• Neuron-Glia interaction and role of Nrf2 in hyperhomocysteinemic retina

      Navneet, Soumya; Biomedical Sciences (Augusta University, 2019-05)
      Elevated level of the excitatory amino acid homocysteine (Hcy) or hyperhomocysteinemia (Hhcy) has been reported in patients with glaucoma, a disease characterized by increased oxidative stress and retinal ganglion cell (RGC) degeneration. Whether Hhcy is causative or merely a biomarker for glaucoma is not known. Primary RGCs exhibit acute sensitivity to Hcy exposure, while in vivo murine models of Hhcy demonstrate a more modest RGC loss (∼20%) over a period of several months. This differential response to Hhcy in isolated cells and the intact retina suggests the presence a buffering mechanism invoked by the retinal milieu. Oxidative stress has been implicated as a mechanism of Hcy-induced neuronal loss. Owing to the key role of Müller glial cells (MCs) in retinal antioxidant defense we hypothesized that MCs protect RGCs under conditions of Hhcy via the NRF2 antioxidant pathway. Compared to RGCs, MCs were less sensitive to Hcy. Hcy exposure increased oxidative stress and induced apoptosis in RGCs, whereas in MCs Hcy evoked several cytoprotective responses including reduced oxidative stress, increased antioxidant levels and improved mitochondrial function. Hcy upregulated the expression of Nrf2 and several downstream antioxidant targets including glutathione in MCs. To investigate the role of NRF2 in Hcy-induced RGC degeneration, we crossed Nrf2-/- mice with two mouse models of chronic Hhcy (Cbs+/- and Mthfr+/- mice) and generated Cbs+/-Nrf2-/- and Mthfr+/-Nrf2-/- mouse models and analyzed their retinas. Absence of NRF2 reduced inner retinal thickness and visual acuity, accelerated RGC loss and increased gliosis in Hhcy mice. To understand the role of Müller glial specific NRF2 in RGC survival, we established an ex-vivo indirect co-culture system using primary RGCs and MCs. Apoptosis induced by Hcy exposure in primary RGCs were abrogated when the RGCs were co-cultured with wild type (WT) MCs but not with Nrf2-/- MCs. Hhcy induced robust mitochondrial and glycolytic response in WT MCs, but not in Nrf2-/- MCs. Altogether, the in vivo and in vitro data here suggest that the deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of retinal antioxidant response mechanism.
    • Neuronal and Astroglial Injury and Recovery from Stroke-Induced Depolarizations

      Risher, William Christopher; Department of Neurosurgery (2010-12)
    • Neurotransmitter Gases as Modulators of GnRH and the Preovulatory LH Surge

      Lamar, Charisee; Institute of Molecular Medicine and Genetics (1998-04)
      Until recently nitric oxide (NO) and carbon monoxide (CO) were viewed only as toxic substances. However, there has been a substantial amount of evidence in the past decade that has redefined these gaseous molecules as physiological messengers. Along these lines, NO and CO are now recognized as modulators of immunological defense, vasodilation, endocrine signaling, and neurotransmission (1-8). As neurotransmitters, NO and CO are unique when compared to classical neurotransmitters. For instance, unlike all other classical neurotransmitters NO and CO are, 1) lipophilic gases with short half lives, 2) not stored in synaptic vesicles, 3) their effects are not mediated through classic receptor proteins - rather their effects result from NO and CO binding to the heme moiety of heme-proteins such as guanylate cyclase and cyclooxygenase, and 4) their effects are terminated by diffusion from target tissues ( 1,2,5,7,9). Production of NO and CO relies on the activity of the enzymes nitric oxide synthase (NOS) and heme oxygenase (HO), respectively. NOS uses the substrate Larginine to generate NO (2, 10), while HO uses the substrate heme to generate CO (9,11-16). NOS exists as three isoforms, macrophage NOS (mNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (17-21). HO also exists as three isoforms, heme oxygenase-1 (HO-1), heme oxygenase-2 (HO-2), and heme oxygenase-3 (HO-3) HO-1 is inducible, while HO-2 and HO-3 are constitutive enzymes; however, HO-3 is currently viewed as a poor heme catalyst (12,13,22). There is a growing body of evidence that suggests that NO and CO regulate hypothalamic function. For example, recent studies have shown that the hypothalamus produces significant quantities of NO, primarily through the actions of nNOS (23,24). Likewise, the hypothalamus possesses one of the largest production rates of CO in the brain (25,26). That NO and CO can regulate neurohormone release from the hypothalamus is evidenced by findings demonstrating that NO and CO regulate corticotropin-releasing hormone (CRH) (27-33), vasopressin (29-31,34,35), and oxytocin secretion (29,36). With respect to reproductive function, numerous studies (23,37-45) have provided evidence for a significant role of NO in the control of the hypothalamic releasing factor, gonadotropin-releasing hormone (GnRH). For instance, it has been demonstrated (37,46,47) that NO neurons are located in close proximity to GnRH generating cells bodies in the hypothalamus and thus strategically located to exert regulatory effects over GnRH neurons. That NO can exert regulatory effects on GnRH neurons was demonstrated in studies where exogenously applied NO markedly stimulated GnRH release from male hypothalamic fragments (1,48,49) and immortalized GnRH (GT1-7) cells in vitro (1,48). A physiological role for NO in the steroid-induced luteinizing hormone (LH) surge has been suggested based on studies in which the LH surge was attenuated by the administration of NOS inhibitors (43,44) or NOS antisense oligonucleotides (38) No studies, however, have reported nNOS mRNA, protein, or NOS activity levels in the hypothalamus during the LH surge so as to verify that an increased NO tone actually occurs during this critical time. To address this deficit, Aim 1 of this study was designed to examine NO tone on proestrus in the cycling female rat-the day the natural preovulatory LH surge occurs. Since heme oxygenase, the enzyme that produces CO from heme molecule metabolism, is located in the hypothalamus (2,11,12,14,15), and CO production in the hypothalamus is one of the highest in the brain (25,26), it is conceivable that CO could play a role in regulating GnRH secretion. This possibility has not been investigated and thus studies on this issue appear warranted. Therefore, Aims 2-4 of this study were designed to assess the possible role of CO in the control of GnRH and LH secretion.
    • Neurotransmitter gases as modulators of GnRH and the preovulatory LH surge

      Lamar, Charisee; School of Graduate Studies (1998-04)
    • Neurotransmitter receptor binding and protein phosphorylation in the rabbit iris smooth muscle

      Taft, William C; Department of Cell and Molecular Biology (1982-06)
    • Neurotransmitters and the phospholipid effect in rabbit iris muscle

      Owen, Mary Pruitt; Department of Cell and Molecular Biology (1976-06)
    • NEUROVASCULAR DEGENERATION FOLLOWING RETINAL ISCHEMIA REPERFUSION INJURY: ROLE OF ARGINASE 2

      Shosha, Esraa; Department of Cellular Biology and Anatomy (2017)
      Ischemic retinopathies such as retinopathy of prematurity, central retinal artery occlusion and diabetic retinopathy are leading causes of visual impairment and blindness. These pathologies share common features of oxidative stress, activation of inflammatory pathways and neurovascular damage. There is no clinically effective treatment for these conditions because the underlying mechanisms are still not fully understood. In the current study, we used a mouse model of retinal ischemia reperfusion (I/R) insult to explore the underlying mechanisms of neurovascular degeneration in ischemic retinopathies. The arginase enzyme utilizes the L-arginine amino acid for the production of L-ornithine and urea. Here, we investigated the role of the mitochondrial arginase isoform, arginase 2 (A2) in retinal I/R induced neurovascular injury. We found that retinal I/R induced neurovascular degeneration, superoxide and nitrotyrosine formation, glial activation, cell death by necroptosis and impairment of inner retinal function in wild type (WT) mice. A2 homozygous deletion (A2-/-) significantly protected against the neurovascular degeneration after retinal I/R. That was attributed to decreased oxidative stress and glial activation. A2 deletion protected against I/R induced retinal function impairment. Using Optical coherence tomography (OCT), we evaluated the retinal structure in live animals and found that A2-/- retinas showed a more preserved structure and less retinal detachment. To investigate the underlying mechanisms of A2 induced vascular damage after I/R, we used an in vitro model of oxygen glucose deprivation/ reperfusion (OGD/R) in bovine retinal endothelial cells (BRECs). Analysis of oxidative metabolism showed impaired mitochondrial function. We also found an increase in dynamin elated protein 1 (Drp1), a mitochondrial fission marker. Mitochondria labeling studies showed fragmented mitochondria after OGD/R. Arginase inhibition reduced mitochondrial fragmentation in OGD/R insult. This dissertation presents A2 as a new therapeutic target in reducing neurovascular damage in ischemic retinopathies.
    • Neutrophil function studies with respect to antibiotic tolerant staphylococcus aureus and patients with recurring infection

      Raynor, Robert H; Depatment of Cell and Molecular Biology (1980-08)
      Polymorphonuclear neutrophils (PMNs) from s~ven patients who experien<;: ed recurring staphylococcal infections, were tested in order to establish if measurements of phagocytic and bactericidal capacity would reveal a functional cause for the repeated episodes of disease. Using a microprocedure developed for this 'purpose, the results for six of these patients were found to be comparable to normal values. PMNs from one patient, however, showed a deficiency in their ability to both phagocytize and kill s. aureus. This defect could not be attributed to serum deficiencies or ascribed to any previously defined class of nel.ltrophil function. In other studies, six clinical isolates of Staphylococcus aureus were compared for their relative susceptibilities to the killing effects of oxacillin. Three of the strains had minimum bactericidal concentrations which were >10 times the minimum bacteriostatic concentration for this antibiotic and were designated tolerant (Tol+). The other st;r-ains had minimum bactericidal concentrations which were comparable to the minimum bacteriostatic concentration (Tol-). Lysis curves of these strains revealed that the Tal+ strains exhibited a diminished rate of lysis when inhibited by oxacillin. This reduced rate of lysis was reflected also in a reduced rate of viability loss when the cells were exposed to oxacillin. Cells of each phenotype, previously labeled with [ 14C]Glycerol, secreted radioactivity when inhibited by oxacillin. However,. the Tal+ strains rele.ased over twice as much labe 1 as the Talstrains. The behavior of 60 to 65%_ of the labeled material released by. inhibited cells during both sodium dodecyl sulfate gel electrophoresis and Sepharose 6B chromatography corresponded to that of lipoteichoic acid. When the major component of secreted material was added to oxacillin-inhibited Tol- strains, an inhibition of the lytic response was observed. These results suggest that oxacillin tolerance in S. aureus could be related to the enhanced secretion of an autolysin inhibitor, such as lipoteichoic acid. Several investigators have recently reported an increase in the severity of infections due to Tol+ strains. Since the enhanced'excretion of LTA by Tol+ staphylococci is the only knownphenotypJ.c difference between these two strains, the effect of this molecule on neutrophil function was measured using the microprocedure. The addition of LTA to the incubation mix·ture res1..1lted in a dose dependent inhibition of phagocytosis •. The decrease~ uptake of. S. aureus in the presence of LTA was accompanied by a corresponding increase in the number of organisms surviving in the presence of the phagocytes. Additional studies suggested that LTA interferes with the process' of opsonization, prior to engulfment.
    • Neutrophil-mediated endothelial dysfunction

      Chen, Xilin; Department of Pharmacology and Toxicology (1992-08)
    • A New Method For Analyzing 1:N Matched Case Control Studies With Incomplete Data

      Jin, Chan; Department of Biostatisctics and Epidemiology (5/8/2017)
      1:n matched case-control studies are commonly used to evaluate the association between the exposure to a risk factor and a disease, where one case is matched to up till n controls. The odds ratio is typically used to quantify such association. Difficulties in estimating the true odds ratio arise, when the exposure status is unknown for at least one individual in a group. In the case where the exposure status is known for all individuals in a group, the true odds ratio is estimated as the ratio of the counts in the discordant cells of the observed two-by-two table. In the case where all data are independent, the odds ratio is estimated using the cross-product ratio from the observed table. Conditional logistic regression estimates are used for incomplete matching data. In this dissertation we suggest a simple method for estimating the odds ratio when the sample consists of a combination of paired and unpaired observations, with 1:n matching. This method uses a weighted average of the odds ratio calculations described above. This dissertation compares the new method to existing methods via simulation.
    • Nitric Oxide Synthase Regulation in Inner Medullary Collecting Duct Cells

      Cai, Zheqing; Department of Internal Medicine (2001-06)
      Nitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that renal tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin-1 (ET-1), express caveolin-1 and protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoform(s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOS 1 was significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET-1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOS 1; furthermore, the ETA receptor antagonist, A 127722, rather than the ETB receptor antagonist, A 192621. inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. express caveolin-1, but none of the NOS isoforms appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS I is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 pM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of NOS 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cnr for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD cells. Therefore, in IMCD cells, NOS1 expression is up-regulated by ET-1 through activation of the ETA receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.
    • Nitric oxide synthase regulation in inner medullary collecting duct cells

      Cai, Zheqing; School of Graduate Studies (2001-06)
      Nitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that ren_al tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin:-1 (ET-1), express caveolin-1 artd protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoforrri.( s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary-collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOSl was . significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET .;.1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOSl; furthermore, the ETA receptor antagonist, A127722, rather than the ETB receptor antagonist, A1926_21,- · inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also express caveolin-1, but none of the NOS isofonns appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting ·that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS 1 is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 μM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of Nos·· 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cm2 for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD . cells. Therefore, in IMCD cells, NOS 1 expression is up-regulated by ET-1 through activation of the ET A receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.
    • Nitroxyl anion as a novel relaxant molecule in the rat pudendal artery and metformin as treatment for angiotensin II-induced erectile dysfunction

      Labazi, Hicham; Department of Physiology (2012)
      Cardiovascular diseases, which are the leading cause of illness and death in the United States, frequently share similar risk factors (hypertension, smoking, aging, etc…) as erectile dysfunction (ED). Hypertension is an important risk factor for both heart failure and ED; hypertension and ED are closely intertwined diseases, which have vascular and endothelial dysfunction as a common cause. Recently it has been shown that ED is an independent predictor of cardiovascular diseases (CVD). Thus, studying mechanisms of erectile function and ED will be of great importance for developing treatments for ED, as well as reducing the burden of CVD. In this dissertation, the mechanisms of angiotensin II (AngII)-induced ED was investigated. In addition, we also investigated the effect of metformin on erectile function in an AngII-hypertensive model of ED. We hypothesized that AngII infusion for 4 weeks results in ED and treatment with metformin improves erectile function in the AngII-infused rats. We observed that AngII infusion resulted in ED, which was accompanied by an increased contraction and decreased relaxation response of the corpus cavernosum and pudendal arteries. Furthermore, it was observed that ERK1/2 activation contributes to ED in the AngIIhypertensive model of ED. Treatment with metformin restored erectile function in AngIIhypertensive rats, with a reversal of the increased contractility and decreased relaxation seen in both the corpus cavernosum and pudendal arteries of the hypertensive rats. Our data suggest that metformin may have potential therapeutic effects in ED, independent of its anti-diabetic effects. Additionally we investigated the contribution of nitroxyl anion (HNO), a NO congener, to erectile function. We observed that the endothelium mediated relaxation in pudendal arteries was partially mediated through HNO, and that this relaxation was soluble guanylyl cyclase-dependent (sGC), resulting in activation of voltage-dependent potassium channels (KV +) and large conductance calcium-activated potassium channel (BKCa). The identification of this novel pathway will enhance our understanding of erectile function and possibly allow for development of therapeutic agents for the treatment of ED.
    • NLRP3 Inflammasome-Mediated Uncoupling of Hippocampal Vasoneuronal Communications in Diabetes: Relevance to Cognitive Impairment and Stroke

      Ward, Rebecca; Department of Neuroscience and Regenerative Medicine (5/22/2018)
      Diabetes is a prevalent chronic disease that affects over 29 million individuals in the United States and 422 million people worldwide as of 2017. Given the high mortality and morbidity associated with diabetes due to its complications including retinopathy, chronic kidney disease, peripheral neuropathy, heart disease and stroke, this increase in incidence of diabetes creates many clinical, social and economic problems. A silent, but unrecognized complication of diabetes is cognitive impairment, which ranges from mild cognitive impairment to dementia. The increased risk and incidence of stroke amplifies these cognitive deficits. The objectives of this dissertation were to 1) determine the role and mechanism(s) by which diabetes worsens cognitive decline and 2) determine the extent and mechanism by which NLRP3 activation contributes to poor cognitive function after stroke in diabetes. To investigate these objectives, feeding rats a high fat diet and administering a low dose of streptozotocin was used as a clinically relevant diet-enhanced model of diabetes. Stroke was induced through either transient MCAO (60 or 90 min) or embolic MCAO. Embolic stroke caused more severe hippocampal neurovascular injury, microglial activation and cognitive decline in diabetes as compared to stroke induced by a shorter 60 min suture occlusion of the MCA. Diabetic females were more sensitive to ischemic injury than males. Furthermore, hippocampal vascularization patterns at baseline and after ischemic injury differed in males and females and despite these sex differences in the extent and patterns of hippocampal neurovascular injury, diabetes worsened cognitive outcomes in both sexes. Collectively, these first studies provide a preclinical foundation for future studies addressing cognitive impairment in diabetes in both sexes. NLRP3 inflammasome, which cleaves IL-1β and IL-18 into their active forms, was upregulated in diabetes and amplified following ischemia. Inhibition of the NLRP3 inflammasome with MCC950, a specific small molecule inhibitor of NLRP3 activation, improved post-stroke cognition, reduced hippocampal cell death, was associated with less leaky vasculature, and blunted chronic inflammation in the hippocampus after 90-min MCAO. MCC950 did not seem to provide neuroprotection to the neuron through the mBDNF, but did reduce cell death after hypoxia/reoxygenation in vitro. These results are the first to provide essential data showing that MCC950 has the potential to become a therapeutic agent to prevent neurovascular remodeling and worsened cognitive decline in diabetic patients following stroke. Collectively, this work may provide a piece of the puzzle explaining how diabetes leads to cognitive impairment and worsens outcome following acute ischemic injury, and it provides a potential therapeutic target to treat cognitive impairment after stroke, especially in diabetic patients.
    • Notch3 Signaling Mediates Heterotypic Cell Interactions During Blood Vessel Formation

      Liu, Hua; Vascular Biology Center (2010-11)
      Blood vessel formation is essential for embryogenesis, wound healing, menstruation, and pregnancy [1, 2]. While much emphasis has been placed on understanding the initial event of endothelial-tube formation, relatively little attention has been paid to the interactions of endothelial cells and the surrounding mural cells (pericytes, smooth muscle cells and fibroblasts). Increasing evidence suggests that the communication of endothelial cells and mural cells is crucial for the assembly, subsequent maturation, and stabilization of blood vessels [3-5]. Abnormal interactions between these two cell types have been implicated in many pathological conditions, including tumor angiogenesis, diabetic microangiopathy, tissue calcification and stroke. However, the molecules mediating the heterotypic interaction are still largely unknown. Our previous studies have shown that in a three-dimensional (3-D) angiogenesis assay, mural cells enhance blood vessel formation and directly interact with endothelial cells [6]. During this process, Notch3 is one gene that is strongly induced in mural cells upon coculture with endothelial cells [6]. Notch3, the causative gene of the neurovascular disorder CADASIL [7], belongs to an evolutionarily conserved family of transmembrane receptors that are known to govern cell fate determination in diverse cell types [8]. Given that Notch receptors and ligands are expressed on both endothelial and mural cells and - 2 - Notch3 is upregulated in mural cells by coculturing with endothelial cells, it is reasonable to assume that the Notch3 receptor might regulate the association of endothelial and mural cells through receptor-ligand interaction during blood vessel formation. The goal of my thesis is to investigate how Notch3 gene expression is regulated in mural cells by endothelial cells and whether the Notch3 receptor is involved in the communication between endothelial and mural cells during blood vessel formation. To achieve these goals, three aims were proposed: Specific Aim 1: To define how Notch3 expression in mural cells is upregulated by endothelial cells. Specific Aim 2: To determine if endothelial cell-induced Notch3 expression is critical for mural cell differentiation. Specific Aim 3: To determine whether Notch3 expression in mural cells modulates blood vessel formation under both physiological and pathological conditions.
    • A Novel Function of ADP-Ribosylation Factor 1 in Prostate Cancer Cell Proliferation through Activating the Mitogen-Activated Protein Kinase Pathway

      Davis, Jason E.; Department of Pharmacology and Toxicology (8/23/2016)
      The enhanced activation of the mitogen-activated protein kinase (MAPK) Raf-MEK-ERK1/2 pathway directly correlates with the growth, androgen-independence, and poor prognosis of prostate cancer. However, the underlying molecular mechanisms remain poorly understood. Here, we have demonstrated that ADP-ribosylation factor 1 (ARF1), a Ras-like small GTPase, was highly expressed in human prostate cancer cells and tissues. In addition, ARF1 was markedly activated in prostate cancer cells. More interestingly, oncogenic G protein-coupled receptors (GPCRs) strongly activated ARF1 and the activation was mediated through Gβγ subunits. These data indicate that GPCRs and heterotrimeric G proteins are the upstream activators of ARF1 in prostate cancer cells. Next, we determined the role of ARF1 in the MAPK activation and proliferation in prostate cancer cells. Lentiviral-mediated overexpression of ARF1 remarkably enhanced, whereas shRNA-mediated depletion of ARF1 dramatically reduced ERK1/2 activation in prostate cancer cells. In addition, disruption of both the Golgi localization of ARF1 and the Golgi structure substantially attenuated ERK1/2 activation in prostate cancer cells. In parallel with their effects on the MAPK activation, ARF1 overexpression greatly enhanced and ARF1 knockdown inhibited the proliferation of prostate cancer cells. These data suggest that ARF1, by its ability to activate the MAPK pathway likely at the Golgi, controls prostate cancer cell proliferation. We then investigated the consequence of pharmacologically inhibiting ARF1 activation. Small molecule inhibitors including brefeldin A, golgicide A, and Exo2 that specifically target Golgi-localized ARF1 markedly reduced both ERK1/2 activation and proliferation in prostate cancer cells. These results further indicate an important role of ARF1 activation in regulating the MAPK pathway and prostate cancer cell proliferation. Altogether, our data suggest a possible GPCR-G-ARF1-MAPK signaling pathway, which may be responsible for the hyperactivation of the MAPK ERK1/2 in prostate cancer and contributes to prostate cancer progression. Our results also imply a novel approach for prostate cancer therapy by targeting ARF1 activation
    • Novel Nitric Oxide Synthase-Dependent Mechanism of Vasorelaxation in Small Arteries from Hypertensive Rats

      Kang, Kyu-Tae; Department of Medicine (2007-10)
      Endothelial dysfunction in hypertension is associated with impaired endothelium-dependent vasorelaxation, which is consistently observed in conduit vessels. However, the controversial observation of either impaired or intact vasorelaxation of small resistance arteries from hypertensive animals suggests that the mechanism(s) of endothelium-dependent vasorelaxation in small resistance arteries may be different from that observed in conduit vessels under hypertensive condition. Vasorelaxation in small resistance arteries is mediated via multiple pathways including nitric oxide synthase (NOS)-, cyclooxygenase (COX)-, and endothelium-derived hyperpolarizing factor (EDHF)-mediated pathway. Therefore, the overall goal of these studies was to determine the mechanism(s) involving vasorelaxation of small arteries from hypertensive rats. For these studies, normotensive (NORM), angiotensin II-infused (ANG), high salt (HS), ANG high salt (ANG/HS), placebo, and deoxycorticosterone acetate-salt (DOCA) rats were studied. The studies with pharmacological blockade of each pathway demonstrated that the NOS-dependent component was increased to maintain acetylcholine (ACh)-induced vasorelaxation in small mesenteric arteries from hypertensive rats. Furthermore, increased NOS-dependent pathway appears to compensate for the dysfunctional Ca2+-activated K+ channel-sensitive EDHF pathway in small mesenteric arteries from ANG compared to NORM. These results led us to design further experiments to test the hypothesis that both NO and H2O2 serve as NOS-dependent mediators to maintain vasorelaxation in small mesenteric arteries from hypertensive rats. In small arteries from ANG, ACh increased NOS-dependent cGMP production. ACh also increased NOS3 phosphorylation at Ser 633 and decreased phosphorylation at Thr 495. While, NOS3 phosphorylation at Ser 1177 was impaired in response to ACh in ANG, which was accompanied by reduced basal and a less extended ACh-stimulated cGMP production in ANG compared to NORM. To investigate the alteration of signal transduction pathways related to impaired NOS3 phosphorylation at Ser 1177 in response to ACh, Akt phosphorylation at Ser 473 and VASP phosphorylation at Ser 239 were tested. These pathways were not changed by ACh in the small mesenteric arteries from ANG. Our results indicate that the NO/cGMP signaling is present in response to ACh in small mesenteric arteries from ANG, however this signaling pathway-mediating vasorelaxation may be facilitated via neither Akt nor PKG. On the other hand, ACh stimulated L-NAME-sensitive H2O2 production in small mesenteric arteries from ANG, but not NORM. H2O2 induced vasorelaxation and catalase blunted ACh-mediated vasorelaxation in small mesenteric arteries from ANG. Reduced BH4/BH2 ratio was observed in small mesenteric arteries from ANG compared to NORM, which might be one of the mechanisms of NOS-mediated H2O2 production. Antioxidant enzyme capacity was also determined in small mesenteric arteries from ANG and NORM. Total superoxide dismutase (SOD) activity and protein expression of CuZn SOD and ecSOD were reduced in ANG compared to NORM, while Mn SOD expression was comparable between groups. Interestingly, both activity and expression of catalase were reduced in ANG compared to NORM, whereas GPx activity and expression were not changed. These results indicate that reduced catalase activity and expression may contribute to the augmentation of H2O2 in small mesenteric arteries from ANG, whereas reduced SOD does not greatly influence the H2O2 production in both basal and ACh-stimulated condition. In conclusion, the NOS pathway appears to be the primary endothelium-derived relaxing factor (EDRF) pathway in small mesenteric arteries from experimental animal models of hypertension. The increased dependence on the NOS pathway in ACh-induced vasorelaxation is mediated by both NOS-derived NO/cGMP signaling and NOS-mediated H2O2.
    • Novel Peroxisomal Localization of Sirtuin 3 and its Biological Implications

      Padia, Ravi N.; Department of Biochemistry and Molecular Biology (2014-04)
      Post translational modifications (PTMs) such as phosphorylation, acetylation, ubiquitination, methylation, SUMOylation etc. serve to add another level of complexity and functional diversity to the proteome. These PTMs serve to generate a distinct signal which can then regulate the functionality of the protein harboring one or more of these PTMs and hence ultimately regulating the associated signaling/biological pathway. Some of the ways in which PTMs can bring about the change in the functionality of proteome is by influencing the sub-cellular localization of proteins, protein-protein, protein-DNA, protein-RNA interactions, activity of enzymes, protein turnover, oligomerization of proteins etc. Further, a combination of two or more PTMs can bring about another layer of regulation where presence of a particular combination of PTMs is obligatory to elicit the regulatory message (Yang and Seto 2008a).
    • A Novel RNA Aptamer-Based Targeted Therapy for Ovarian Cancer with PRKCI Gene Amplification

      Rehmani, Hina Salam; Biomedical Sciences (Augusta University, 08-2019)
      Ranked fifth in cancer death among women, ovarian cancer cure rates have remained low over the past two decades due to unsuccessful detection of early-stage disease, stagnant methodologies of treatment, and high relapse rates. Stage IV invasive epithelial ovarian cancer patients have a meager 17% relative 5-year survival rate and around 70% of patients relapse within the first two years of diagnosis. Accounting for more deaths than any other cancer of the female reproductive system, it is paramount to strategically offer ovarian cancer patients targeted therapies to improve outcome. Ovarian cancer contains a host of copy number aberrations (CNA) that can lead to the silencing or amplification of tumor suppressor genes, oncogenes, or non-coding RNAs that modify the expression of genes. Clinically, gene amplifications have prognostic and diagnostic usefulness as they can be a mechanism to promote tumorigenesis and/or attainment of drug resistance. Protein kinase C iota (PKCiota), a cytoplasmic serine-threonine protein kinase, is highly amplified and overexpressed within the 3q26 amplicon in ovarian cancer and is identified as an oncogene in multiple cancers. However, selectively targeting the iota isoform with small molecule inhibitors is challenging since it shares a 72% overall homology with another atypical PKC isoform, zeta. We found that small-molecule inhibitors currently being investigated do not offer selective inhibition nor specificity in ovarian cancer cell lines. We hypothesize that PRKCI amplification offers a unique opportunity to stratify patients into different risk categories and that specifically targeting PRKCI using a siRNA-aptamer can offer therapeutic benefits by impairing ovarian cancer cell tumorigenesis. We expect this approach to provide potent and selective anti-tumor activity compared to targeting using other pharmaceutical approaches. To test this hypothesis, we identified ovarian cancer cell lines with PRKCI gene amplification, identified a pattern of onco-addiction specificity in cell lines with amplification, and created an RNA-based aptamer that efficiently becomes internalized in ovarian cancer cells and mediates PRKCI mRNA silencing. In conclusion, we have identified a rationale to target, specifically, the PKCiota gene amplification in ovarian cancer and our RNA-based aptamer prevents ovarian tumorigenesis both in vitro and in vivo, opening a door for future therapies.