays a key role in renal failure then curcumin should be effective in improving CRF. The effectiveness of curcumin was compared with enalapril, a compound known to ameliorate human and experimental CRF. Investigation was conducted in Sprague-Dawley rats where CRF was induced by 5/6 nephrectomy (Nx). The Nx animals were divided into untreated (Nx), curcumin-treated (curcumin), and enalapril-treated (enalapril) groups. Sham-operated animals served as a control. Renal dysfunction in the Nx group, as evidenced by elevated blood urea nitrogen, plasma creatinine, proteinuria, segmental sclerosis, and tubular dilatation, was significantly reduced by curcumin and enalapril treatment. However, only enalapril significantly improved blood pressure. Compared with the control, the Nx animals had significantly higher plasma and kidney TNF-alpha, which was associated with NF-kappaB activation and macrophage infiltration in the kidney. These changes were effectively antagonized by curcumin and enalapril treatment. The decline in the anti-inflammatory peroxisome proliferator-activated receptor gamma (PPARgamma) seen in Nx animals was also counteracted by curcumin and enalapril. Studies in mesangial cells were carried out to further establish that the anti-inflammatory effect of curcumin in vivo was mediated essentially by antagonizing TNF-alpha. Curcumin dose dependently antagonized the TNF-alpha-mediated decrease in PPARgamma and blocked transactivation of NF-kappaB and repression of PPARgamma, indicating that the anti-inflamatory property of curcumin may be responsible for alleviating CRF in Nx animals.

Macrophage cholesteryl ester mobilization and atherosclerosis

Accumulation of cholesteryl esters (CE) stored as cytoplasmic lipid droplets is the main characteristic of macrophage foam cells that are central to the development of atherosclerotic plaques. Since only unesterified or free cholesterol (FC) can be effluxed from the cells to extracellular cholesterol acceptors, hydrolysis of CE is the obligatory first step in CE mobilization from macrophages. This reaction, catalyzed by neutral cholesteryl ester hydrolase (CEH), is increasingly being recognized as the rate-limiting step in FC efflux. CEH, therefore, regulates the process of reverse cholesterol transport and ultimate elimination of cholesterol from the body. In this review, we summarize the earlier controversies surrounding the identity of CEH in macrophages, discuss the characteristics of the various candidates recognized to date and examine their role in mobilizing cellular CE and thus regulating atherogenesis. In addition, physiological requirements to hydrolyze lipid droplet-associated substrate and complexities of interfacial catalysis are also discussed to emphasize the importance of evaluating the biochemical characteristics of candidate enzymes that may be targeted in the future to attenuate atherosclerosis.

Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg2+ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg2+. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg2+. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg2+ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF.It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg2+.

Reoxygenating microvascular endothelium exhibits temporal dissociation of NF-κB and AP-1 activation

Alterations of cellular redox balance in microvascular endothelium results in changes of essential cell functions. These alterations may arise, in part, due to modifications in the pattern of gene expression produced by transcription factor activation. Endothelium subjected to hypoxia/reoxygenation becomes redox imbalanced, thereby leading to activation and perhaps production of a proinflammatory state. A human dermal microvascular endothelial cell line (HMEC-1) was exposed to 6 h of hypoxia (3% O(2)) followed by return to normoxia atmospheric conditions. Reactive oxygen species (ROS) generation (dichlorofluoroscein epifluorescence) was immediate and significant following reoxygenation. Electrophoretic mobility shift assays revealed activation of the oxidant sensitive transcription factors NFkappaB and AP-1, though importantly, peak activation of each factor was separated temporally by greater than 60 min. NFkappaB activation occurred without degradation of the inhibitory protein IkappaBalpha. Reoxygenating HMEC-1 exhibited a greater than 500-fold increase in polymorphonuclear neutrophil (PMN) adhesion when compared to normoxic controls. Exposure of reoxygenating HMEC-1 to the antioxidant pyrrolidine dithiocarbamate produced complete abrogation of NFkappaB activation and the intensive PMN adhesion observed in untreated, posthypoxic HMEC-1. Though rexoygenation stress induced significant upregulation of PMN adhesion, no upregulation of interleukin-8 production was observed. Our results suggest that ROS generation occurring in endothelium following onset of reoxygenation stress signals activation of key transcription factors and that their activation takes place in a temporal fashion. The temporal feature of transcription factor activation may be key to production of a postischemic proinflammatory state.

AEG-1 Regulates Retinoid X Receptor and Inhibits Retinoid Signaling

Retinoid X receptor (RXR) regulates key cellular responses such as cell growth and development, and this regulation is frequently perturbed in various malignancies, including hepatocellular carcinoma (HCC). However, the molecule(s) that physically govern this deregulation are mostly unknown. Here, we identified RXR as an interacting partner of astrocyte-elevated gene-1 (AEG-1)/metadherin (MTDH), an oncogene upregulated in all cancers. Upon interaction, AEG-1 profoundly inhibited RXR/retinoic acid receptor (RAR)-mediated transcriptional activation. Consequently, AEG-1 markedly protected HCC and acute myelogenous leukemia (AML) cells from retinoid- and rexinoid-induced cell death. In nontumorigenic cells and primary hepatocytes, AEG-1/RXR colocalizes in the nucleus in which AEG-1 interferes with recruitment of transcriptional coactivators to RXR, preventing transcription of target genes. In tumor cells and AEG-1 transgenic hepatocytes, overexpressed AEG-1 entraps RXR in cytoplasm, precluding its nuclear translocation. In addition, ERK, activated by AEG-1, phosphorylates RXR that leads to its functional inactivation and attenuation of ligand-dependent transactivation. In nude mice models, combination of all-trans retinoic acid (ATRA) and AEG-1 knockdown synergistically inhibited growth of human HCC xenografts. The present study establishes AEG-1 as a novel homeostatic regulator of RXR and RXR/RAR that might contribute to hepatocarcinogenesis. Targeting AEG-1 could sensitize patients with HCC and AML to retinoid- and rexinoid-based therapeutics.

Genetic deletion of AEG-1 prevents hepatocarcinogenesis.

Activation of the oncogene AEG-1 (MTDH, LYRIC) has been implicated recently in the development of hepatocellular carcinoma (HCC). In mice, HCC can be initiated by exposure to the carcinogen DEN, which has been shown to rely upon activation of NF-κB in liver macrophages. Because AEG-1 is an essential component of NF-κB activation, we interrogated the susceptibility of mice lacking the AEG-1 gene to DEN-induced hepatocarcinogenesis. AEG-1-deficient mice displayed resistance to DEN-induced HCC and lung metastasis. No difference was observed in the response to growth factor signaling or activation of AKT, ERK, and β-catenin, compared with wild-type control animals. However, AEG-1-deficient hepatocytes and macrophages exhibited a relative defect in NF-κB activation. Mechanistic investigations showed that IL6 production and STAT3 activation, two key mediators of HCC development, were also deficient along with other biologic and epigenetics findings in the tumor microenvironment, confirming that AEG-1 supports an NF-κB-mediated inflammatory state that drives HCC development. Overall, our findings offer in vivo proofs that AEG-1 is essential for NF-κB activation and hepatocarcinogenesis, and they reveal new roles for AEG-1 in shaping the tumor microenvironment for HCC development.

Curcumin and chronic kidney disease (CKD): major mode of action through stimulating endogenous intestinal alkaline phosphatase.

Curcumin, an active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma longa), has significant anti-inflammatory properties. Chronic kidney disease (CKD), an inflammatory disease, can lead to end stage renal disease resulting in dialysis and transplant. Furthermore, it is frequently associated with other inflammatory disease such as diabetes and cardiovascular disorders. This review will focus on the clinically relevant inflammatory molecules that play a role in CKD and associated diseases. Various enzymes, transcription factors, growth factors modulate production and action of inflammatory molecules; curcumin can blunt the generation and action of these inflammatory molecules and ameliorate CKD as well as associated inflammatory disorders. Recent studies have shown that increased intestinal permeability results in the leakage of pro-inflammatory molecules (cytokines and lipopolysaccharides) from gut into the circulation in diseases such as CKD, diabetes and atherosclerosis. This change in intestinal permeability is due to decreased expression of tight junction proteins and intestinal alkaline phosphatase (IAP). Curcumin increases the expression of IAP and tight junction proteins and corrects gut permeability. This action reduces the levels of circulatory inflammatory biomolecules. This effect of curcumin on intestine can explain why, despite poor bioavailability, curcumin has potential anti-inflammatory effects in vivo and beneficial effects on CKD.

High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide

A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency.

Nanomedicines for dysfunctional macrophage-associated diseases

Macrophages play vital functions in host inflammatory reaction, tissue repair, homeostasis and immunity. Dysfunctional macrophages have significant pathophysiological impacts on diseases such as cancer, inflammatory diseases (rheumatoid arthritis and inflammatory bowel disease), metabolic diseases (atherosclerosis, diabetes and obesity) and major infections like human immunodeficiency virus infection. In view of this common etiology in these diseases, targeting the recruitment, activation and regulation of dysfunctional macrophages represents a promising therapeutic strategy. With the advancement of nanotechnology, development of nanomedicines to efficiently target dysfunctional macrophages can strengthen the effectiveness of therapeutics and improve clinical outcomes. This review discusses the specific roles of dysfunctional macrophages in various diseases and summarizes the latest advances in nanomedicine-based therapeutics and theranostics for treating diseases associated with dysfunctional macrophages.