• Role of GluN2C-containing N-methyl-D-aspartate (NMDA) receptor in Oligodendrocyte Differentiation and Myelination

      Luo, Tong; Department of Neuroscience & Regenerative Medicine (1/25/2018)
      Myelination by oligodendrocytes (OLs) is critical for rapid nerve signal conduction. Abnormalities of OLs mediate a variety of central nervous system (CNS) diseases, including multiple sclerosis. N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors expressed in neurons and are key regulators for neuron survival and normal brain functions. Recently, NMDA receptors were identified in OLs and contribute to OL migration, differentiation and myelination. However, the exact function of NMDA receptors on OLs remains unclear. Previous studies have shown that GluN2C is one of the predominant NMDA receptor subunits expressed in OLs. Here we report that NMDA treatment promotes OL differentiation in vitro, but fails to increase mature OL percentage in the absence of GluN2C. In addition, we observed an early developmental myelination delay and long-term recovery in the optic nerve of GluN2C-knockout (KO) mice in vivo, which was closely related to the impairment of OL differentiation. Overall, these results indicate a functional involvement of GluN2C-containing NMDA receptors in OL differentiation and myelination.
    • The role of glutathione in the process of amino acid uptake in the mammalian kidney

      Pillion, Dennis J.; Department of Cell and Molecular Biology (1976-06)
    • The role of gonadal hormones on the gonadotropin secretion of the rat

      Ryan, Gail Eileen; Department of Endocrinology (1976-09)
    • The Role of GPR109A in NAD+ Metabolism in Aging RPE

      Fuller, Jasmine; Department of Biochemistry and Cancer Biology (Augusta University, 2018-12-28)
      Age-related macular degeneration (AMD) is the leading cause of blindness in people over the age of 50 worldwide. The retinal pigment epithelium (RPE), located in the back of the eye, is most affected in AMD. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme common to most metabolic pathways. Reductions in NAD+ and NAD+- dependent enzymes (e.g., SIRT1) have been linked causally to the development/progression of many age-related pathologies. None, however, have evaluated NAD+ directly or the mechanisms governing its biosynthesis and related availability in RPE. In our previous study, we have shown that NAD+ levels decline with age in the RPE. This correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression. SIRT1 expression and activity was also significantly reduced. Using the human RPE cell line, ARPE-19, primary mouse RPE cells and FK866, a highly specific, noncompetitive NAMPT inhibitor, we simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence. Using this model, we demonstrated the positive impact that therapies that provide supplemental or alternate energy sources such as nicotinamide mononucleotide (NMN) and β-hydroxybutyrate (B-HB) have on RPE viability and the possible role of G-protein coupled receptor, GPR109A in this process.
    • The Role of Iron Induced Oxidative Stress in Acute Ischemic Stroke and the Potential Role for Fasciculations in their Therapy

      Mehta, Shyamal H.; Department of Physiology (2003-07)
      (Introductory Paragraphs) Stroke accounts for about one of every 15 deaths in the United States. It is the third leading cause of death behind heart disease and cancer in the United States and the second most common cause of death worldwide, according to the National Center for Health Statistics (1,2). Stroke is also the leading cause of serious disability in the United States; four million people are coping with the debilitating consequences of surviving a stroke which adds to the significant public financial burden (3). Based on the Framingham Heart Study 500,000 people suffer a new or recurrent stroke each year, of whom one third die over the next year, one-third remain permanently disabled and the remaining one-third make a reasonable recovery (1,4). Stroke is a sudden loss of brain function resulting from a disruption in the supply of blood and oxygen to the central nervous system (CNS) giving rise to hypoxic-ischemic conditions within the tissue. Acute stroke can be classified either as: 1. Ischemic stroke involves an interruption in blood supply to the CNS secondary to a vaso-occlusive phenomenon, accounting for 80% of the stroke cases. On basis of its etiology it can be further arbitrarily classified to extra-cranial or intracranial thrombosis and embolism (5). 2. Hemorrhagic stroke involves an interference in blood supply secondary to vascular disruption, accounting for 20% of the cases, which can be further classified to intracranial hemorrhage and subarachnoid hemorrhage (5). A progressing stroke or a stroke in evolution is an extremely complex event whose etiopathogenesis is poorly understood. Its multifactorial etiology makes it difficult to predict and treat by means of clinical, imaging and laboratory data currently available in clinical practice. The hemodynamic changes in the cerebral milieu and the biochemical mechanisms that hasten the progression of neurological injury are crucial to understand in order to reduce neurological morbidity and to design clinically effective interventions. In cerebral ischemia there is an ischemic gradient which can be divided into the core, which is the central ischemic zone and the penumbra, which is the area peripheral to the core. In the penumbra, functional impairment occurs in the neurons and the glia, with the neurons being more susceptible to ischemic injury due their dependence on oxidative metabolism (5). A better understanding of the pathologic mechanisms in ischemic injury would help limit the neurological injury in the penumbra through therapeutic intervention. The major pathogenic mechanisms include energy failure and excitotoxicity, loss of protein translation in the susceptible neurons, apoptotic mechanisms, inflammation and lastly, injury mediated by oxidative stress through the generation of reactive oxygen species (ROS) (6). Many of the above mentioned mechanisms are influenced by the generation of ROS. It has been directly demonstrated in numerous studies that ROS are involved in oxidative damage through peroxidation of lipids, proteins and nucleic acids in ischemic tissues (7). In addition, ROS also function as signaling molecules in cellular ischemia and reperfusion. In this dissertation we tried to elucidate the role of ROS in exacerbation of neurological injury in acute ischemic stroke. In order to gain a better understanding of the pathophysiological mechanisms underlying oxidative stress, we studied iron induced oxidative stress, as iron generates ROS through the Fenton reaction. We believe that ROS exacerbate ischemic injury, hence we wanted to demonstrate the neuroprotective ability of various antioxidants. In the end, we present a model of neuronal behavior in vitro that may have possible implications in post-injury remodeling and repair. Chapter 1 will review the literature in the field of antioxidants and ROS in stroke. In addition, the prevailing theories on the role of iron-induced oxidative stress and the various antioxidant agents used in stroke will be critically reviewed.
    • The Role of Lateral Diffusion in G-Protein-Coupled Receptor Signaling

      Lober, Robert M.; Department of Pharmacology and Toxicology (2006-05)
      In the standard model of G-protein-coupled receptor (GPCR) signaling, receptors and G-proteins are free to diffuse laterally within the plane of the plasma membrane, and these molecules encounter each other by random collision. It is possible that formation of the receptor-G-protein (R-G) complex precedes receptor activation, although the dynamics of this process have been challenging to observe in live cells. We have approached this problem by measuring the membrane diffusion and binding reactions of receptors and Gproteins. We examined the functional consequences of immobilizing receptors, G-proteins, and inwardly rectifying potassium (GIRK) channels at the cell surface by biotinylation and avidin crosslinking, and monitored intermolecular binding events reflected by receptor-imposed constraints on G-protein diffusion, as measured by fluorescence recovery after photobleaching. In whole-cell voltage-clamp recordings, we found that both mobile and immobile heterologous p-opioid receptors (MOR) activated endogenous GIRK channels in cerebellar granule neurons with kinetics and agonist sensitivity resembling native synaptic responses. In HEK 293 cells, immobile GIRK channels were activated by multiple populations of immobilized receptors and Gproteins. Immobilization of MOR constrained the apparent mobility of freely diffusing fluorescent Gaj/o-family proteins, indicating measurable binding reactions between the two protein types, but had no effect on the diffusion of unrelated membrane proteins or Gaq subunits. Transient binding reactions were highly specific, as determined by competition with unlabeled binding partners. RG binding was disrupted by receptor agonist or GTPase-deficient G-protein mutants. Neither receptor antagonists nor pertussis toxin blocked basal R-G binding. Furthermore, the Ga subunit amino terminus (amino acids 1-31) was sufficient for mediating R-G binding. Our results provide evidence for the free diffusion of receptors and Gproteins, as well as a pre-signaling binding-dissociation equilibrium between them that is altered upon activation. The frequency of collisions between receptors and G-proteins does not limit the rate of signaling in neurons, but by diffusion receptors can “swap” G-proteins that are not stably associated with GIRK channels. A three-stage sequential fit model of R-G coupling is suggested.
    • The role of lateral diffusion in G-protein-coupled receptor signaling

      Lober, Robert M.; School of Graduate Studies (2006-05)
    • The Role of MHC-II Dependent Events in the Suppression Mediated by CD4+Foxp3+ Regulatory T cells

      Mmanywa, Faith Daima; Department of Medicine (2008-08)
      CD4+Foxp3+ regulatory T cells (Tregs) and antigen presenting cells (APCs) play an important role in maintaining peripheral tolerance but are otherwise exploited by tumors to create a state of unresponsiveness towards tumor antigens. The mechanisms of Treg mediated suppression are still not well understood. This work seeks to elucidate the role of major histocompatibility complex class II (MHC-II) dependent events in CD4+Foxp3+ Treg mediated suppression. The studies described here take advantage of novel conditional MHC-II deficient mice, which lack expression of MHC-II on peripheral APCs but still maintain their own naïve CD4 T cells and Tregs. In an in vitro system antigen-specific Tregs suppress CD8 T cell proliferation and effector molecule production in an antigen-specific and MHC-II dependent manner. In vivo, MHC-II deficiency resulted in a delay in tumor progression that was CD8 T cell dependent. We further describe two in vivo models in which the role of MHC-II dependent events in Treg mediated suppression can be tested. Therefore, a better understanding of Treg mediated suppression in the context of tumor-induced tolerance could provide potential strategies that could be utilized for anti-tumor immunotherapy.
    • Role of microtubules and motor proteins in mRNA localization

      Sanghavi, Paulomi; Department of Cellular Biology and Anatomy (2015-08)
      Establishment of polarity is essential for many cell types to perform their functions. A common mechanism that is used to establish polarity is localization of mRNAs at specific sites. This results in spatial restriction of protein expression. mRNA localization is a widespread phenomenon, occurring in most species. However, the mechanism by which mRNAs are localized is poorly understood. Using Drosophila as the model system, we investigated the localization of one such localized transcript, oskar mRNA. Studying the mechanism by which oskar mRNA is localized is important because many factors involved in localizing this transcript also function in localizing mRNAs in mammalian neurons. oskar mRNA localizes at the posterior pole of the Drosophila oocyte. This results in the posterior restriction of Oskar protein, which is turn functions in establishment of polarity in the oocyte and the future embryo. Localization of oskar mRNA is microtubule-dependent. We, therefore, characterized the polarity of microtubules in the oocyte. Our findings suggest that the posterior region is highly enriched in microtubule plus ends. However, this polarization is not essential for oskar mRNA localization. Secondly, the posterior localization of oskar mRNA was shown to be mediated primarily by the Kinesin-1 motor. Our findings demonstrate the role of an additional motor, Dynein, in this pathway. We found that Dynein associates with oskar mRNA in vivo and depletion of Dynein caused a significant delocalization of oskar mRNA. Next, we examined the role of a Dynein adaptor, Egalitarian (Egl), in the oskar mRNA localization pathway. Egl has been shown to recruit localized mRNAs to the Dynein motor in Drosophila embryos. Our results suggest that Egl associates with oskar mRNA in vivo and is required for the posterior localization of this transcript. Interestingly, one of the mechanisms by which Egl affects the localization of oskar mRNA is by affecting the microtubule polarity in the oocyte. Additionally, depletion of Egl caused precocious translation of oskar mRNA in the oocyte. Thus, our findings revealed a novel function for Egl in organizing oocyte microtubules and in regulating the translation of a localized mRNA.
    • The Role of Muscle Activity in Embryonic Death of Motor Neurons

      Creazzo, Tony Louis; Department of Anatomy (1980-06)
      N/A
    • The Role of Myostatin (GDF-8) in Chondrogenesis and Fracture Healing

      Elkasrawy, Moataz N.; Department of Medicine (2010-11)
      Traumatic musculoskeletal injuries frequently include damage to both muscle and bone where muscle injury itself can delay bone healing. Myostatin (GDF-8) is a member of the transforming growth factor-β (TGF-β) superfamily, and a negative regulator of skeletal muscle growth. Loss of myostatin function leads to a doubling of skeletal muscle mass, a general increase in bone density, and an increase in fracture callus bone volume. Myostatin is highly expressed during the first twenty-four hours after fracture, yet nothing is known about its role in fracture repair. We hypothesize that myostatin is a key regulator in the process of bone regeneration, and is a major therapeutic target for enhancement of fracture healing. Pharmacological inhibition of myostatin may therefore improve the regenerative capacity of both muscle and bone.
    • Role of NADPH Oxidase following Traumatic Brain Injury

      Ma, Merry Wenlan; Department of Neuroscience and Regenerative Medicine (5/22/2018)
      Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Despite intense investigation, no neuroprotective agents for TBI have yet translated to the clinic. Recent efforts have focused on identifying potential therapeutic targets that underlie the secondary TBI pathology that evolves minutes to years following the initial injury. Oxidative stress is a key player in this complex cascade of secondary injury mechanisms and prominently contributes to neurodegeneration and neuroinflammation. In addition, the NLRP3 inflammasome, which produces pro-inflammatory signals, can become activated in response to oxidative stress and may exacerbate secondary pathology. NADPH oxidase (NOX) is a unique family of enzymes whose primary function is to produce reactive oxygen species (ROS). Human post-mortem and animal studies have identified elevated NOX2 and NOX4 levels in the injured brain, suggesting that NOX is involved in the pathogenesis of TBI. Our experiments demonstrate that targeting NOX, specifically NOX2 and NOX4, can reduce oxidative stress, attenuate neuroinflammation, reduce lesion size, and promote neuronal survival following TBI. In particular, deletion of NOX2 or inhibition of NOX can attenuate the increased expression and activation of the NLRP3 inflammasome via TXNIP- mediated pathway and decrease the production of pro-inflammatory factors, such as caspase-1 and IL-1β. We also demonstrate the novel findings that deletion of NOX4 can reduce neuronal oxidative damage evidenced by decreased DNA oxidation, lipid peroxidation, and protein nitration in the injured cerebral cortex. Mice lacking NOX4 also showed reduced cell death and neurodegeneration following TBI. Collectively, our results support the notion that targeting NOX enzymes can suppress neuroinflammatory secondary TBI pathology in addition to alleviating oxidative damage following injury. In addition, our inhibitor studies extend the critical window of efficacious TBI treatment, which further supports the pursuit of NOX as therapeutic targets.
    • Role of NEK1 in VHL and Cell Cycle Regulation

      Patil, Mallikarjun; Department of Cellular Biology and Anatomy (2013)
      Nekl is the member of NIMA (Never in mitosis gene A) related protein kinase family that is widely expressed in mammals. Nekl is an essential protein because loss of function in Nekl gene causes polycystic kidney disease in mice, which is similar to ADPKD (Autosomal Dominant Polycystic Kidney Disease) in humans. In Humans Nekl mutations also cause short rib polydactyl syndrome characterized by renal cysts and other developmental defects. At the cellular level Nekl thought to regulate ciliogenesis, centrosome duplication and DNA damage response.Nekl mutations leading to PKD have long been attributed to its role in ciliogenesis. Interestingly, VHL (Von hippel lindau) protein a known tumor suppressor is also involved in ciliogenesis.VHL mutations cause cystic kidney disease and renal clear cell carcinoma. Since Nekl and VHL are involved in ciliogenesis and cystic kidney disease, my overall goal was to investigate if Nekl and VHL are part of common regulatory pathway and also to investigate the role of Nekl in cell cycle regulation. My results indicate that Nekl phosphorylates VHL and this has important role in cilia regulation. Nekl phosphorylates VHL on multiple sites and S168 of VHL a site phosphorylated by Nekl significantly affects its stability. Importantly renal cells expressing S168A VHL that cannot be phosphorylated by Nekl grow cilia that are resistant to serum stimulation and Nocodazole treatment. Surprisingly I also found that Nekl is an essential regulator of S phase. Nekl knockdown in HEK cells blocks cell cycle progression. Further characterization Nekl showed that Nekl is needed for S phase progression and DNA replication. Nekl deficient cells have replication stress and activate cell cycle check point. Nekl loads on to chromatin and this increases during replication stress. We have also identified that Nekl interacts with and affects Ku80 loading on to chromatin. These findings have provided novel insights into the Nekl functions, which help in understanding the pathophysiology and development of polycystic kidney disease in mice and short rib polydactyl syndrome mejawski in humans.
    • Role of Oxidative Stress in High Endothelin Models of Hypertension

      Elmarakby, Ahmed A.; Department of Medicine (2004-07)
      Recent studies have shown that the potent vasoconstrictor peptide endothelin-1 (ET-1) stimulates superoxide production in vivo and in vitro. We hypothesized that ET-1 induced hypertension, at least in part, is due to an increase in oxidative stress. In the initial experiments, we hypothesized that ETA receptor stimulation contributes to the elevated blood pressure and superoxide production in ETB receptor deficient rats as an example of a high endothelin model of hypertension. Experiments were conducted on homozygous {si/si) ETB deficient and wild type {wt) rats fed a high salt diet for three weeks. Separate groups of rats were given normal drinking water or water containing the ETA receptor antagonist, ABT 627. On a normal salt diet, sl/sl rats had a significantly elevated systolic blood pressure (SBP) compared to wt. High salt caused a significant increase in SBP in sl/sl compared with wt rats. ETA receptor blockade decreased SBP in sl/sl rats on high salt without affecting the blood pressure in wt rats. Plasma 8-isoprostane levels, an indirect measure of oxidative stress, were significantly higher in sl/sl rats compared with the wt. ETa receptor blockade significantly attenuated the elevation in plasma 8- isoprostane levels in sl/sl rats'. These findings suggest that ET-1, through the ETA receptor, contributes to salt-induced hypertension and superoxide production in ETB deficient rats. We hypothesized that ET-1 increases superoxide production via the stimulation of the NADPH oxidase system. Chronic ET-1 infused rats were fed a high salt diet and either allowed to drink tap water, water containing the SOD mimetic, tempol, or the NADPH oxidase inhibitor, apocynin, for two weeks. Infusion of ET-1 increased mean arterial pressure (MAP) when compared to baseline values. Neither tempol nor apocynin treatment had any effect on the increase in MAP produced by ET 1. Plasma 8-isoprostane was increased significantly in ET-1 infused rats compared to rats on a high salt diet alone. Both tempol and apocynin treatment significantly attenuated the ET-1 induced increase in plasma 8-isoprostane. These data provide evidence that chronic ET-1 infusion increases vascular NADPH oxidase dependent superoxide production, but does not account for chronic ET-1-induced hypertension. Finally, experiments were performed to determine if increased kinins and/or decreased superoxide attenuates the elevation in blood pressure in chronic Ang II hypertensive rats. Four groups of rats, all given Ang II, were studied and allowed to drink tap water, water containing enalapril, tempol, or both for two weeks. Ang II infusion significantly increased SBP when compared with the baseline. Neither enalapril nor tempol treatment alone was able to attenuate the elevation in SBP. Combined administration of tempol and enalapril prevented the increase in SBP. Plasma 8-isoprostane was elevated significantly in Ang II infused rats when compared with control untreated rats. Tempol treatment alone or tempol plus enalapril significantly attenuated the increase in plasma 8-isoprostane. These studies support the hypothesis that an antioxidant alone is not effective in preventing Ang II hypertension. However, administration of an ACE inhibitor with an antioxidant enhances antioxidant efficiency in preventing Ang II hypertension. Overall, these studies showed that ETA receptor stimulation participates in superoxide production via the stimulation of NADPH oxidase and that antioxidant treatment alone is not sufficient to lower blood pressure in high endothelin models of hypertension.
    • The Role of Phospholipase D2 and Its Interaction with Aquaporin 3 in Primary

      Qin, Haixia; Department of Physiology (2010-04)
      Regulated keratinocyte proliferation and differentiation is necessary for normal skin function. In mouse keratinocytes phospholipase D2 (PLD2) colocalizes with aquaporin 3 (AQP3), probably via a direct protein-protein interaction as seen in Sf9 cells. Since AQP3 can transport glycerol, a substrate of PLD2, we hypothesized that AQP3 and PLD2 function together to form phosphatidylglycerol (PG), a lipid second messenger which inhibits keratinocyte proliferation and promote differentiation. First, adenovirusmediated PLD2 overexpression enhanced keratinocyte proliferation under control conditions and inhibited differentiation induced by a moderately elevated calcium level. However, PG synthesis was inhibited with PLD2 overexpression; this decrease may result from disruption of the endogenous PLD2 and AQP3 interaction, and/or reduced AQP3 activity following overexpression of PLD2. Next, AQP3 was overexpressed. Overexpression of either PLD2 or AQP3 inhibited the activity of transglutaminase (TGase), a marker of keratinocyte differentiation. However, co-overexpression of AQP3 and PLD2 returned TGase activity to control levels, under both control and calciumstimulated conditions. Similarly, PG synthesis was inhibited by either PLD2 or AQP3 overexpression, but PG levels were returned to control values with co-overexpression. These results are consistent with our hypothesis that PG is a differentiation signal: less PG leads to proliferation and inhibition of differentiation. The caveolin-1 scaffolding domain peptide has been found to interact functionally with PLD2 in low-density membrane microdomains. We propose that reduced AQP3 and PLD2 interaction resulting from disruption of lipid rafts by the caveolin-1 scaffolding domain peptide results in less PG synthesis and the inhibition of calcium-induced keratinocyte differentiation. Mouse keratinocytes were treated with cell-permeable caveolin-1 scaffolding domain peptide (CSDP) and cell differentiation was stimulated using a moderately elevated extracellular calcium concentration. The CSDP had no effect itself on PG synthesis, differentiation or proliferation, but it prevented the changes induced by a moderate calcium concentration, whereas a negative control peptide did not. The CSDP altered PLD2 distribution within membrane microdomains, but had little or no effect on AQP3 distribution. Finally, we showed that the CSDP disrupted lipid rafts in cell membranes by itself, while when applied with calcium simultaneously it prevented the changes induced by moderate calcium. We conclude that the CSDP regulates both calcium-inhibited proliferation and -stimulated differentiation, at least in part, through effects on PG production.
    • The role of proinflammatory cytokines on taste function

      Kumarhia, Devaki; Institute of Molecular Medicine and Genetics (2015)
    • Role of Protein Kinase D in Keratinocyte Proliferation and Epidermal Tumorigenesis

      Arun, Senthil N.; Department of Physiology (2010-05)
      Skin is an excellent model to study carcinogenesis as the cells that comprise the outer layer of the skin, the epidermal keratinocytes exhibit an inherent pattern of proliferation and differentiation. Any deviation from this physiological process results in hyperproliferative diseases such as non-melanoma skin cancers and psoriasis. Protein kinase D (PKD) is a serine/threonine kinase that has been implicated in numerous cellular processes. Previous studies performed in our laboratory show that PKD levels are upregulated in human basal cell carcinomas (BCC) and in a neoplastic mouse keratinocyte line, supporting a possible tumorigenic role for PKD in epidermis. In addition, PKD activity appears to be correlated with epidermal keratinocyte proliferation. Thus, compounds that inhibit PKD and protein kinase C (PKC), but not those that inhibit only PKC, stimulate differentiation. Mouse keratinocytes treated with the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), exhibit an initial decrease in PKD activation followed by a recovery. Thus, the biphasic response of PKD activation after TPA treatment seems to mirror the biphasic response of initial differentiation followed by proliferation and tumor promotion seen in TPA-treated keratinocytes and epidermis in vivo, and we hypothesized that PKD may contribute to TPA-induced tumorigenesis. Indeed, we found that the PKC/PKD inhibitor G66976 blocked the increase in DNA specific activity induced by chronic TPA without affecting the initial TPA-elicited differentiation. In contrast, the PKC inhibitor G66983 prevented both the initial induction of differentiation and the subsequent promotion of proliferation in response to TPA. Our results support the idea that PKD may be involved in tumor formation in the epidermis.The biggest risk factor for the development of skin tumors in humans is sun exposure, leading us to investigate the ability of ultraviolet B (UVB) exposure to activate PKD. Our data suggest that irradiation of primary mouse keratinocytes with UVB resulted in activation of PKD in a time- and dose-dependent manner, via ROS generation. In addition, PKD overexpression protected keratinocytes from UVB-induced apoptosis. Collectively, our data suggest that PKD plays an important role in protecting keratinocytes from UVB-induced apoptosis and provide a link between sun exposure, BCC and PKD.
    • The Role of PTP-1B in Vascular Insulin Resistance

      Ketsawatsomkron, Pimonrat; Vascular Biology Center (2008-02)
      Recent studies have suggested that insulin resistance in the vasculature can be linked to cardiovascular complications. However, the mechanism of insulin resistance which occurs in blood vessels is not well understood. Previous studies have shown that Protein Tyrosine Phosphatase -IB (PTP-1B) is a negative regulator of insulin signaling, however, the role of PTP-1B in regulating insulin signaling in the vasculature has never been explored. We hypothesized that PTP-1B plays an important role in vascular insulin resistance both in vitro and in vivo. For in vitro experiments, we utilized the model of angiotensin II (Ang II)-induced insulin resistance in vascular smooth muscle cells (VSMC) and hypothesized that Ang II-induced activation of PTP-1B is the underlying mechanism. Using standard Western techniques, we found that Ang II significantly inhibited insulin-induced phosphorylation of IRS-1 and Akt, downstream members the insulin-induced anti-mitogenic pathway. Furthermore, Ang II enhanced the insulin-induced activation of p42/p44 MAPK, a mitogenic pathway. In addition, we found that PTP-1B is involved in the insulin-induced blockade of Ang II-induced VSMC growth. Finally, we also showed that Ang II induced activation of PTP-1B in VSMC was through a PKA/JAK2 dependent mechanism. Therefore, from these in vitro studies, we conclude that Ang II modulates both anti-mitogenic and mitogenic pathways stimulated by insulin via activation of PTP-1B. For the in vivo studies, we hypothesized that PTP- 1B is an underlying mechanism of vascular insulin resistance in animal models. Experiments were conducted on PTP-1B knockout (PTP-1B KO) mice compared to wild type (WT) mice in different insulin resistant conditions. In high fat feeding induced obesity, we showed that the activation of Akt following insulin stimulation ex vivo was significantly decreased in high fat fed WT mice which was restored by deletion of PTP- 1B. However, the expression of PTP-1B was not different between WT mice on either regular or high fat diet. We concluded that PTP-1B partly plays a role in vascular insulin resistance in high fat fed model. We next examined the roles of PTP-1B and vascular insulin resistance in a new double transgenic obese model. We showed here that the expression of PTP-1B was increased significantly in obese control mice (K^HPTP-IB) compared to lean control mice. Activation of Akt following insulin injection was impaired in aorta of obese KdbHpTP-iB mice and was not restored by deletion of PTP-1B. Therefore, our data suggest that other insulin induced signaling molecules in the aortamay be involved in the regulation of Akt and not PTP-1B. Overall, our studies in this thesis suggest both an in vivo and in vitro contributionof PTP-1B to vascular insulin resistance. The overall goal of the study was to determine the significance of PTP-1B in the development of vascular insulin resistance particularly in vascular smooth muscle cell (VSMC). We hypothesized that PTP-1B plays an important role in vascular insulin resistance both in vitro and in vivo.
    • The Role of RYBP in the Regulation of Apoptosis

      Novak, Rachel Lynn; Georgia Cancer Center (6/5/2014)
      The tumor suppressor Tp53 is the most frequently mutated gene in human cancer. Tp53 encodes a sequence specific transcription factor termed p53 that activates a number of biological programs contributing to tumor suppression, most notably, the promotion of cell cycle arrest and apoptosis. To identify new regulators of p53’s transcriptional activity, we performed a yeast 2-hyrbid screen and have identified Ring 1 YY1 Binding Protein (Rybp) as a novel p53-interacting partner. Consistent with its role as a transcriptional repressor, we have demonstrated that Rybp inhibits p53-mediated transcription. In addition, Rybp forms a trimeric complex with the critical negative regulator of p53, Mdm2. Mdm2 is an E3 ligase that ubiquitinates p53, targeting it for degradation, and expression of Rybp enhances the Mdm2-mediated ubiquitination. To further investigate the role of Rybp in the regulation of endogenous p53 stability we constructed a recombinant adenovirus expressing Rybp (Ad-Rybp). Ad-Rybp infection inhibited the accumulation of p53 and the induction of p53 target genes in response to genotoxic stress. However, interpretation of the results was confounded by Ad-Rybp infection reducing global mRNA levels. Despite inhibition of p53, Ad-Rybp was a powerful inducer of apoptosis, and we investigated this in more detail. Analysis of a panel of tumor cell and untransformed cell types revealed that Ad-Rybp infection specifically induces apoptosis in tumor cells but not in normal diploid cells. Furthermore, at a low multiplicity of infection, Ad-Rybp sensitizes tumor cells to apoptosis in the presence of the death receptor ligands, Tumor Necrosis Factor alpha (TNFα) and TNF related apoptosis inducing ligand (TRAIL). These results suggest that the tumor-specific killing properties of Rybp may be exploited for therapeutic advantage.
    • The role of RYBP in the regulation of apoptosis

      Novak, Rachel; School of Graduate Studies (2008-05)
      The tumor suppressor Tp53 is the most frequently mutated gene in human cancer. Tp53 encodes a sequence specific transcription factor termed p53 that activates a number of biological programs contributing to tumor suppression, most notably, the pro~qtion of cell cycle arrest and apoptosis. ·To identify new regulators of p5 3 's transcriptional activity, we performed a yeast 2-hyrbid screen and have identified Ring 1 YYl Binding Protein (Rybp) as a novel p53-interacting partner. Consistent with· its role as a transcriptional repressor, we have demonstrated that Rybp inhibits p53-mediated transcription. In addition, Rybp forms a trimeric complex with the critical negative regulator of p53, Mdm2. Mdm2 is an E3 ligase that ubiquitinates p53, targeting it for degradation, and expression of Rybp enhances the Mdm2-mediated ubiquitination. To further investigate the role of Rybp in the regulation of endogenous p53 stability we constructed a recombinant adenovirus expressing Rybp (Ad-Rybp ). Ad-Rybp infection inhibited the accumulation of p53 and the induction of p53 target genes in response to genotoxic stress. However, interpretation of the results was confounded by Ad-Rybp infection reducing global mRNA levels. Despite inhibition of p53; Ad-Rybp was a powerful inducer of apoptosis, and we investigated this in more detail. Analysis of a panel of tumor cell and untransformed cell types revealed that Ad-Rybp infection specifically induces apoptosis in tumor cells but not in normal diploid cells. Furthermore, at a low multiplicity of infection, Ad-Rybp sensitizes tumor cells to apoptosis in the presence of the death receptor ligands, Tumor Necrosis Factor alpha (TNFa) and TNF related apoptosis inducing ligand (TRAIL). These results suggest that the tumor-specific killing properties of Rybp may be exploited for therapeutic advantage.