Biomedical subjects

Latha P Ganesan

Publications and source records attributed to Latha P Ganesan.

12 recordsLinked to original sources

PubMed2006

Akt/Protein kinase B modulates macrophage inflammatory response to Francisella infection and confers a survival advantage in mice.

The Gram-negative bacterium Francisella novicida infects primarily monocytes/macrophages and is highly virulent in mice. Macrophages respond by producing inflammatory cytokines that confer immunity against the infection. However, the molecular details of host cell response to Francisella infection are poorly understood. In this study, we demonstrate that F. novicida infection of murine macrophages induces the activation of Akt. Inhibition of Akt significantly decreases proinflammatory cytokine production in infected macrophages, whereas production of the anti-inflammatory cytokine IL-10 is enhanced. Analysis of the mechanism of Akt influence on cytokine response demonstrated that Akt promotes NF-kappaB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with phagosomal escape. Infection with mglA mutants of F. novicida elicited sustained activation of Akt in comparison to cells infected with wild-type F. novicida. Concomitantly, there was significantly higher proinflammatory cytokine production and lower IL-10 production in cells infected with the mglA mutant. Finally, transgenic animals expressing constitutively active Akt displayed a survival advantage over their wild-type littermates when challenged with lethal doses of F. novicida. Together, these observations indicate that Akt promotes proinflammatory cytokine production by F. novicida-infected macrophages through its influence on NF-kappaB, thereby contributing to immunity against F. novicida infection.

Animals↗

PubMed2006

A mitotically inheritable unit containing a MAP kinase module.

Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate.

Alleles↗

PubMed2006

FcgammaR-induced production of superoxide and inflammatory cytokines is differentially regulated by SHIP through its influence on PI3K and/or Ras/Erk pathways.

Phagocytosis of IgG-coated particles via FcgammaR is accompanied by the generation of superoxide and inflammatory cytokines, which can cause collateral tissue damage in the absence of regulation. Molecular mechanisms regulating these phagocytosis-associated events are not known. SHIP is an inositol phosphatase that downregulates PI3K-mediated activation events. Here, we have examined the role of SHIP in FcgammaR-induced production of superoxide and inflammatory cytokines. We report that primary SHIP-deficient bone marrow macrophages produce elevated levels of superoxide upon FcgammaR clustering. Analysis of the molecular mechanism revealed that SHIP regulates upstream Rac-GTP binding, an obligatory event for superoxide production. Likewise, SHIP-deficient macrophages displayed enhanced IL-1beta and IL-6 production in response to FcgammaR clustering. Interestingly, whereas IL-6 production required activation of both PI3K and Ras/Erk pathways, IL-1beta production was dependent only on Ras/Erk activation, suggesting that SHIP may also regulate the Ras/Erk pathway in macrophages. Consistently, SHIP-deficient macrophages displayed enhanced activation of Erk upon FcgammaR clustering. Inhibition of Ras/Erk or PI3K suppressed the enhanced production of IL-6 in SHIP-deficient macrophages. In contrast, inhibition of Ras/Erk, but not PI3K, suppressed IL-1beta production in these cells. Together, these data demonstrate that SHIP regulates phagocytosis-associated events through the inhibition of PI3K and Ras/Erk pathways.

Animals↗

PubMed2006

Attenuation of myocardial ischemia-reperfusion injury by trimetazidine derivatives functionalized with antioxidant properties.

Trimetazidine (TMZ), an anti-ischemic metabolic drug, is used to treat chest pain (angina pectoris). We hypothesized that derivatives of TMZ with antioxidant functions may improve the cardiac dysfunction caused by ischemia-reperfusion (I/R) above that observed with TMZ alone. Isolated rat hearts perfused with Krebs-Henseleit buffer according to the Langendorff method were subjected to 30 min of global ischemia followed by 45 min of reperfusion. Trimetazidine, TMZ-NH (TMZ modified with a pyrroline moiety), or TMZ-PhiNH (TMZ-NH with a phenyl substitute) were infused (50 microM) for 1 min before the onset of ischemia. Untreated (control) hearts at the end of 45 min of reperfusion showed a significant decrease in the recovery of coronary flow (42%), left ventricular-developed pressure (22%), and rate-pressure product (25%) compared with preischemic baseline values. The I/R hearts also showed markedly increased lactate dehydrogenase and creatine kinase activities in the coronary effluent, significant myocardial infarction (46% of risk area), and activation of Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase. Pretreatment of hearts with TMZ-NH or TMZ-PhiNH significantly enhanced the recovery of heart function and decreased infarct size. The I/R-induced activation of Akt was further enhanced by TMZ-PhiNH. The present study demonstrated that TMZ-NH and TMZ-PhiNH significantly protected hearts against I/R-mediated cardiac dysfunction and injury. The protective effect of the TMZ derivatives could be due to the combined effects of antioxidant and anti-ischemic activities as well as enhanced pro-survival Akt activity.

Animals↗

PubMed2006

Macrophage pro-inflammatory response to Francisella novicida infection is regulated by SHIP.

Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida-induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida-induced cytokine production through the inhibition of NFkappaB. Consistently, macrophages lacking SHIP displayed enhanced NFkappaB-driven gene transcription, whereas overexpression of SHIP led to decreased NFkappaB activation. Thus, we propose that SHIP negatively regulates F. novicida-induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFkappaB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia.

Animals↗

PubMed2005

C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling.

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.

Animals↗

PubMed2005

Lipopolysaccharide-induced production of interleukin-10 is promoted by the serine/threonine kinase Akt.

The bacterial endotoxin lipopolysaccharide (LPS), is a potent inducer of the inflammatory response. Previous studies demonstrated that LPS-induced toxicity is reversed upon FcgammaR clustering by IgG immune complexes (IC) through upregulation of the anti-inflammatory cytokine IL-10. The PI3K-Akt pathway is also reported to reverse LPS-induced inflammation. In this study, we have examined the role of Akt in LPS-induced IL-10 production. First, we compared Akt activation in macrophages stimulated with either LPS alone, or with a combination of LPS and ICs. Our experiments revealed that while Akt was activated under both conditions, the level of activation was significantly higher in cells stimulated with LPS and ICs, suggesting that Akt may be involved in IC-induced upregulation of IL-10 production. Using several independent models we have then tested the notion that enhanced Akt activation may lead to enhanced LPS-induced IL-10 production. Over-expression of constitutively active Myr-Akt in the mouse macrophage cell line Raw 264.7 led to significant increase in IL-10 production in response to LPS. In addition, down-regulation of Akt by siRNA resulted in a decrease in LPS-induced IL-10 production. Peritoneal macrophages from transgenic mice with macrophage-specific expression of Myr-Akt produced significantly higher levels of IL-10 when stimulated with LPS, compared to their wild-type counterparts. Consistent with this observation, serum levels of IL-10, post-LPS challenge, was higher in the Myr-Akt transgenic mice compared to the wild-type mice. Taken together, these data demonstrate that Akt plays a critical role in LPS-induced production of IL-10.

Animals↗

PubMed2005

Molecular analysis of expression and function of hFcgammaRIIbl and b2 isoforms in myeloid cells.

The inhibitory receptor FcgammaRIIb becomes tyrosine phosphorylated and associates with the inositol phosphatase SHIP to downregulate phagocytosis. The two splice variants of FcgammaRIIb, b1 and b2, are differentially expressed in hematopoetic cells. Both isoforms of FcgammaRIIb are expressed in human myeloid cells although FcgammaRIIb2 predominates. In murine B cells FcgammaRIIb2 associates with clathrin-coated pits and undergoes endocytosis, whereas FcgammaRIIbl is excluded from the coated pits, indicating that the two isoforms serve partially differing functions. In humans, there are conflicting reports with regard to the ability of FcgammaRIIb2 to become tyrosine phosphorylated, and the functional capacities of the two isoforms are poorly understood. We, and others, have previously reported that the expression of FcgammaRIIb is upregulated in human monocytes by the anti-inflammatory cytokine IL-4. Here, we extend these findings to demonstrate that the IL-4-induced upregulation of FcgammaRIIb is synergistically enhanced by the addition of IL-10, both at the protein and the mRNA level. The upregulated receptors are functional as assessed by their ability to become tyrosine phosphorylated and to downregulate phagocytosis. Interestingly, both b1 and b2 isoforms are upregulated by anti-inflammatory cytokines. Transfection experiments expressing human FcgammaRIIbl or b2 in Raw 264.7 murine macrophage cells revealed that both isoforms are tyrosine phosphorylated and promote SHIP phosphorylation. Finally, both b1 and b2 isoforms of FcgammaRIIb downregulate phagocytosis to a similar extent. Thus we conclude that FcgammaRIIbl and b2 are both functional inhibitory receptors in the phagocytic process.

Animals↗

PubMed2004

The serine/threonine kinase Akt Promotes Fc gamma receptor-mediated phagocytosis in murine macrophages through the activation of p70S6 kinase.

Fc gamma receptor (Fc gamma R) clustering by immune complexes activates multiple signaling pathways leading to phagocytosis. We and others have previously reported that Akt is phosphorylated in response to Fc gamma R clustering. However, the functional consequence of Akt activation by Fc gamma R is not known. Using Raw 264.7 macrophage cells transfected to overexpress either constitutively active myristoylated (Myr)-Akt or a dominant-negative CAAX-Akt and bone marrow macrophages (BMMs) from wild-type and transgenic mice expressing macrophage-specific Myr-Akt, we analyzed the function of Akt in phagocytosis. We report that overexpression of Myr-Akt resulted in significant increase in phagocytic efficiency, whereas CAAX-Akt down-regulated phagocytosis in Raw 264.7 cells. Likewise BMMs expressing Myr-Akt displayed enhanced phagocytic ability. Analyzing the downstream effectors of Akt, we demonstrate that p70S6 kinase is constitutively phosphorylated in Myr-Akt-expressing BMMs. p70S6 kinase is reported to influence actin cytoskeleton and cell migration, suggesting that Akt may influence phagocytosis through the activation of p70S6 kinase. Consistent with this, overexpression of either wild-type or constitutively active but not a kinase-inactive p70S6 kinase in Raw 264.7 cells significantly enhanced phagocytosis. Likewise suppression of p70S6 kinase with rapamycin down-regulated phagocytic efficiency conferred by the expression of constitutively active Akt. These findings demonstrate a novel role for Akt in phagocytosis through the activation of p70S6 kinase.

Animals↗

PubMed2004

Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP.

LPS stimulates monocytes/macrophages through TLR4, resulting in the activation of a series of signaling events that potentiate the production of inflammatory mediators. Recent reports indicated that the inflammatory response to LPS is diminished by PI3K, through the activation of the serine/threonine kinase Akt. SHIP is an inositol phosphatase that can reverse the activation events initiated by PI3K, including the activation of Akt. However, it is not known whether SHIP is involved in TLR4 signaling. In this study, we demonstrate that LPS stimulation of Raw 264.7 mouse macrophage cells induces the association of SHIP with lipid rafts, along with IL-1R-associated kinase. In addition, SHIP is tyrosine phosphorylated upon LPS stimulation. Transient transfection experiments analyzing the function of SHIP indicated that overexpression of a wild-type SHIP, but not the SHIP Src homology 2 domain-lacking catalytic activity, up-regulates NF-kappaB-dependent gene transcription in response to LPS stimulation. These results suggest that SHIP positively regulates LPS-induced activation of Raw 264.7 cells. To test the validity of these observations in primary macrophages, LPS-induced events were compared in bone marrow macrophages derived from SHIP(+/+) and SHIP(-/-) mice. Results indicated that LPS-induced MAPK phosphorylation is enhanced in SHIP(+/+) cells, whereas Akt phosphorylation is enhanced in SHIP(-/-) cells compared with SHIP(+/+) cells. Finally, LPS-induced TNF-alpha and IL-6 production was significantly lower in SHIP(-/-) bone marrow-derived macrophages. These results are the first to demonstrate a role for SHIP in TLR4 signaling, and propose that SHIP is a positive regulator of LPS-induced inflammation.

Animals↗

PubMed2003

The protein-tyrosine phosphatase SHP-1 associates with the phosphorylated immunoreceptor tyrosine-based activation motif of Fc gamma RIIa to modulate signaling events in myeloid cells.

Fc gamma RIIa is a low affinity IgG receptor uniquely expressed in human cells that promotes phagocytosis of immune complexes and induces inflammatory cytokine gene transcription. Recent studies have revealed that phagocytosis initiated by Fc gamma RIIa is tightly controlled by the inositol phosphatase SHIP-1, and the protein-tyrosine phosphatase SHP-1. Whereas the molecular nature of SHIP-1 involvement with Fc gamma RIIa has been well studied, it is not clear how SHP-1 is activated by Fc gamma RIIa to mediate its regulatory effect. Here we report that Fc gamma RIIa clustering induces SHP-1 phosphatase activity in THP-1 cells. Using synthetic phosphopeptides, and stable transfectants expressing immunoreceptor tyrosine-based activation motif (ITAM) tyrosine mutants of Fc gamma RIIa, we demonstrate that SHP-1 associates with the phosphorylated amino-terminal ITAM tyrosine of Fc gamma RIIa, whereas the tyrosine kinase Syk associates with the carboxyl-terminal ITAM tyrosine. Association of SHP-1 with Fc gamma RIIa ITAM appears to suppress total cellular tyrosine phosphorylation. Furthermore, Fc gamma RIIa clustering results in the association of SHP-1 with key signaling molecules such as Syk, p85 subunit of PtdIns 3-kinase, and p62dok, suggesting that these molecules may be substrates of SHP-1 in this system. Finally, overexpression of wild-type SHP-1 but not catalytically deficient SHP-1 led to a down-regulation of NF kappa B-dependent gene transcription in THP-1 cells activated by clustering Fc gamma RIIa.

Antigens, CD↗

PubMed2003

SHIP-2 inositol phosphatase is inducibly expressed in human monocytes and serves to regulate Fcgamma receptor-mediated signaling.

SHIP-2, a recently identified inositol 5'-phosphatase, shares high level homology with SHIP-1. Although the role of SHIP-1 has been extensively studied, the role of SHIP-2 in myeloid cell functions is not known. Here, we have analyzed the expression patterns, molecular mechanism of activation, and function of SHIP-2 in human myeloid cell Fcgamma receptor (FcgammaR) signaling. We report that SHIP-2 is expressed in transformed myeloid cells and in primary macrophages, but not in peripheral blood monocytes. Treatment of peripheral blood monocytes with bacterial lipopolysaccharide induced expression of SHIP-2 in a dose-dependent manner. FcgammaRIIa clustering in THP-1 cells induced SHIP-2 tyrosine phosphorylation, suggesting a role for SHIP-2 in modulating FcgammaR-mediated function. Consistent with this notion, overexpression of wild-type SHIP-2 (but not catalytically deficient SHIP-2) in THP-1 cells almost completely abrogated NFkappaB-mediated gene transcription in response to FcgammaRIIa clustering. Furthermore, FcgammaRIIa-induced Akt activation was blocked by wild-type SHIP-2, but not by a catalytically deficient mutant of SHIP-2. Additional experiments analyzing the molecular mechanism of SHIP-2 induction by FcgammaRIIa revealed that SHIP-2 associated with the phosphorylated FcgammaRIIa immunoreceptor tyrosine-based activation motif via the SHIP-2 SH2 domain. Thus, an SH2 domain mutant of SHIP-2 failed to associate with FcgammaRIIa or to become tyrosine-phosphorylated upon FcgammaRIIa clustering. Finally, we also demonstrate that SHIP-2 phosphorylation was induced by FcgammaRI clustering in THP-1 cells. These findings unravel a novel level of regulation of FcgammaR-mediated activation of human myeloid cells by the expression and function of the inositol phosphatase SHIP-2.

Animals↗