Gautam Shrikhande

The BCR/ABL Tyrosine Kinase Induces Production of Reactive Oxygen Species in Hematopoietic Cells

The BCR/ABL oncogene causes chronic myelogenous leukemia, a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and myeloid cells. It is shown here that transformation of the hematopoietic cell lines Ba/F3, 32Dcl3, and MO7e with BCR/ABL results in an increase in reactive oxygen species (ROS) compared with quiescent, untransformed cells. The increase in ROS was directly due to BCR/ABL because it was blocked by the ABL-specific tyrosine kinase inhibitor STI571. Oxidative stress through ROS is believed to have many biochemical effects, including the potential ability to inhibit protein-tyrosine phosphatases (PTPases). To understand the significance of increased production of ROS, a model system was established in which hydrogen peroxide (H2O2) was added to untransformed cells to mimic the increase in ROS induced constitutively by BCR/ABL. H2O2 substantially reduced total cellular PTPase activity to a degree approximately equivalent to that of pervanadate, a well known PTPase inhibitor. Further, stimulation of untransformed cells with H2O2 or pervanadate increased tyrosine phosphorylation of each of the most prominent known substrates of BCR/ABL, including c-ABL, c-CBL, SHC, and SHP-2. Treatment of the BCR/ABL-expressing cell line MO7/p210 with the reducing agents pyrrolidine dithiocarbamate orN-acetylcysteine reduced the accumulation of ROS and also decreased tyrosine phosphorylation of cellular proteins. Further, treatment of MO7e cells with H2O2 or pervanadate increased the tyrosine kinase activity of c-ABL. Drugs that alter ROS metabolism or reactivate PTPases may antagonize BCR/ABL transformation.

BCR/ABL Directly Inhibits Expression of SHIP, an SH2-Containing Polyinositol-5-Phosphatase Involved in the Regulation of Hematopoiesis

ABSTRACT The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP−/−mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.

Forkhead transcription factors inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to atherosclerosis, postangioplasty restenosis, and transplant vasculopathy. Forkhead transcription factors belonging to the FoxO subfamily have been shown to inhibit growth and cell cycle progression in a variety of cell types. We hypothesized that forkhead proteins may play a role in VSMC biology. Under in vitro conditions, platelet-derived growth factor (PDGF)-BB, tumor necrosis factor-α, and insulin-like growth factor 1 stimulated phosphorylation of FoxO in human coronary artery smooth muscle cells via MEK1/2 and/or phosphatidylinositol 3-kinase-dependent signaling pathways. PDGF-BB, tumor necrosis factor-α, and insulin-like growth factor 1 treatment resulted in the nuclear exclusion of FoxO, whereas PDGF-BB alone down-regulated the FoxO target gene, p27kip1, and enhanced cell survival and progression through the cell cycle. These effects were abrogated by overexpression of a constitutively active, phosphorylation-resistant mutant of the FoxO family member, TM-FKHRL1. The anti-proliferative effect of TM-FKHRL1 was partially reversed by small interfering RNA against p27kip1. In a rat balloon carotid arterial injury model, adenovirus-mediated gene transfer of FKHRL1 caused an increase in the expression of p27kip1 in the VSMC and inhibition of neointimal hyperplasia. These data suggest that FoxO activity inhibits VSMC proliferation and activation and that this signaling axis may represent a therapeutic target in vasculopathic disease states.

The phosphatidylinositol polyphosphate 5-phosphatase SHIP and the protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells which can be regulated by BCR/ABL and growth factors.

We report here that interleukin-3 (IL-3) and erythropoietin (EPO) induce formation of a complex composed of two SH2-containing phosphatases, the tyrosine phosphatase SHP-2 and the SH2 containing inositol 5-phosphatase (SHIP). Both SHP-2 and SHIP are known to be involved in growth factor signal transduction, but their potential interaction in the same pathway is novel. SHIP has previously been shown to associate with SHC, and potentially to be involved in regulating apoptosis. In contrast, in some model systems, SHP-2 has been demonstrated to positively regulate cell growth. Both phosphatases in the complex were tyrosine phosphorylated, and the amount of SHIP coprecipitating with SHP-2 was inversely related to the amount of SHIP coprecipitating with SHC. In hematopoietic cells transformed by the BCR/ABL oncogene, this phosphatase complex was found to be constitutively present with both components heavily tyrosine phosphorylated. Also, other proteins were detected in the complex, including BCR/ABL itself and c-CBL. However, transformation by BCR/ABL was associated with a reduced SHIP protein expression, which could further affect the accumulation of various inositol polyphosphates in these leukemic cells. These data suggest that the function of SHIP and SHP-2 in normal cells are linked and that BCR/ABL alters the function of this signaling complex.

A20, a modulator of smooth muscle cell proliferation and apoptosis, prevents and induces regression of neointimal hyperplasia

A20 is a NF‐κB‐dependent gene that has dual anti‐inflammatory and antiapoptotic functions in endothelial cells (EC). The function of A20 in smooth muscle cells (SMC) is unknown. We demonstrate that A20 is induced in SMC in response to inflammatory stimuli and serves an anti‐inflammatory function via blockade of NF‐κB and NF‐κB‐dependent proteins ICAM‐1 and MCP‐1. A20 inhibits SMC proliferation via increased expression of cyclin‐dependent kinase inhibitors p21waf1 and p27kip1. Surprisingly, A20 sensitizes SMC to cytokine‐ and Fas‐mediated apoptosis through a novel NO‐dependent mechanism. In vivo, adenoviral delivery of A20 to medial rat carotid artery SMC after balloon angioplasty prevents neointimal hyperplasia by blocking SMC proliferation and accelerating re‐endothelialization, without causing apoptosis. However, expression of A20 in established neointimal lesions leads to their regression through increased apoptosis. This is the first demonstration that A20 exerts two levels of control of vascular remodeling and healing. A20 prevents neointimal hyperplasia through combined anti‐inflammatory and antiproliferative functions in medial SMC. If SMC evade this first barrier and neointima is formed, A20 has a therapeutic potential by uniquely sensitizing neointimal SMC to apoptosis. A20‐based therapies hold promise for the prevention and treatment of neointimal disease.—Patel, V. I., Daniel, S., Longo, C. R., Shrikhande, G. V., Scali, S. T., Czismadia, E., Groft, C. M., Shukri, T., Motley‐Dore, C., Ramsey, H. E., Fisher, M. D., Grey, S. T., Arvelo, M. B., Ferran, C. A20, a modulator of smooth muscle cell proliferation and apoptosis, prevents and induces regression of neointimal hyperplasia. FASEB J. 20, 1418–1430 (2006)

A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1‐dependent mechanism

The liver has a remarkable regenerative capacity, allowing recovery following injury. Regeneration after injury is contingent on maintenance of healthy residual liver mass, otherwise fulminant hepatic failure (FHF) may arise. Understanding the protective mechanisms safeguarding hepatocytes and promoting their proliferation is critical for devising therapeutic strategies for FHF. We demonstrate that A20 is part of the physiological response of hepatocytes to injury. In particular, A20 is significantly upregulated in the liver following partial hepatectomy. A20 protects hepatocytes from apoptosis and ongoing inflammation by inhibiting NF‐κB. Hepatic expression of A20 in BALB/c mice dramatically improves survival following extended and radical lethal hepatectomy. A20 expression in the liver limits hepatocellular damage hence maintains bilirubin clearance and the liver synthetic function. In addition, A20 confers a proliferative advantage to hepatocytes via decreased expression of the cyclin‐dependent kinase inhibitor p21waf1. In conclusion, A20 provides a proliferative advantage to hepatocytes. By combining anti‐inflammatory, antiapoptotic and pro‐proliferative functions, A20‐based therapies could be beneficial in prevention and treatment of FHF. (HEPATOLOGY 2005;42:156–164.)

O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.

MARCKS silencing differentially affects human vascular smooth muscle and endothelial cell phenotypes to inhibit neointimal hyperplasia in saphenous vein

Intimal hyperplasia (IH) limits the patency of all cardiovascular vein bypass grafts. We previously found the myristoylated alanine‐rich C kinase substrate (MARCKS), a key protein kinase C (PKC) substrate, to be up‐regulated in canine models of IH. Here, we further characterize the role of MARCKS in IH and examine the phenotypic consequences of MARCKS silencing by small interfering RNA (siRNA) transfection in human vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) in vitro and use a rapid 10‐min nonviral siRNA transfection technique to determine the effects of MARCKS silencing in human saphenous vein cultured ex vivo. We demonstrate MARCKS silencing attenuates VSMC migration and arrests VSMC proliferation in part through the up‐regulation of the cyclin‐dependent kinase inhibitor p27kip1. Conversely, MARCKS silencing had little or no effect on EC migration or proliferation. These phenotypic changes culminated in reduced neointimal formation in cultured human saphenous vein. These data identify MARCKS as a pathogenic contributor to IH and indicate therapeutic MARCKS silencing could selectively suppress the “atherogenic,” proliferative phenotype of VSMCs without collateral harm to the endothelium. This approach could be readily translated to the clinic to silence MARCKS in vein bypass grafts prior to implantation.— Monahan, T. S., Andersen, N. D., Martin, M. C, Malek, J. Y., Shrikhande, G. V., Pradhan, L., Ferran, C, LoGerfo, F. W. MARCKS silencing differentially affects human vascular smooth muscle and endothelial cell phenotypes to inhibit neointimal hyperplasia in saphenous vein. FASEB J. 23, 557–564 (2009)

A20 Modulates Lipid Metabolism and Energy Production to Promote Liver Regeneration

Background Liver Regeneration is clinically of major importance in the setting of liver injury, resection or transplantation. We have demonstrated that the NF-κB inhibitory protein A20 significantly improves recovery of liver function and mass following extended liver resection (LR) in mice. In this study, we explored the Systems Biology modulated by A20 following extended LR in mice. Methodology and Principal Findings We performed transcriptional profiling using Affymetrix-Mouse 430.2 arrays on liver mRNA retrieved from recombinant adenovirus A20 (rAd.A20) and rAd.βgalactosidase treated livers, before and 24 hours after 78% LR. A20 overexpression impacted 1595 genes that were enriched for biological processes related to inflammatory and immune responses, cellular proliferation, energy production, oxidoreductase activity, and lipid and fatty acid metabolism. These pathways were modulated by A20 in a manner that favored decreased inflammation, heightened proliferation, and optimized metabolic control and energy production. Promoter analysis identified several transcriptional factors that implemented the effects of A20, including NF-κB, CEBPA, OCT-1, OCT-4 and EGR1. Interactive scale-free network analysis captured the key genes that delivered the specific functions of A20. Most of these genes were affected at basal level and after resection. We validated a number of A20s target genes by real-time PCR, including p21, the mitochondrial solute carriers SLC25a10 and SLC25a13, and the fatty acid metabolism regulator, peroxisome proliferator activated receptor alpha. This resulted in greater energy production in A20-expressing livers following LR, as demonstrated by increased enzymatic activity of cytochrome c oxidase, or mitochondrial complex IV. Conclusion This Systems Biology-based analysis unravels novel mechanisms supporting the pro-regenerative function of A20 in the liver, by optimizing energy production through improved lipid/fatty acid metabolism, and down-regulated inflammation. These findings support pursuit of A20-based therapies to improve patients’ outcomes in the context of extreme liver injury and extensive LR for tumor treatment or donation.

Diabetes and peripheral vascular disease

Diabetes and peripheral vascular disease : , Diabetes and peripheral vascular disease : , کتابخانه دیجیتال جندی شاپور اهواز