Anil K. Bhunia

PKD1 Induces p21waf1 and Regulation of the Cell Cycle via Direct Activation of the JAK-STAT Signaling Pathway in a Process Requiring PKD2

Autosomal dominant polycystic kidney disease is characterized by cyst formation in the kidney and other organs and results from mutations of PKD1 or PKD2. Previous studies suggest that their gene products have an important role in growth regulation. We now show that expression of polycystin-1 activates the JAK-STAT pathway, thereby upregulating p21(waf1) and inducing cell cycle arrest in G0/G1. This process requires polycystin-2, a channel protein, as an essential cofactor. Mutations that disrupt polycystin-1/2 binding prevent activation of the pathway. Mouse embryos lacking Pkd1 have defective STAT1 phosphorylation and p21(waf1) induction. These results suggest that one function of the polycystin-1/2 complex is to regulate the JAK/STAT pathway and explain how mutations of either gene can result in dysregulated growth.

Hydrogen Sulfide as Endothelium-Derived Hyperpolarizing Factor Sulfhydrates Potassium Channels

Rationale: Nitric oxide, the classic endothelium-derived relaxing factor (EDRF), acts through cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelium-derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H2S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine &ggr;-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine. Objective: The purpose of this study was to determine if H2S is a major physiological EDHF. Methods and Results: We now show that H2S is a major EDHF because in blood vessels of CSE-deleted mice, hyperpolarization is virtually abolished. H2S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H2S-elicited hyperpolarization. The endothelial intermediate conductance (IKCa) and small conductance (SKCa) potassium channels mediate in part the effects of H2S, as selective IKCa and SKCa channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide-insensitive, H2S-induced vasorelaxation. Conclusions: H2S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. Because EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H2S biosynthesis offer therapeutic potential.

Cleavage of polycystin-1 requires the receptor for egg jelly domain and is disrupted by human autosomal-dominant polycystic kidney disease 1-associated mutations

Polycystin-1 plays an essential role in renal tubular morphogenesis, and disruption of its function causes cystogenesis in human autosomal-dominant polycystic kidney disease (ADPKD). We demonstrated that polycystin-1 undergoes cleavage at G protein coupled receptor proteolytic site in a process that requires the receptor for egg jelly domain. Most of the N-terminal fragment remains tethered at the cell surface, although a small amount is secreted. PKD1-associated mutations in the receptor for egg jelly domain disrupt cleavage, abolish the ability of polycystin-1 to activate signal transducer and activator of transcription-1, and induce tubulogenesis in vitro. We conclude that the cleavage of polycystin-1 is likely essential for its biologic activity.

Polycystin-1, the gene product of PKD1, induces resistance to apoptosis and spontaneous tubulogenesis in MDCK cells.

The major form of autosomal dominant polycystic kidney disease (ADPKD) results from mutation of a gene (PKD1) of unknown function that is essential for the later stages of renal tubular differentiation. In this report, we describe a novel cell culture system for studying how PKD1 regulates this process. We show that expression of human PKD1 in MDCK cells slows their growth and protects them from programmed cell death. MDCK cells expressing PKD1 also spontaneously form branching tubules while control cells form simple cysts. Increased cell proliferation and apoptosis have been implicated in the pathogenesis of cystic diseases. Our study suggests that PKD1 may function to regulate both pathways, allowing cells to enter a differentiation pathway that results in tubule formation.

Redox-regulated Signaling by Lactosylceramide in the Proliferation of Human Aortic Smooth Muscle Cells

Previously, our laboratory reported that lactosylceramide (LacCer) stimulated human aortic smooth muscle cell proliferation via specific activation of p44 mitogen-activated protein kinase (MAPK) in the p21 ras /Raf-1/MEK2 pathway and induced expression of the transcription factor c-fos downstream to the p44 MAPK signaling cascade (Bhunia A. K., Han, H., Snowden, A., and Chatterjee S. (1996) J. Biol. Chem. 271, 10660–10666). In the present study, we explored the role of free oxygen radicals in LacCer-mediated induction of cell proliferation. Superoxide levels were measured by the lucigenin chemiluminescence method, MAPK activity was measured by immunocomplex kinase assays, and Western blot analysis and c-fos expression were measured by Northern blot assay. We found that LacCer (10 μm) stimulates endogenous superoxide production (7-fold compared with control) in human aortic smooth muscle cells specifically by activating membrane-associated NADPH oxidase, but not NADH or xanthine oxidase. This process was inhibited by an inhibitor of NADPH oxidase, diphenylene iodonium (DPI), and by antioxidants,N-acetyl-l-cysteine (NAC) or pyrrolidine dithiocarbamate. NAC and DPI both abrogated individual steps in the signaling pathway leading to cell proliferation. For example, the p21 ras ·GTP loading, p44 MAPK activity, and induction of transcription factor c-fos all were inhibited by NAC and DPI as well as an antioxidant pyrrolidine dithiocarbamate or reduced glutathione (GSH). In contrast, depletion of GSH byl-buthionine (S,R)-sulfoximine up-regulated the above described signaling cascade. In sum, LacCer, by virtue of activating NADPH oxidase, produces superoxide (a redox stress signaling molecule), which mediates cell proliferation via activation of the kinase cascade. Our findings may explain the potential role of LacCer in the pathogenesis of atherosclerosis involving the proliferation of aortic smooth muscle cells.

Rtp801, a suppressor of mTOR signaling, is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema

Rtp801 (also known as Redd1, and encoded by Ddit4), a stress-related protein triggered by adverse environmental conditions, inhibits mammalian target of rapamycin (mTOR) by stabilizing the TSC1-TSC2 inhibitory complex and enhances oxidative stress–dependent cell death. We postulated that Rtp801 acts as a potential amplifying switch in the development of cigarette smoke–induced lung injury, leading to emphysema. Rtp801 mRNA and protein were overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The regulation of Rtp801 expression by cigarette smoke may rely on oxidative stress–dependent activation of the CCAAT response element in its promoter. We also found that Rtp801 was necessary and sufficient for nuclear factor-κB (NF-κB) activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF-κB activation, alveolar inflammation, oxidative stress and apoptosis of alveolar septal cells. In contrast, Rtp801 knockout mice were markedly protected against acute cigarette smoke–induced lung injury, partly via increased mTOR signaling, and, when exposed chronically to cigarette smoke, against emphysema. Our data support the notion that Rtp801 may represent a major molecular sensor and mediator of cigarette smoke–induced lung injury.

Hypoxia and chronic lung disease

The lung is both the conduit for oxygen uptake and is also affected by hypoxia and hypoxia signaling. Decreased ventilatory drive, airway obstructive processes, intra-alveolar exudates, septal thickening by edema, inflammation, fibrosis, or damage to alveolar capillaries will all interpose a significant and potentially life-threatening barrier to proper oxygenation, therefore enhancing the alveolar/arterial pO2 gradient. These processes result in decreased blood and tissue oxygenation. This review addresses the relationship of hypoxia with lung development and with lung diseases. We particularly focus on molecular mechanisms underlying hypoxia-driven physiological and pathophysiological lung processes, specifically in the infant lung, pulmonary hypertension, and chronic obstructive pulmonary disease.

OxLDL-dependent activation of arginase II is dependent on the LOX-1 receptor and downstream RhoA signaling

AIMS Arginase II regulates NOS activity by competing for the substrate l-arginine. Oxidized LDL (OxLDL) is a proatherogenic molecule that activates arginase II. We tested the hypotheses that OxLDL-dependent arginase II activation occurs through a specific receptor, and via a Rho GTPase effector mechanism that is inhibited by statins. METHODS AND RESULTS Arginase II activation by OxLDL was attenuated following preincubation with the LOX-1 receptor-blocking antibody JTX92. This also prevented the dissociation of arginase II from microtubules. LOX-1(-/-) mice failed to exhibit the increased arginase II activity seen in WT mice fed a high cholesterol diet. Furthermore, endothelium from LOX-1(-/-) mice failed to demonstrate the diet-dependent reduction in NO and increase in ROS that were observed in WT mice. OxLDL induced Rho translocation to the membrane and Rho activation, and these effects were inhibited by pretreatment with JTX92 or statins. Transfection with siRNA for RhoA, or inhibition of ROCK both decreased OxLDL-stimulated arginase II activation. Preincubation with simvastatin or lovastatin blocked OxLDL-induced dissociation of arginase II from microtubules and prevented microtubule depolymerization. CONCLUSIONS This study provides a new focus for preventive therapy for atherosclerotic disease by delineating a clearer path from OxLDL through the endothelial cell LOX-1 receptor, RhoA, and ROCK, to the activation of arginase II, downregulation of NO, and vascular dysfunction.

Polycystin-1 Induces Resistance to Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

Polycystin-1 (PC-1), the PKD1 gene product, is a large receptor whose expression in renal epithelial cells results in resistance to apoptosis and tubulogenesis, a model consistent with the phenotype observed in patients. This study links PC-1 expression to a signaling pathway that is known to be both antiapoptotic and important for normal tubulogenesis. This study found that PC-1 expression results in phosphorylation of Akt and downstream effectors and that phosphatidylinositol 3-kinase (PI3-K) inhibitors prevent this process. In addition, it is shown that dominant negative Akt can revert PC-1-induced protection from apoptosis. Furthermore, it was observed that increased PI3-K beta activity in PC-1-expressing MDCK cells seems to be dependent on both tyrosine-kinase activity and heterotrimeric G proteins. It also was found that PC-1-induced tubulogenesis is inhibited by PI3-K inhibitors. Taken together, these data suggest that the PI3-K/Akt cascade may be a central modulator of PC-1 function and that its deregulation might be important in autosomal dominant polycystic kidney disease.

GD3 Recruits Reactive Oxygen Species to Induce Cell Proliferation and Apoptosis in Human Aortic Smooth Muscle Cells

Sialic acid containing glycosphingolipids (gangliosides) are expressed on the surface of all mammalian cells and have been implicated in regulating various biological phenomena; however, the detailed signaling mechanisms involved in this process are not known. We report here a novel aspect of disialoganglioside, GD3-mediated regulation of cell proliferation and cell death via the recruitment of reactive oxygen species (ROS). A low concentration (2.5–10 μm) of GD3, incubated with human aortic smooth muscle cells for a short period of time (10–30 min), stimulates superoxide generation via the activation of both NADPH oxidase and NADH oxidase activity. This leads to downstream signaling leading to cell poliferation and apoptosis. However, [3H]GD3 incubated with the cells under such conditions was found in a trypsin-sensitive fraction that was separable from endogenous GD3. The exact mechanism causing ROS generation and downstream signaling remains to be elucidated. The uptake of GD3 was accompanied by a 2.5-fold stimulation in the activity of mitogen-activated protein (MAP) kinase and 5-fold stimulation in cell proliferation. Preincubation of cells with membrane-permeable antioxidants, pyrrolidine dithiocarbamate, andN-acetylcysteine abrogated the superoxide generation and cell proliferation. In contrast, at higher concentrations (50–200 μm) GD3 inhibited the generation of superoxides but markedly stimulated the generation of nitric oxide (NO) (10-fold compared with control). This in turn stimulated mitochondrial cytochrome c release and intrachromosomal DNA fragmentation, which lead to apoptosis. In sum, at a low concentration, GD3 recruits superoxides to activate p44 MAPK and stimulates cell proliferation. In contrast, at high concentrations GD3 recruits nitric oxide to scavenge superoxide radicals that triggered signaling events that led to apoptosis. These observations might have relevance in regard to the potential role of GD3 in aortic smooth muscle cell proliferation and apoptosis that may contribute to plaque rupture in atherosclerosis.