Bin Xian Zhang

Loss of manganese superoxide dismutase leads to abnormal growth and signal transduction in mouse embryonic fibroblasts.

Manganese superoxide dismutase (MnSOD) in the mitochondria plays an important role in cellular defense against oxidative damage. Homozygous MnSOD knockout (Sod2(-/-)) mice are neonatal lethal, indicating the essential role of MnSOD in early development. To investigate the potential cellular abnormalities underlying the aborted development of Sod2(-/-) mice, we examined the growth of isolated mouse embryonic fibroblasts (MEFs) from Sod2(-/-) mice. We found that the proliferation of Sod2(-/-) MEFs was significantly decreased compared with wild-type MEFs despite the absence of morphological differences. The Sod2(-/-) MEFs produced less cellular ATP, had lower O(2) consumption, generated more superoxide, and expressed less Prdx3 protein. Furthermore, the loss of MnSOD dramatically altered several markers involved in cell proliferation and growth, including decreased growth stimulatory function of mTOR signaling and enhanced growth inhibitory function of GSK-3β signaling. Interestingly, the G-protein-coupled receptor-mediated intracellular Ca(2+) signal transduction was also severely suppressed in Sod2(-/-) MEFs. Finally, the ratio of microtubule-associated protein light chain 3 (LC3)-II/LC3-I, an index of autophagic activity, was increased in Sod2(-/-) MEFs, consistent with a reduction in mTOR signal transduction. These data demonstrate that MnSOD deficiency results in alterations in several key signaling pathways, which may contribute to the lethal phenotype of Sod2(-/-) mice.

Role of β-adrenergic receptors in regulation of hepatic fat accumulation during aging

Excessive fat accumulation in liver (hepatic steatosis) predisposes to hepatic functional and structural impairment and overall metabolic risk. Previous studies noted an association between hepatic steatosis and age in humans and rodents. However, the mechanisms leading to age-associated hepatic fat accumulation remain unknown. Earlier work from our group showed that β-adrenergic receptor (β-AR) levels and β-AR-stimulated adenylyl cyclase activity increase in rat liver during aging. Here we investigated whether age-associated increases in β-AR signaling play a role in augmenting hepatic lipid accumulation. We demonstrate an increase in hepatic lipid content during senescence and a significant correlation between hepatic fat content and stimulation of adenylyl cyclase activity by the β-AR agonist isoproterenol in rat liver. Isoproterenol administration to young and old rodents in vivo increased hepatic lipid accumulation. Furthermore, in vitro overexpression of β1- and β2-AR subtypes in hepatocytes from young rodents increased cellular lipid content, whereas inhibition of β-ARs by receptor subtype-specific inhibitors reduced lipid levels in hepatocytes from senescent animals. Isoproterenol-induced hepatic lipid accumulation in vivo was prevented by the β-AR nonselective blocker propranolol, suggesting a novel therapeutic effect of this class of drugs in hepatic steatosis. Acipimox, which inhibits adipose tissue lipolysis, did not alter isoproterenol-mediated hepatic fat accumulation; thus β-AR responsive hepatic lipid accumulation does not appear to be related primarily to altered lipolysis. These findings suggest that augmented hepatic β-AR signaling during aging may increase lipid accumulation in liver and advocate a possible role for β-adrenergic blockers in preventing or retarding the development of hepatic steatosis.

The role of mitochondrial complex III in melatonin-induced ROS production in cultured mesangial cells.

Abstract:  Melatonin is a potent scavenger of reactive oxygen (ROS) and reactive nitrogen species (RNS). At pharmacological concentrations, however, melatonin is documented to cause ROS/RNS production, especially in cultured cancerous cells. Currently, the mechanism responsible for melatonin‐induced ROS generation remains elusive. In this study, we provided evidence that melatonin, at micromolar concentrations, induced rapid ROS generation by a mitochondrial‐dependent mechanism in primary human mesangial (HM) cells. The melatonin‐induced ROS production occurred independent of changes in Ca2+ concentrations in the cytosol and/or in mitochondria. In mitochondria isolated from HM cells and mice kidney tissues, melatonin caused ROS production; this melatonin response was completely blocked by the complex III inhibitor antimycin A. In contrast, both the mitochondrial complex I inhibitor, rotenone, and another complex III inhibitor, myxothiazol, which interacts with complex III at a distinct site, had no significant inhibitory effect on melatonin‐induced ROS generation. These results demonstrate that melatonin induced rapid ROS generation via the antimycin A‐sensitive site of mitochondrial complex III.

Role for notch signaling in salivary acinar cell growth and differentiation

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ‐secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes‐1 expression, and Hes‐1 expression was inhibited by γ‐secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes‐1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation. Developmental Dynamics 238:724–731, 2009.

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line.

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+ ([Ca2+]i by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms-pilocarpine acting via M3 receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC-converging on the ERK pathway.

Impaired mitochondrial complex III and melatonin responsive reactive oxygen species generation in kidney mitochondria of db/db mice.

Abstract:  We have previously demonstrated that melatonin, at pharmacological concentrations, causes rapid reactive oxygen species (ROS) generation at the antimycin‐A sensitive site of mitochondrial complex III (MC‐3). In the current work, we used this melatonin response to investigate the role of mitochondrial dysfunction in the development of diabetic nephropathy. We find that the development of diabetic nephropathy, as indicated by hyperfiltration and histopathological lesions in the kidney of db/db mice, is associated with diminished melatonin‐induced ROS generation and MC‐3 activity, indicating impaired MC‐3 at the antimycin‐A site. The MC‐3 protein level in the renal mitochondria was equivalent in db/db and the nondiabetic db/m mice, whereas mitochondrial complex I (MC‐1) protein was dramatically upregulated in the db/db mice. This differential regulation in mitochondrial complexes may alter the equilibrium of the electron transport in renal mitochondria and contribute to ROS overproduction. The study provides one mechanism of enhanced oxidative stress that may be involved in the pathogenesis of diabetic nephropathy in db/db mice.

Geldanamycin Derivative Ameliorates High Fat Diet-Induced Renal Failure in Diabetes

Diabetic nephropathy is a serious complication of longstanding diabetes and its pathogenesis remains unclear. Oxidative stress may play a critical role in the pathogenesis and progression of diabetic nephropathy. Our previous studies have demonstrated that polyunsaturated fatty acids (PUFA) induce peroxynitrite generation in primary human kidney mesangial cells and heat shock protein 90β1 (hsp90β1) is indispensable for the PUFA action. Here we investigated the effects of high fat diet (HFD) on kidney function and structure of db/db mice, a widely used rodent model of type 2 diabetes. Our results indicated that HFD dramatically increased the 24 h-urine output and worsened albuminuria in db/db mice. Discontinuation of HFD reversed the exacerbated albuminuria but not the increased urine output. Prolonged HFD feeding resulted in early death of db/db mice, which was associated with oliguria and anuria. Treatment with the geldanamycin derivative, 17-(dimethylaminoehtylamino)-17-demethoxygeldanamycin (17-DMAG), an hsp90 inhibitor, preserved kidney function, and ameliorated glomerular and tubular damage by HFD. 17-DMAG also significantly extended survival of the animals and protected them from the high mortality associated with renal failure. The benefit effect of 17-DMAG on renal function and structure was associated with a decreased level of kidney nitrotyrosine and a diminished kidney mitochondrial Ca2+ efflux in HFD-fed db/db mice. These results suggest that hsp90β1 is a potential target for the treatment of nephropathy and renal failure in diabetes.

Epidermal Growth Factor-induced Depletion of the Intracellular Ca2+ Store Fails to Activate Capacitative Ca2+Entry in a Human Salivary Cell Line

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cγ and mobilization of intracellular Ca2+ ([Ca2+] i ). Generally, agonist-mediated Ca2+ mobilization involves both Ca2+ release from internal stores and Ca2+influx activated by store depletion (i.e. capacitative or store-operated Ca2+ influx). However, the role of capacitative Ca2+ entry in EGF-mediated Ca2+mobilization is still largely unknown. In this study, we compared [Ca2+] i signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca2+entry. Unlike carbachol and Tg, EGF (5 nm) elicited a transient [Ca2+] i signal without a plateau phase in the presence of extracellular Ca2+ and also failed to accelerate Mn2+ entry. Repletion of extracellular Ca2+ to cells stimulated with EGF in the absence of Ca2+ elicited an increase in [Ca2+] i , indicating that EGF indeed stimulates Ca2+ influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca2+ release. Interestingly, the phospholipase C inhibitorU73122 completely inhibited Ca2+ release induced by both EGF and carbachol and also reduced Ca2+ influx responsive to carbachol but had no effect on Ca2+ influx induced by EGF. EGF-induced Ca2+ influx was potentiated by low concentrations (<5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca2+ influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca2+influx responsive to carbachol but did not increase EGF-activated Ca2+ influx. Both EGF and carbachol depleted internal Ca2+ stores. Our results demonstrate that EGF-induced Ca2+ release from internal stores does not activate capacitative Ca2+ influx. Rather, EGF stimulates Ca2+ influx via a mechanism distinct from capacitative Ca2+ influx induced by carbachol and Tg.

G-protein signaling abnormalities mediated by CD95 in salivary epithelial cells

Salivary epithelial cells from patients with primary Sjögrens syndrome (SS) undergo Fas-mediated apoptosis. Bcl-2 and Bcl-xL are apoptosis suppressing oncogenes. Very little is known about the role of these oncogene molecules in salivary epithelial cells. To investigate the possible prevention of salivary glandular destruction in SS by Bcl-2 and Bcl-xL, stable transfectants expressing these molecules were made from HSY cells, a human salivary epithelial cell line. HSY cells were transfected with an expression vector for human Bcl-2 or Bcl-xL. Stable transfectants were selected and apoptosis was induced by anti-Fas antibody. Apoptosis was quantified by propidium iodide staining followed by flow cytometry. Caspase activity was detected by immunohistochemical analysis and enzyme cleavage of DEVD-AMC, a fluorescent substrate. Response to carbachol, a muscarinic receptor agonist, and EGF was measured by Ca2+ mobilization and influx. Fas-mediated apoptosis was significantly inhibited in Bcl-2 and Bcl-xL transfectants compared to wild-type and control transfectants (empty vector). Surprisingly, caspase activity was not inhibited in Bcl-2 and Bcl-xL transfectants. Activation of the Fas pathway in the Bcl-2 and Bcl-xL transfectants by antibody also inhibited carbachol and EGF responsiveness (i.e., Ca2+ mobilization and/or influx) by 50–60%. This Fas-mediated inhibition of cell activation was partially or completely restored by specific peptide interference of caspase enzyme activity. The prevention of Fas-mediated apoptosis by the overexpression of Bcl-2 and Bcl-xL in salivary gland epithelial cells results in injured cells expressing caspase activity and unable to respond normally to receptor agonists. Such damaged cells may exist in SS patients and could explain the severe dryness out of proportion to the actual number of apoptotic cells seen on salivary gland biopsy. Cell Death and Differentiation (2000) 7, 1119–1126

Linoleic Acid-Induced Mitochondrial Ca2+ Efflux Causes Peroxynitrite Generation and Protein Nitrotyrosylation

It is well known that excessive non-esterified fatty acids in diabetes contribute to the pathogenesis of renal complications although the mechanism remains elusive. Enhanced oxidative stress has been hypothesized as a unified factor contributing to diabetic complications and increased protein nitrotyrosylation has been reported in the kidneys of diabetic patients. In the current manuscript we described that linoleic acid (LA) caused mitochondrial Ca2+ efflux and peroxynitrite production, along with increased nitrotyrosine levels of cellular proteins in primary human mesangial cells. The peroxynitrite production by LA was found to depend on mitochondrial Ca2+ efflux. Downregulation of hsp90β1, which has been previously shown to be essential for polyunsaturated fatty acid-induced mitochondrial Ca2+ efflux, significantly diminished LA-responsive mitochondrial Ca2+ efflux and the coupled peroxynitrite generation, implicating a critical role of hsp90β1 in the LA responses. Our results further demonstrated that mitochondrial complexes I and III were directly involved in the LA-induced peroxynitrite generation. Using the well established type 2 diabetic animal model db/db mice, we observed a dramatically enhanced LA responsive mitochondrial Ca2+ efflux and protein nitrotyrosylation in the kidney. Our study thus demonstrates a cause-effect relationship between LA and peroxynitrite or protein nitrotyrosylation and provides a novel mechanism for lipid-induced nephropathy in diabetes.