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K Irani

Publications and source records attributed to K Irani.

34 records · Page 2Linked to original sources
PubMed1998

A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy.

We have used adenoviral-mediated gene transfer of a constitutively active (V12rac1) and dominant negative (N17rac1) isoform of rac1 to assess the role of this small GTPase in cardiac myocyte hypertrophy. Expression of V12rac1 in neonatal cardiac myocytes results in sarcomeric reorganization and an increase in cell size that is indistinguishable from ligand-stimulated hypertrophy. In addition, V12rac1 expression leads to an increase in atrial natriuretic peptide secretion. In contrast, expression of N17rac1, but not a truncated form of Raf-1, attenuated the morphological hypertrophy associated with phenylephrine stimulation. Consistent with the observed effects on morphology, expression of V12rac1 resulted in an increase in new protein synthesis, while N17rac1 expression inhibited phenylephrine-induced leucine incorporation. These results suggest rac1 is an essential element of the signaling pathway leading to cardiac myocyte hypertrophy.

Adenoviridae↗
PubMed1998

Ras, superoxide and signal transduction.

The superoxide anion has been associated with the bactericidal activity of phagocytes. Produced by an enzymatic complex, NADPH oxidase, bactericidal superoxide is released within phagolysosomes where bacteria are being degraded. The activity of NADPH oxidase is regulated by Rac, a small GTP binding protein of the Ras family. Recent evidence indicates that, in addition to its bactericidal activity, superoxide seems to function as a signal-transduction messenger, mediating the downstream effects of Ras and Rac in nonphagocytic cells. As such, superoxide contributes to the unchecked proliferation of Ras-transformed cells. In the nitric oxide (NO) system, low concentrations of NO transduce signals within vessels and neurons, while high concentrations of NO can produce damage to cells and microorganisms. By analogy, superoxide and probably other oxidants serve as messengers at low concentrations, while larger amounts are required for inducing damage. The activity of oxidants as messengers opens new avenues for pharmacological intervention against Ras-mediated pathways in mammalian cells.

Animals↗
PubMed1998

Protection from reoxygenation injury by inhibition of rac1.

We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.

Aerobiosis↗
PubMed1998

Bcl-2 regulates nonapoptotic signal transduction: inhibition of c-Jun N-terminal kinase (JNK) activation by IL-1 beta and hydrogen peroxide.

We have explored the role of bcl-2 as a potential modulator of intracellular signal transduction. Stable expression of bcl-2 in fibroblasts inhibited the activation of the c-jun amino terminal kinase (JNK) by the nonapoptotic cytokine interleukin-1 beta (IL-1 beta). This effect appeared to be selective for JNK activation as bcl-2 did not appear to alter the other aspects of IL-1 beta signal transduction. Similarly, bcl-2 did not inhibit all all activators of JNK as it had no effect on JNK activation by the protein synthesis inhibitor anisomycin. Treatment with nonlethal concentrations of H2O2, which resulted in the simultaneous stimulation of mitogen-activated protein kinase (MAPK) and JNK, demonstrated that bcl-2 appeared to alter the balance of activation of these two kinase cascades. The pathway by which bcl-2 inhibits JNK activation is demonstrated to be independent of the rac1 GTPase. In contrast, the reduction in JNK activity in cells expressing bcl-2 can be restored by costimulation with a calcium ionophore. This suggests that bcl-2 can regulate certain nonapoptotic signaling pathways. Such results therefore expand the functions of bcl-2 and may have important implication in the understanding of the role of this protein in a variety of human diseases.

Activating Transcription Factor 2↗
PubMed1998

A requirement for rac1 in the PDGF-stimulated migration of fibroblasts and vascular smooth cells.

Rac1 is a member of the Rho family of small GTPases. Although rac1 has been demonstrated to regulate the cytoskeleton, relatively little is known regarding its role in directional migration of mammalian cells. To address this issue, we have used recombinant adenoviruses to transiently express in fibroblasts either a dominant negative (N17rac1) or constitutively active (V12rac1) isoform of the small GTP-binding protein rac1. Expression of N17rac1 is demonstrated to inhibit PDGF-stimulated migration of rat fibroblasts. Surprisingly, expression of V12rac1 also inhibited, albeit to a lesser degree, the chemotactic response to PDGF. In contrast, expression of N17rac1 had no effect on PDGF stimulation of mitogen activated protein kinase (MAPK) or the adherence of cells to plastic or fibronectin coated surfaces. Similar to what was observed in fibroblasts, expression of N17rac1 inhibited the PDGF-stimulated migration of primary vascular smooth muscle cells. These results identify rac1 as an important downstream mediator of PDGF-induced chemotaxis.

Adenoviridae↗
PubMed1998

Priming of platelet alphaIIbbeta3 by oxidants is associated with tyrosine phosphorylation of beta3.

Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H2O2, a major cellular oxidant. Exposure to H2O2 triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced99032416 required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO4) and was dependent on the participation of integrin alphaIIbbeta3 (glycoprotein IIb-IIIa). A specific inhibitor of alphaIIbbeta3 blocked platelet aggregation induced by H2O2 and NaVO4, thus confirming that aggregation requires this receptor. In the presence of H2O2 and NaVO4, multiple platelet substrates were phosphorylated on tyrosine. Such tyrosine kinase response was necessary but not sufficient to activate alphaIIbbeta3, as detected by binding of soluble fibrinogen to platelets. Stirring of the platelets exposed to H2O2 and NaVO4 was also needed to allow for binding of fibrinogen to alphaIIbbeta3. The tyrosine kinase inhibitor genistein was able to block platelet aggregation induced by H2O2 and NaVO4, thus confirming that tyrosine kinase activity was needed to trigger alphaIIbbeta3 activation on stirring. N-Acetyl-L-cysteine, a cell-permeant antioxidant, blocked the tyrosine phosphorylation of platelet substrates and also the platelet aggregation induced by H2O2 and NaVO4. We found that beta3 was phosphorylated on tyrosine in platelets exposed to H2O2 and NaVO4, even in the absence of aggregation. Hence, tyrosine phosphorylation of beta3 might contribute to the "priming" of alphaIIbbeta3 induced by H2O2 and NaVO4, whereby the receptor can become activated on stirring of the platelets.

Humans↗
PubMed1997

Rac1 is required for cell proliferation and G2/M progression.

We have transiently expressed a dominant negative form of rac1 (N17rac1) using adenoviral-mediated gene transfer. The level of N17rac1 expression is demonstrated to be proportional to the multiplicity of infection. Expression of N17rac1 in Rat 2 fibroblasts results in cytostatic growth arrest. Cell-cycle analysis demonstrates that cells expressing N17rac1 accumulate in G2/M. These results suggest that rac1 is required for cell proliferation and provide the first demonstration in mammalian cells of a role for small GTP-binding proteins in the G2/M transition.

Adenoviridae↗
PubMed1997

Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

3T3 Cells↗
PubMed1997

Regulation of endothelial cell adhesion by profilin.

BACKGROUND: Although profilin is believed to be an essential regulator of the actin cytoskeleton in most cells, its precise role in mammalian cells remains unknown. We have used replication-incompetent adenovirus carrying the human profilin I cDNA as a means rapidly to increase the concentration of profilin in human aortic endothelial cells 12-31-fold above baseline--levels never before achieved in mammalian cells. RESULTS: The concentration of filamentous actin was not detectably affected by profilin overexpression. Actin stress fibers were, however, absent from areas of high profilin content in overexpressing cells, and the bulk of filaments was located at the periphery of the cells. We observed a gradient in the distribution of overexpressed profilin in migrating endothelial cells, with most profilin molecules concentrated near the advancing edge where focal contacts are being formed and focal adhesion proteins are located. Profilin overexpression resulted in increased recruitment of fibronectin receptors to the plasma membrane. Adhesion of endothelial cells to fibronectin was markedly and selectively increased by profilin overexpression. CONCLUSIONS: We conclude that an important role for profilin in mammalian cells may be its contribution to the formation of focal contacts, particularly those involving the fibronectin receptor.

Actins↗
PubMed1996

Superoxide-mediated actin response in post-hypoxic endothelial cells.

The mechanism leading to changes in the superstructure of endothelial cells exposed to ischemia and reperfusion remains uncharacterized. We show that in post-hypoxic endothelial cells, the simple re-addition of oxygen induces a profound reorganization of the actin cytoskeleton. The total filamentous actin pool increases by 41% and translocation of actin filaments to the submembranous network is observed. Concurrent with the actin polymerization, increased tyrosine phosphorylation of endothelial cell substrates is detected on Western blots. Overexpression of superoxide dismutase using replication incompetent adenovirus inhibits the actin and tyrosine phosphorylation responses to reoxygenation. Inhibition of tyrosine kinases with the isoflavone genistein also suppressed the actin polymerization response to reoxygenation, but unlike superoxide dismutase, genistein also induced the collapse of the superstructure of endothelial cells upon reoxygenation. These experiments support the concept that reoxygenation following a period of hypoxia can induce the remodeling of the actin cytoskeleton in endothelial cells. Such a response requires the intact coupling of superoxide producing pathway(s) with tyrosine kinase pathway(s).

Actins↗
PubMed1996

Regulation of reactive-oxygen-species generation in fibroblasts by Rac1.

In a variety of non-phagocytic cell types, there is a marked increase in intracellular levels of reactive oxygen species (ROS), including superoxide and H2O2, after ligand stimulation. We demonstrate that in NIH 3T3 cells transient expression of constitutively activated forms of the small GTP-binding proteins Ras or Rac1 leads to a significant increase in intracellular ROS. An increase in intracellular ROS is also demonstrated after growth factor [platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)] or cytokine [tumour necrosis factor-alpha (TNF-alpha) or interleukin (IL)-1 beta] stimulation of NIH 3T3 cells. Expression of a dominant negative allele of Rac1 inhibits the rise in ROS seen after Ras expression or after stimulation by either growth factors or cytokines. These results provide the first demonstration of the pathway by which ligand stimulation of ROS occurs in non-phagocytic cells and suggest that the family of Ras-related small GTP-binding proteins may function as regulators of the intracellular redox state.

3T3 Cells↗
PubMed1996

rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation.

The signal transduction pathway leading to the activation of the transcription factor NF-kappaB remains incompletely characterized. We demonstrate that in HeLa cells, transient expression of a constitutively active mutant of the small GTP-binding protein rac1 (V12rac1) leads to a significant increase in NF-kappaB transcriptional activity. In addition, expression of a dominant-negative rac1 mutant (N17rac1) inhibits basal and interleukin 1beta-stimulated NF-kappaB activity. Gel shift analysis using nuclear extract prepared from HeLa cells infected with a recombinant adenovirus encoding N17rac1 (Ad.N17racl) showed reduced levels of cytokine-stimulated DNA binding to a consensus NF-kappaB binding site. We demonstrate that rac proteins function downstream of ras proteins in the activation of NF-kappaB. In addition, V12rac1 stimulation of NF-kappaB activity is shown to be independent of the ability of rac proteins to activate the family of c-jun amino-terminal kinases. In an effort to further explore how rac proteins might regulate NF-kappaB activity, we demonstrate that expression of V12rac1 in HeLa cells or stimulation with cytokine results in a significant increase in intracellular reactive oxygen species (ROS). Treatment of cells with either of two chemically unrelated antioxidants inhibits the rise in ROS that occurs following V12rac1 expression as well as the ability of V12rac1 to stimulate NF-kappaB activity. These results suggest that in HeLa cells, rac1 regulates intracellular ROS production and that rac proteins function as part of a redox-dependent signal transduction pathway leading to NF-kappaB activation.

Cytokines↗
PubMed1995

Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.

Stimulation of rat vascular smooth muscle cells (VSMCs) by platelet-derived growth factor (PDGF) transiently increased the intracellular concentration of hydrogen peroxide (H2O2). This increase could be blunted by increasing the intracellular concentration of the scavenging enzyme catalase or by the chemical antioxidant N-acetylcysteine. The response of VSMCs to PDGF, which includes tyrosine phosphorylation, mitogen-activated protein kinase stimulation, DNA synthesis, and chemotaxis, was inhibited when the growth factor-stimulated rise in H2O2 concentration was blocked. These results suggest that H2O2 may act as a signal-transducing molecule, and they suggest a potential mechanism for the cardioprotective effects of antioxidants.

Acetylcysteine↗
PubMed1994

Ras proteins regulate multiple mitogenic pathways in A10 vascular smooth muscle cells.

We examined the role of ras proteins in mitogenesis and proliferation of A10 smooth muscle cells. An assay was developed in which a dominant negative ras gene could be transiently expressed in vascular smooth muscle cells. As opposed to cells transfected with a control plasmid, those transfected with a dominant negative ras expression plasmid exhibited a decrease in thymidine uptake in response to serum, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor and thrombin stimulation. In addition, expression of dominant negative ras blocked smooth muscle cell proliferation as assessed by the number of surviving colonies after transfection and neomycin selection. Our data is the first to show that ras is essential for smooth muscle cell proliferation and is a mediator in multiple smooth muscle cell mitogenic signalling pathways.

Animals↗
PubMed1992

von Willebrand factor interacts with malignant hematopoietic cell lines: evidence for the presence of specific binding sites and modification of von Willebrand factor structure and function.

von Willebrand factor (vWf) mediates the adherence of platelets to exposed vascular subendothelium. During hematogenous metastasis in certain model systems, platelets are also deposited at endothelial surfaces in heterotypic aggregates with tumor cells. The role of vWf in this metastatic event was investigated by examining the interaction between purified iodine 125-labeled vWf and the human tumor cell lines, U937 and CA46. vWf-tumor cell binding was specific, partially reversible, and saturable at 4 degrees C. At 37 degrees C, however, U937 cell binding sites could not be saturated, suggesting endocytosis as a possible mechanism of interaction. When constant amounts of vWf were added, interactions with tumor cells were complete after 60 minutes, but rapidly dissociated over the next 2 hours. With the addition of specific proteinase inhibitors, vWf remained in stable association with tumor cells throughout a 3-hour time course. Loss of vWf antigen in supernatant samples complemented the 125I-labeled vWf bound directly to tumor cells. However, disproportionately large reductions in vWf functional activity were observed and correlated with alterations in multimeric structure. These data suggest an initial binding of vWf to some tumor cells, followed by a time-dependent loss of structural and functional integrity. VWf may contribute to tumor cell arrest within the microvasculature and may influence other tumor cell-subendothelial interactions as the adhesive function of vWf is subsequently modified.

Amino Acid Sequence↗