Sita Subbaram

Albany Medical College, Albany, New York, United States

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Publications (13)31.9 Total impact

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    ABSTRACT: Aberrant matrix metalloproteinase-1 (MMP-1) expression contributes to the pathogenesis of many degenerative disease processes that are associated with increased oxidative damage or stress. We and others have established that shifts in steady-state H2O2 production resulting from enforced antioxidant gene expression, senescence or UV irradiation control MMP-1 expression. Here we establish that histone deacetylase-2 (HDAC2) protein levels and its occupancy of the MMP-1 promoter are decreased in response to enforced manganese superoxide dismutase expression (Sod2). Inhibition of histone deacetylase (HDAC) activity further accentuates the redox-dependent expression of MMP-1. Sod2-dependent decreases in HDAC2 are associated with increases in a proteasome-sensitive pool of ubiquitinylated HDAC2 and MMP-1 specific histone H3 acetylation. Sod2 overexpression also enhanced recruitment of Ets-1, c-Jun, c-Fos, and the histone acetyltransferase (HAT) PCAF to the distal and proximal region of the MMP-1 promoter. Furthermore, the Sod2-dependent expression of MMP-1 can be reversed by silencing the transcriptional activator c-Jun. All of the above Sod2-dependent alterations are largely reversed by catalase coexpression, indicating that the redox-control of MMP-1 is H2O2-dependent. These findings identify a novel redox-regulation of MMP-1 transcription that involves site specific promoter recruitment of both activating factors and chromatin modifying enzymes, which converge to maximally drive MMP-1 gene expression.
    Free radical biology & medicine. 06/2014;
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    ABSTRACT: Recent studies have shown that alterations in mRNA content, achieved through post-transcriptional mechanisms such as alternative splicing or polyadenylation, are critical for regulation of cancer-promoting genes by determining transcript susceptibility to mRNA degradation pathways. However, it remains unclear how cues from the tumor microenvironment trigger this regulation to control genes that drive malignant growth. Expression of integrin α3β1 in breast cancer cells promotes tumor growth and invasion, in part through induction of cyclooxygenase-2 (Cox-2). In the current study, we used RNAi to suppress α3β1 in human MDA-MB-231 breast cancer cells, then utilized exon microarrays to compare global gene expression between control and α3β1-deficient cells. This analysis identified numerous mRNAs, including Cox-2, that show altered expression and/or alternate exon usage (AEU) in α3β1-deficient cells. AEU included patterns predicted to render a mRNA susceptible to degradation, such as 3'-UTR variations or retention of elements that target it for nonsense-mediated decay (NMD). PCR-based analysis of α3β1-deficient cells confirmed changes in Cox-2 mRNA that may target it for NMD, including retention of an intron that harbors premature termination codons and changes within the 3'-UTR. Consistently, Cox-2 mRNA stability was reduced in α3β1-deficient cells, and siRNA-mediated knockdown of UPF1 (an essential NMD factor) in these cells led to Cox-2 mRNA accumulation. Our study identifies α3β1-mediated AEU as a novel paradigm of integrin-dependent gene regulation that may be exploitable as a therapeutic target to inhibit breast cancer.
    Journal of Cell Science 01/2014; · 5.88 Impact Factor
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    ABSTRACT: The pro-inflammatory cytokine Interleukin-1α (IL-1α) has recently emerged as a susceptibility marker for a wide array of inflammatory diseases associated with oxidative stress including Alzheimer's, arthritis, atherosclerosis, diabetes and cancer. In the present study, we establish that expression and nuclear localization of IL-1α are redox-dependent. Shifts in steady-state H2O2 concentrations (SS-[H2O2]) resulting from enforced expression of manganese superoxide dismutase (SOD2) drive IL-1α mRNA and protein expression. The redox-dependent expression of IL-1α is accompanied by its increased nuclear localization. Both IL-1α expression and its nuclear residency are abrogated by catalase co-expression. Sub-lethal doses of H2O2 also cause IL-1α nuclear localization. Mutagenesis revealed IL-1α nuclear localization does not involve oxidation of cysteines within its N terminal domain. Inhibition of the processing enzyme calpain prevents IL-1α nuclear localization even in the presence of H2O2. H2O2 treatment caused extracellular Ca(2+) influx suggesting oxidants may influence calpain activity indirectly through extracellular Ca(2+) mobilization. Functionally, as a result of its nuclear activity, IL-1α overexpression promotes NF-kB activity, but also interacts with the histone acetyl transferase (HAT) p300. Together, these findings demonstrate a mechanism by which oxidants impact inflammation through IL-1α and suggest that antioxidant-based therapies may prove useful in limiting inflammatory disease progression.
    Redox biology. 01/2013; 1(1):218-25.
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    Sita Subbaram, C Michael Dipersio
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    ABSTRACT: INTRODUCTION: Integrin receptors for cell adhesion to the extracellular matrix have important roles in all stages of cancer progression and metastasis. Since the integrin family was discovered in the early 1980's, many studies have identified critical adhesion and signaling functions for integrins expressed on tumor cells, endothelial cells and other cell types of the tumor microenvironment, in controlling proliferation, survival, migration and angiogenesis. In recent years, the laminin-binding integrin α3β1 has emerged as a potentially promising anti-cancer target on breast cancer cells. AREAS COVERED: Studies from the past decade that implicate integrins as promising anti-cancer targets and the development of integrin antagonists as anti-cancer therapeutics. Recent preclinical studies that have identified the laminin-binding integrin α3β1 as an appealing anti-cancer target and the knowledge gaps that must be closed to fully exploit this integrin as a therapeutic target for breast cancer. EXPERT OPINION: Although the tumor-promoting functions of α3β1 implicate this integrin as a promising therapeutic target on breast cancer cells, successful exploitation of this integrin as an anti-cancer target will require a better understanding of the molecular mechanisms whereby it regulates specific tumor cell behaviors and the identification of the most appropriate α3β1 functions to antagonize on breast cancer cells.
    Expert Opinion on Therapeutic Targets 08/2011; 15(10):1197-210. · 4.90 Impact Factor
  • Free Radical Biology and Medicine - FREE RADICAL BIOL MED. 01/2011; 51.
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    ABSTRACT: Many degenerative disease processes associated with aging result from enhanced extracellular matrix (ECM) breakdown. Concomitant with aberrant matrix destruction are alterations in levels of reactive oxygen species (ROS) generating and detoxification systems. ROS function as second messengers due to their ability to react with wide range of biomolecules resulting in modification of an array of signaling networks. ROS can activate upstream kinases (MKK) responsible for MAPK activation and restrict the activity of their inhibitory phosphatases. Here we focus on the redox-sensitive signaling components that control the expression of MMP-1, which is largely responsible for maintaining ECM homeostasis. Numerous disease processes are associated with shifts in steady state ROS levels that influence overall ECM degradation. This review highlights the redox-sensitive regulatory signals that control the expression of the primary initiating protease MMP-1 and provides strong rational for the use of antioxidant based therapies for treatment of degenerative disorders associated with aberrant matrix destruction.
    Respiratory Physiology & Neurobiology 12/2010; 174(3):299-306. · 2.05 Impact Factor
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    ABSTRACT: Alternative splicing plays an important role in regulation of normal cellular function. Alternative splicing of pre-mRNA leads to the diversity of downstream protein products in the cell. The Affymetrix Exon arrays allow for a high throughput evaluation of the differences in spliced mRNA expressed in a biological system. In this study, we describe a method using this technology to study the generation of alternative mRNA transcripts in breast cancer cells that differ in the levels of a particular integrin, alpha3beta1.
    Methods in molecular biology (Clifton, N.J.) 01/2010; 632:63-72. · 1.29 Impact Factor
  • Free Radical Biology and Medicine - FREE RADICAL BIOL MED. 01/2010; 49.
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    ABSTRACT: Cellular systems produce reactive oxygen species during the process of metabolism. Oxidative stress results in the activation or repression of many genes in important signaling pathways. DNA microarrays allow for a high throughput evaluation of the changes in gene expression levels in any biological system. In this study, we describe a method to employ gene expression microarrays to study the transcriptional changes in redox-engineered cell lines that will overexpress MnSOD and/or catalase in the mitochondria.
    Methods in molecular biology (Clifton, N.J.) 02/2009; 477:357-68. · 1.29 Impact Factor
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    ABSTRACT: Reactive oxygen species have been shown to play an important role in the regulation of distinct signaling cascades, many of which act upon the production of matrix metalloproteinases (MMP). Using a series of redox-engineered cell lines we have previously demonstrated that MMP-1 expression is sensitive to the alterations in the steady state production of H2O2 (Ranganathan, A. C., Nelson, K. K., Rodriguez, A. M., Kim, K. H., Tower, G. B., Rutter, J. L., Brinckerhoff, C. E., Epstein, C. J., Huang, T. T., Jeffrey, J. J., and Melendez, J. A. (2001) J. Biol. Chem. 276, 14264-14270). In the present study, we investigate the molecular mechanisms involved in the H2O2-mediated induction of MMP-1. Mutational analysis of an MMP-1 promoter indicates that both the single nucleotide polymorphism creating an Ets binding site at -1607 and a proximal AP-1 site at -1602 are required for maximal H2O2-dependent transcription. The redox-sensitive MMP-1 protein expression requires activation of both ERK1/2 and JNK pathways. Importantly, JNK signaling is largely responsible for the H2O2 sensitivity of the MMP-1 promoter, whereas ERK1/2 contributes to both its basal and H2O2 dependence. H2O2 control of Ets-1 expression was ERK1/2-dependent whereas that of c-Jun requires both ERK1/2 and JNK signaling. Chromatin immunoprecipitation assays indicate that binding of the histone acetyltransferase, p300, and the transcription factors Ets-1 and c-Jun to the MMP-1 promoter is redox sensitive. The redox sensitivity of MMP-1 expression is also associated with an increase in the abundance of oxidatively inactivated protein-tyrosine phosphatases. Targeted cytosolic or mitochondrial scavenging of H2O2 prevented all of the aforementioned signals. These studies provide substantial insight into the mechanisms underlying the redox-dependent control of MMP-1 and may lead to the development of novel targeted antioxidant-based inhibitory therapies for controlling MMP-1 expression during degenerative disease processes.
    Journal of Biological Chemistry 06/2006; 281(20):14100-10. · 4.65 Impact Factor
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    ABSTRACT: Manganese superoxide dismutase (SOD2) has been well established to be essential for protection from a variety of apoptotic stimuli. Here we demonstrate that the antiapoptotic effects of SOD2 are attributed to its ability to generate H(2)O(2) and that its efficient removal resensitizes cells to tumor necrosis factor (TNF)-alpha-induced apoptosis. SOD2 overexpression in HT-1080 cells leads to a decrease in the fluorescence of the superoxidesensitive fluorophore, dihydroethidium, and a concomitant increase in oxidation of the H2O2-sensitive dye, dichlorodihydrofluorescein diacetate (DCFDA). The rate of aminotriazole-inhibited catalase activity also was increased when SOD2 is overexpressed and reflects a 1.6-fold increase in the steady-state production of H(2)O(2). The increase in H(2)O(2) was associated with decreased sensitivity to TNF-alpha-mediated apoptosis, as measured by monitoring the loss of mitochondrial membrane potential (MMP), caspase activation, poly-ADP ribose polymerase (PARP) cleavage, and accumulation of hypodiploid DNA content. Both the increase in H2O2 and resistance to TNF-mediated apoptosis were reversed by coexpression of catalase. The lipid hydroperoxide scavengers, beta-hydroxytoluene and trolox, and the iron chelator, desferroxamine, showed partial recovery of TNF-induced apoptosis. These findings indicate that increases in the intracellular steady-state production of H(2)O(2) by SOD2 can block the activation of key processes fundamental to the process of programmed cell death.
    Antioxidants and Redox Signaling 01/2006; 8:1295-1305. · 7.19 Impact Factor
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    ABSTRACT: Recent studies have demonstrated that the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10), the antagonist of the phosphosphoinositol-3-kinase (PI3K) signaling cascade, is susceptible to H2O2-dependent oxidative inactivation. This study describes the use of redox-engineered cell lines to identify PTEN as sensitive to oxidative inactivation by mitochondrial H2O2. Increases in the steady state production of mitochondrial derived H2O2, as a result of manganese superoxide dismutase (Sod2) overexpression, led to PTEN oxidation that was reversed by the coexpression of the H2O2-detoxifying enzyme catalase. The accumulation of an oxidized inactive fraction of PTEN favored the formation of phosphatidylinositol 3,4,5-triphosphate at the plasma membrane, resulting in increased activation of Akt and modulation of its downstream targets. PTEN oxidation in response to mitochondrial H2O2 enhanced PI3K signaling, leading to increased expression of the key regulator of angiogenesis, vascular endothelial growth factor. Overexpression of PTEN prevented the H2O2-dependent increase in vascular endothelial growth factor promoter activity and immunoreactive protein, whereas a mutant PTEN (G129R), lacking phosphatase activity, did not. Furthermore, mitochondrial generation of H2O2 by Sod2 promoted endothelial cell sprouting in a three-dimensional in vitro angiogenesis assay that was attenuated by catalase coexpression or the PI3K inhibitor LY2949002. Moreover, Sod2 overexpression resulted in increased in vivo blood vessel formation that was H2O2-dependent as assessed by the chicken chorioallantoic membrane assay. Our findings provide the first evidence for the involvement of mitochondrial H2O2 in regulating PTEN function and the angiogenic switch, indicating that Sod2 can serve as an alternative physiological source of the potent signaling molecule, H2O2.
    Journal of Biological Chemistry 05/2005; 280(17):16916-24. · 4.65 Impact Factor
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