ORAL PRESENTATIONSKhaled Moussawi Adnan Al-Ayoubi Linda Heffernan-Stroud
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POSTER PRESENTATIONSDanielle Clark Michael DeFee Joseph Palatinus Rebecca Weber
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ACUTE CROSSTALK BETWEEN RETINOID AND TGFβ SIGNALING PATHWAYS
Loretta L. Hoover, Elizabeth Burton, Kimberly M. Sauls, Bonnie A. Brooks, Christopher Daniel and
Steven W. Kubalak
The molecular events controlling cardiac development are of critical importance for the eventual development of medical therapies for congenital heart diseases. It is known that transforming growth factor (TGF)-β and retinoid signaling, two pathways important in cardiovascular development, regulate each other on a long-term timescale, secondary to transcriptional and translational events. Previous studies by the Kubalak Lab show germline loss of retinoid X receptor (RXR)-α results in increased TGFβ2 in the midgestational heart, particularly in the outflow tract (OFT). We originally hypothesized that increased TGFβ2 leads to abnormally high levels of canonical TGFβ-mediated events that are causal for OFT defects seen in this model. To test this, we first examined the levels of TGFβ’s most immediate downstream signal, SXS motif phosphorylated Smad2 (pSmad2), in the RXRα knockout (RXRα-/-) heart. Expecting to see increased pSmad2 in the knockout we were surprised to see less nuclear-localized and less abundant pSmad2 in OFT cushion mesenchymal cells. This unexpected finding led to further studies which suggest that acute and direct interactions occur between retinoid and TGFβ signaling. We have found that retinoid treatment, which causes a pharmacologic knockdown of cytosolic RXRα, leads to increased pSmad2 accumulation under conditions of TGFβ stimulation. Similarly, TGFβ treatment of RXRα-/- mouse embryonic fibroblasts (MEFs) and trypsin-dispersed hearts leads to increased pSmad2 levels when compared to those seen in wildtype (WT) cells. We have found that RXRα interacts with Smad2 and propose that it regulates activities of Smad2 by controlling its nuclear uptake and consequently, its dephosphorylation, which occurs in the nucleus. We hypothesize that the increased TGFβ2 seen in RXRα-/- mice is result of loss of negative feedback inhibition on TGFβ2 production, which is normally controlled by transcriptionally active (i.e. nuclear) pSmad2 and pSmad3. We propose this increased TGFβ2 likely signals non-canonically, (i.e. via p38 MAP kinase) to yield elevated apoptosis which is at least partially responsible for the OFT defects seen in RXRα-/- mice. We have also determined that RXRα-/- MEFs are less migratory than WT MEFs, a finding which could lead to the re-analysis of the cellular events of OFT remodeling in RXRα-/- hearts.
MECHANISMS OF TGF-?–INDUCED ENDOTHELIAL PAXILLIN DEPHOSPHORYLATION
Jarrett E. Walsh 1 , M. Rita Young 2,3
1Department of Microbiology & Immunology, Medical University of South Carolina; 2Research Services, Ralph H. Johnson VAMC; 3Departments of Otolaryngology and Medicine, Medical University of South Carolina
The regulation of angiogenesis has become an exciting prospect for many future therapies in diseases as wide-ranging as macular degeneration and solid tumors of many types. A single therapy has yet been derived that can effectively eliminate the diversity of signals that contribute to this highly regulated process. In fact, the best current therapies are those that are targeting signals broadly. To that end, understanding the signaling pathways influencing cellular migration in response to angiogenic factors could be used to create new therapies with broader coverage. The work presented here investigates the role of the angiogenic growth factor TGF ß on protein phosphatase regulation and subsequent changes in the phosphorylation of the focal adhesion protein, paxillin. Phosphorylation regulation of paxillin is crucial in its localization to focal adhesions and coordination of signals to other focal adhesion members. While regulation by the serine/threonine phosphatase PP-2A has been demonstrated, its regulation by PP-1 has not. This work demonstrates the novel dephosphorylation of paxillin by PP-1, in vitro and in vivo . These studies provide the basis for future studies to examine the specific function of these phosphorylations in angiogenesis- related cellular motility.
A noradrenergic lesion exacerbates memory loss and neurodegeneration in a mouse model of Down syndrome
Jason Lockrow 1 , Heather Boger 1 , Greg Gerhardt 2 , and Ann-Charlotte Granholm 1,3
Individuals with Down syndrome (DS) acquire an Alzheimer's-like dementia earlier and at greater rates than the population at large, and exhibit Alzheimer's disease (AD) neuropathology with near uniformity. However, the biological mechanisms for these alterations have not been discovered and there is currently no effective therapy for either DS- or AD-related dementia. Partial trisomy 16 (Ts65Dn) mice provide a useful model for many of the degenerative changes which occur with age in DS individuals, including cognitive deficits, neuroinflammation, and degeneration of basal forebrain cholinergic neurons. Loss of noradrenergic locus coeruleus (LC) neurons is an early event in AD and in DS individuals, and may contribute to neuroinflammation and neuronal degeneration. We report that Ts65Dn mice exhibit progressive loss of NE phenotype in LC neurons. In order to determine whether the LC degeneration contributes to memory loss and neurodegeneration in Ts65Dn mice, we administered the specific noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 2 doses of 50 mg/kg) to Ts65Dn mice at four months of age. At eight months of age, Ts65Dn mice treated with DSP-4 exhibited an 80% reduction in hippocampal NE levels, coupled with a marked increase in hippocampal neuroinflammation. Noradrenergic depletion also resulted in accelerated cholinergic neuron degeneration and a further impairment of memory function in Ts65Dn mice. In contrast, DSP-4 had minimal effects on their normosomic littermates. These data suggest that noradrenergic degeneration may play a role in the progressive memory loss, neuroinflammation, and cholinergic loss occurring in DS individuals.
Antagonism of the Angiotensin 2 Type 1 Receptor with Losartan or SII Reduces Bradykinin-mediated Calcium Responsiveness of the Bradykinin Type 2 Receptor
Parker C. Wilson, Louis M. Luttrell, Ayad A. Jaffa
The risk factors and mechanisms that contribute to progression of diabetic nephropathy (DN) are still undefined . Renal and vascular expression of the bradykinin type 2 receptor (B2R) is upregulated (3-fold) in the early stages of streptozotocin-induced type 1 diabetes, and remains elevated at 6 months. B2R activation promotes renal injury by increasing the expression of a family of pro-fibrotic growth factors and extracellular matrix components, whereas genetic deletion of the B2R slows the progression of DN. Calcium response following BK stimulation is an upstream signal in the mitogenic cascade. Our laboratory has shown that antagonism of the AT1R receptor decreases BK-mediated calcium responsiveness in VSMC. The small molecule antagonist, losartan, and the ß -arrestin selective peptide agonist, SII, both reduce the amplitude of a BK-mediated calcium signal. Heterodimerization acts as a mechanism for diversifying the cellular response to physiological ligands, and can have both synergistic and antagonistic effects. We hypothesize that ligand binding to the AT1R induces a conformational change in the proportion of B2R that is heterodimerized with the AT1R. The reduction in BK-mediated calcium response following SII, or losartan pretreatment indicates that as much as 40% of endogenous B2R exists as B2-AT1 heterodimers.
CD44 Membrane Dynamics in Metastatic Breast Cancer Cells
Daniel Grass, Mark G. Slomiany, and Bryan P. Toole
The plasma membrane, a dynamically heterogeneous interface between the extracellular matrix and the cytosol, aids in the turnover of surface proteins and associated lipids. Evidence now indicates that the plasma membrane is composed of numerous heterogeneous compartments termed ‘lipid rafts' or ‘membrane domains'. These rafts are dynamic silos for signaling complexes and represent a major compartment for internalization of surface proteins. Rafts associate transiently with the underlying cytoskeleton and raft-associated proteins, including proteins that act as receptors for the extracellular matrix. Certain pools of CD44, the major receptor for the matrix molecule, Hyaluronan (HA), associate in raft domains; this association is important for both turnover of matrix HA and surface CD44 via internalization. Currently, the only modification known to promote CD44 raft-association is palmitoylation. CD44 complexes with many proteins, including: growth factor receptors, multi-drug efflux pumps and lactate transporters. In addition, CD44 has been shown to associate with the immunoglobulin superfamily molecule, EMMPRIN, on the cell surface. EMMPRIN is a multifunctional protein that participates in matrix metalloproteinase production, inflammation, therapy-resistance, lactate efflux, and HA synthesis. The mechanisms that regulate CD44-EMMPRIN interactions are unclear, but appear to depend on intact HA-CD44 interactions. A molecule that associates with both CD44 and EMMPRIN is Annexin II (AnxA2). AnxA2 is a multifunctional protein that binds negatively-charged phospholipids in a Ca2+ -dependent manner and mediates surface protein trafficking and endocytosis. Employing metastatic breast cancer cells, we have demonstrated that CD44 and EMMPRIN are internalized when constitutive HA-CD44 interactions are disrupted with HA oligosaccharides (o-HA), which displace high affinity multi-valent high molecular weight HA-CD44 interactions with low affinity mono-valent HA-CD44 interactions. In addition, these cancer cells demonstrate surface colocalization of CD44, EMMPRIN, AnxA2, and Actin; these interactions are disrupted when cells are depleted of cholesterol or if HA-CD44 interactions are perturbed. AxnA2-EMMPRIN colocalization is also seen in perinuclear compartments, indicating a potential role of AnxA2 in transporting EMMPRIN to the cell surface. It is clear that a complex association may exist between CD44, EMMPRIN, AnxA2, Actin, and lipid rafts. These studies show great promise in targeting breast cancer stem-like cells, which are enriched in CD44 and are therapy-resistant.
THE INVOLVEMENT OF CALPAIN IN CD4 + T HELPER CELL BIAS
Jonathan Butler*, Naren Banik † , Craig Beeson ‡
*Department of Molecular and Cellular Biology and Pathobiology,† Department of Neuroscience/Neurology Division, ‡Department of Pharmaceutical Sciences, Medical University of South Carolina , Charleston , South Carolina
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that affects approximately 400,000 people in the United States every year and results in neurological deficits. Inflammatory events associated with MS include activation of auto-reactive T cells and migration of these and other immune cells into the CNS, leading to a coordinated attack upon oligodendrocytes and demyelination. Most current therapies only treat the symptoms of disease, not the cause, which is still in large part unknown. Therefore, the identification of the etiology of this complex disease and the development of new therapies is of great importance. Improvement in clinical signs has occurred in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, with the use of calpain inhibitors. Calpain is a calcium-mediated neutral protease involved in many normal physiological as well as pathological events. We hypothesize that calpain plays a role in several cellular processes involving T cells including subtype bias. This hypothesis was tested by exposing myelin basic specific T cells to various concentrations of calpain inhibitors and testing the proliferation, cytokine profile and assessing the protein levels and localization of various signaling pathways involved with subtype bias. The results indicate that calpain is a negative regulator of the Th2 cell type.
NOVEL PKC ISOFORMS, PKCε AND δ, REGULATE PI3K/MTOR COMPLEX-2-DEPENDENT AKT ACTIVATION INDEPENDENT OF C-RAF/ERK PATHWAY
Phillip Moschella, and Dhandapani Kuppuswamy
Department of Medicine: Division of Cardiology, MUSC Charleston SC
Activation of mammalian target of rapamycin (mTOR) has been shown to be critical during hypertrophic growth of the myocardium. Recently mTOR has been shown to act through two unique complexes, mTOR complexed with Raptor (mTOR complex 1 or mTORC1) and mTOR complexed to Rictor (mTOR complex 2 or mTORC2) onto S6K1 and AKT respectively. Selective rapamycin blockade of mTORC1 has been shown to blunt and even regress TAC-induced cardiac hypertrophic growth. We have previously characterized the nPKC isoforms on mTORC1-dependent activation of S6K1 downstream of both PI3K-dependent/independent pathways in adult cardiomyocytes. As PI3K-medaited AKT S473 phosphorylation, is known to contribute to physiologic hypertrophy, possibly downstream of mTORC2, we sought to explore the role of the nPKCs. AKT phosphorylation following phenylephrine (PE) or insulin (Ins) treatment in cardiomyocytes was significantly reduced by wortmannin blockade of PI3K or following adenoviral expression of either dominant negative PKCε or δ (dnPKCε, and dnPKCδ). However, AKT S473 phosphorylation was not blocked by either Gö6976 blockade of the classical PKC isoforms or MEK inhibition using either U0126 or dominant negative c-RAF expression. Treatment with rapamycin disrupted mTORC1 assembly but also augmented AKT S473 activation. Rapamycin augmentation of AKT activation was also blocked by dnPKCε, and dnPKCδ adenoviral expression. Recipricol coimmunoprecipitation experiments show PKCε, and Rictor in a unique complex that also contains AKT and mTOR during Ins stimulation. We also show increased S473 phosphorlyation of AKT bound to Rictor during rapamycin potentiation of Ins stimulation. These data establish i) the necessity of the novel PKC isoforms during PI3K dependent AKT activation ii) disruption of mTORC1 and increased S473 AKT bound to mTORC2 during rapamycin treatment, and iii) a unique signaling complex containing Rictor, PKCε, mTOR and AKT during insulin stimulation that regulates AKT activation in adult cardiomyocytes. Given the role of AKT in physiologic growth, these studies indicate that the novel PKCs may play a pivotal role during growth and protection upstream of AKT activation in hypertrophying cardiomyocytes.
ROLE FOR SPHINGOSINE KINASE 1 ONCOGENIC TRANSFORMATION
Christopher R. Gault, Ashley J. Snider, Alexa K. Orr, and Lina M. Obeid
Ralph H. Johnson Veterans Administration Research Service and the Department of Medicine & the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina
Sphingosine kinase 1 (SK1) is an important enzyme involved in the production of the bioactive lipid sphingosine-1-phosphate (S1P) and also acts as an important regulator of sphingolipid degradation. SK1 is overexpressed in many forms of cancer and promotes cellular growth and angiogenesis both in vitro and in xenograft models of cancer progression. Despite this, the contribution SK1 provides in cancer progression is still unclear. It is generally accepted that human cancers arise through inactivating mutations in tumor suppressor genes or by activating mutations in tumor promoting genes. One of the best characterized mutations found in several forms of human cancer is a point mutation in the Ras oncogene which disrupts its GTPase activity and leads to stimulation of the MEK/ERK pathway. Since SK1 activity and subcellular localization has been shown to be regulated by ERK, we wished to investigate the effect of oncogenic Ras on activity of SK1, sphingolipid metabolism, and tumor growth. Here we show that K-RasG12V expression in mouse embryonic fibroblasts (MEFs) increases SK1 activity in both cytosolic and membrane fractions. In addition, K-RasG12V transformed SK1 -/- MEFs are unable to form colonies on soft agar suggesting that SK1 is also necessary for anchorage independent growth. Treatment of A549, a human lung adenocarcinoma cell line carrying the oncogenic K-RasG12V allele, with SK1 RNAi lead to an increase in cellular ceramide, and a decrease in the cellular proliferation rate. A549 cells were unable to form colonies in soft agar in the presence of a SK inhibitor suggesting that SK activity is essential for anchorage independent growth. To investigate the role that SK1 plays in a mouse model of early lung tumor formation, we crossed the KRasLA2 mouse with the Sphk1 -/- mouse. KRasLA2 Sphk1 -/- mice showed no difference in lung adenoma formation as compared with their KRasLA2 Sphk1 +/+ littermates. Together these data suggest that SK1 is not essential for K-RasG12V induced lung adenoma formation. A role for SK1 in later stages of tumor progression is currently being explored. Future studies will investigate how SK1 activity contributes to anchorage independent growth and Ras oncogenesis.
*This work could not have been completed without the help of the MUSC Lipidomics Core Facility, support from the NIH National Research Service Award Individual Fellowship F30 HL093991-01A1, support from the Hollings Cancer Center, the Abney Cancer Foundation, and support from the program project grant P01 NIH/NCI CA097132.
ABSENCE OF SPHINGOSINE KINASE 1 INHIBITS JOINT EROSIONS AND LOCAL INFLAMMATION IN TNF-ALPHA INDUCED ARTHRITIS
DeAnna A. Baker*, Lina M. Obeid, MD† §, Gary Gilkeson, MD
‡ §* MD/PhD Student, Department of Microbiology and Immunology, MUSC, Charleston, SC 29425, † Department of Medicine, MUSC, Charleston, SC 29425 ‡ Department of Medicine, Division of Rheumatology and Immunology, MUSC, Charleston, SC 29425, §Department of Veterans Affairs, Ralph H. Johnson Medical Center, 109 Bee St., Charleston, SC 29401
Background/Purpose: Sphingolipids are constituents of the plasma membrane. Variations in their cellular levels lead to alterations of cellular functions. Sphingosine 1 phosphate (S1P) in vitro is required for TNFα induced production of COX-2 and PGE2. Additionally, stimulation with TNFα and S1P together leads to higher production of COX-2 and PGE2 than either alone. Both sphingosine kinase (SphK) 1 and 2 are upregulated in the rheumatoid synovium compared to osteoarthritis synovium. S1P1R (EDG1), one of the receptors for S1P, is also upregulated in the joints of rheumatoid arthritis patients. Fibroblast-like synoviocytes (FLS), found in the synovial lining, proliferate in response to proinflammatory cytokines and produce COX-2 and PGE2 in response to TNFα and S1P. We hypothesized that S1P, induced by TNFα, is a critical mediator of inflammation and joint damage in the rheumatoid joint. The following experiments were performed to test this hypothesis.
Methods: Transgenic hTNFα mice were crossed with SphK1-/- mice and genotyped by PCR. Arthritis in these mice develops independent of antigen, T cells or B cells. The mice were observed weekly for disease activity, while CT images and microarray analysis were used to evaluate disease activity in the joint and evaluate genetic profiles respectively. Mouse synoviocytes were isolated from the knee joints of WT and SphK1-/- mice, cultured, and stimulated with TNFα. OA and RA human synoviocytes were cultured, and stimulated with hTNFα.
Results: hTNF/Sphk1-/- mice (n=15) had significantly decreased clinical joint disease compared to hTNF/SphK1+/+ mice (n=18), with average arthritis scores of 1+/-0.5 vs. 5+/- 1.2 respectively at 5 months (based on joint swelling and deformity). An erosion Index, measured quantitatively from 3D CT images of the ankles was significantly decreased in hTNF/SphK1-/- mice at 4 and 5 months, with a 2 fold decrease in erosions in hTNF/SphK1-/- vs. hTNF/SphK1+/+ mice. Microarray analysis of ankle joint synovium, with RT-PCR confirmation, demonstrated significant modulation of a cluster of genes regulated by SOCS3 in hTNF/SphK1-/- mice compared to hTNF/SphK1+/+ mice. Synoviocytes from SphK1-/- mice, stimulated with TNFα, produced significantly less IL-6 and PGE2 than synoviocytes from WT mice. Similarly, human RA synoviocytes stimulated with TNFα and treated with a specific SphK inhibitor produced significantly less IL-6 and PGE2 than cells treated with TNFα alone.
Conclusion: Genetic deletion of SphK1 significantly decreased the severity of hTNFα induced arthritis, decreased erosions and led to upregulation of SOCS3 with impact on expression of SOCS3 related genes. Lack of SphK1 resulted in decreased PGE2 and IL6 production by mouse and human synoviocytes in response to TNFα. These data indicate that S1P plays a key role in TNFα induced joint inflammation and erosions and is a potential target for therapeutic intervention in inflammatory arthritis.
HEAT SHOCK INHIBITS CISPLATIN-INDUCED HAIR CELL DEATH IN THE ADULT MOUSE UTRICLE
Tiffany Baker, Mona Taleb, Shimon Francis, Carlene Brandon, Keely Morris, Fu Shing Lee and Lisa Cunningham
MUSC Department of Pathology and Laboratory Medicine, Charleston , SC
Cisplatin is used to treat a wide variety of cancers; however, a significant proportion of patients receiving cisplatin experience permanent hearing loss. The ototoxic side effects of cisplatin result in part from damage to sensory hair cells, leading to the apoptotic death of those cells. We previously showed that upregulation of heat shock proteins (Hsps) inhibits aminoglycoside-induced hair cell death, and that Hsp70 accounts for most of the protective effect of heat shock (Taleb et al., 2008). We have now examined whether Hsp induction can inhibit cisplatin-induced hair cell death. Adult mouse utricles were cultured at 37°C overnight and then were either heat shocked (43°C for 30 min) or maintained at 37°C (control utricles). Six hours after heat shock, utricles were exposed to either control media or media containing cisplatin at a variety of doses for 24 hours. Our results indicate a protective effect of heat shock against cisplatin-induced hair cell death across the dose-response curve (2-way ANOVA, F6,160 = 5.778, p<0.0001). Our data also indicate that Hsp70, the most inducible and widely-conserved heat shock protein, is necessary for the protective effect of heat shock against cisplatin-induced hair cell death. In addition, Hsp70 overexpression is protective against cisplatin-induced hair cell death. Furthermore, pharmacological induction of another Hsp, Hsp32, also protected hair cells from from cisplatin-induced hair cell death.
THE EFFECTS OF PEPA, AN AMPAKINE, ON BEHAVIORAL ACTIVITY AND EXPRESSION OF BDNF IN RATS FOLLOWING AN ACUTE ADMINISTRATION OF AMPHETAMINE
Laura Briggs, Sarah Eisenstein, Timothy Whitfield, Adrian Gomez, Jacqueline McGinty
Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and new synaptic growth through its activation of signaling pathways, including the MAP kinase, Jun kinase and PI-3 kinase cascades. By enhancing long term potentiation in projections from the prefrontal cortex (PFC) to the nucleus accumbens (NAc), BDNF infusions within the prefrontal cortex (PFC) reduce cocaine reinforcement and the reinstatement of cocaine-seeking behavior. BDNF expression has been shown to be upregulated by intracranial ampakine administration, a class of compounds that potentiate glutamatergic AMPA receptors. This study aims to determine whether a systemic administration of PEPA, an ampakine, can alter BDNF expression and activity within the prefrontal cortex, the nucleus accumbens and striatum when coupled with amphetamine. Rats were administered an acute dose of amphetamine (1 m/kg, i.p.) followed by an acute administration of PEPA (1mg/kg, i.p.) or saline vehicle. Behavioral activity was recorded for 3 hours after which rats were sacrificed.
In situ and ELISA data in progress
POLY-N-ACETYLGLUCOSAMINE NANOFIBERS FROM A MARINE DIATOM PROMOTE WOUND HEALING AND DEFENSIN EXPRESSION VIA AN AKT1/ETS1-DEPENDENT PATHWAY
Haley Buff1 , Elizabeth Perkins1 Aiguo Zhang3 Juanita Eldridge1 , Marina Demcheva2 , Arun Seth3,
John Vournakis2 and Robin C. Muise-Helmericks1
1Department of Cell Biology and Anatomy and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA, 2Marine Polymer Technologies, Inc., Danvers, MA 01923 and 3Sunnybrook Research Institute, University of Toronto, Ontario, Canada, M5G 1G6
Recent findings show that treatment of cutaneous wounds with poly-N-acetyl-glucosamine nanofibers (pGlcNAc/Taliderm), a novel polysaccharide material derived from a marine diatom, results in an increased kinetics of wound healing that can be attributed, in part, by a marked increase in angiogenesis. Our published data suggests that treatment of primary endothelial cells (EC) with this nanofiber results in a marked increase in cell migration, which is due to an integrin-dependent up-regulation of the Ets1 transcription factor. We show that pGlcNAc stimulation of Ets1 results from the activation of Akt1 by these nanofibers. Nanofiber treatment results in marked increases in the expression of genes involved in cellular recruitment, such as IL-1 (a known Ets1 target), VEGF and several defensins (ß3, a1, a4, and a5), small anti-microbial peptides recently shown to act as chemo attractants. Both pharmacological inhibition of the PI3K/Akt1 pathway and Akt1 knockdown using shRNAs results in decreased expression of these chemotactic factors. Akt1 null mice exhibit a delayed wound healing phenotype that is partially rescued by Taliderm nanofibers. Taliderm treated wounds also show an increase in defensin expression that is Akt1 dependent. Taken together our findings suggest a central role of the Akt1?Ets1 pathway in the regulation of cutaneous wound healing by pGlcNAc nanofibers and support the use of these fibers as a novel and effective method for enhancing wound healing.
TRANSCRIPTIONAL CONTROL OF ACID CERAMIDASE IN PROSTATE CANCER CELLS
Joseph C. Cheng, Wenjun Zhou, Lorianne S. Turner, Thomas H. Beckham,
Marion Sewer, Xiang Liu,
James S. Norris
In recent years, studies of the regulation, aberration, and manipulation of biologically active sphingolipids, particularly ceramide and sphingosine-1-phosphate, have contributed to our knowledge of the biology of cancer. Our lab recently uncovered a role for the ceramide-metabolizing enzyme, acid ceramidase, in the development of prostate cancer cell resistance to ionizing radiation: the study demonstrated that gamma-irradiation of prostate cancer cells induced acid ceramidase expression, ceramide metabolism, sphingosine-1-phosphate production (Mahdy and Cheng et al. 2009 Molecular Therapy 17:430). Targeted inhibition of acid ceramidase both reduced ceramide metabolism and rendered prostate cancer cells susceptible to previously sublethal doses of ionizing radiation. Identification of the mechanisms controlling acid ceramidase expression and activity would benefit our understanding of this new target for cancer therapy. In the present study, we demonstrate that administration of C6 -ceramide also induces acid ceramidase expression. Collectively, our results lead us to the hypothesis that cell stressors that induce long-chain ceramide generation, such as ionizing radiation and exogenous, short-chain ceramide, up-regulate acid ceramidase expression via a common pathway based on intracellular ceramide accumulation. We tested this hypothesis with luciferase reporter constructs bearing variable-length acid ceramidase-promoter sequences. Exposure of human embryonic kidney 293 cells and PPC-1 prostate cancer cells to ceramide-generating cell stress stimuli demonstrate differential expression of the acid ceramidase-promoter-reporters based on sequence length. Promoter sequence analyses reveal candidate cis- acting transcription factors that bind consensus motifs within sequence fragments that contribute most to ceramide-regulated acid ceramidase expression.
THE USE OF FLUORESCENT PROBES TO REVEAL THE ROLE OF ER-IPLA2gIN OXIDANT-INDUCED LIPID PEROXIDATION, CA2+ RELEASE, AND NECROTIC CELL DEATH
Andre C. Eaddy and Rick G. Schnellmann, Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina, USA, 29401
Oxidant injury and necrotic cell death are major components of ischemia/reperfusion-induced acute kidney injury (AKI). We have previously shown that inhibition of Ca2+-independent phospholipase A2g (iPLA2γ), present in both endoplasmic reticulum (ER) and mitochondria, potentiates oxidant-induced lipid peroxidation and necrotic cell death in renal proximal tubule cells (RPTC). We hypothesized that ER-iPLA2γprevents and/or mediates repair of oxidant-induced ER lipid damage and, thereby, blocks ER Ca2+ release, an important trigger of necrotic cell death. We investigated the role of iPLA2γ in oxidant-induced lipid peroxidation and Ca2+ release in isolated kidney cortex microsomes and the relationship between ER-iPLA2γ and ER Ca2+ release in oxidant-induced necrotic cell death in RPTC. Microsomes loaded with cis-parinaric acid to monitor lipid peroxidation were exposed to the model oxidant tert-butyl hydroperoxide (TBHP) in the presence and absence of iPLA2γ inhibition using bromoenol lactone (BEL). TBHP induced lipid peroxidation and iPLA2γ inhibition potentiated the lipid peroxidation. We developed an assay to monitor ER Ca2+ release using a membrane impermeant Ca2+ indicator, fluo-4. After ATP-dependent Ca2+ loading, microsomes were treated with TBHP in the presence and absence of BEL. TBHP caused ER Ca2+ release after the initiation of lipid peroxidation, and iPLA2γinhibition potentiated the ER Ca2+ release. In RPTC, depletion of ER Ca2+ stores with thapsigargin, an ER Ca2+ pump inhibitor, prior to TBHP exposure reduced RPTC necrotic cell death and completely blocked the potentiation of TBHP-induced necrotic cell death by BEL. Confocal imaging of RPTC ER Ca2+ stores and mitochondrial membrane potential using Fluo-5F AM, and tetramethyl rhodamine methyl ester, respectively, revealed oxidant-induced ER Ca2+ release occurs prior to mitochondrial depolarization and cell death. We suggest that ER-iPLA2γ protects renal cells by reducing and/or repairing oxidant-induced ER lipid peroxidation, preventing subsequent membrane disruption and necrotic cell death triggered by ER Ca2+ release.
NIH NIEHS F30 ES015964-01
MAP KINASE PHOSPHATASE-1 NEGATIVELY REGULATES OSTEOCLASTOGENESIS
Alfred C. Griffin, Carlos Rossa, Keith L. Kirkwood
Department of Craniofacial Biology, College of Dental Medicine, Medical University of South Carolina
Negative regulation of MAPK activity is provided by MAPK phosphatases (MKPs) that dephosphorylate MAPK proteins. MKP-1 is a founding member of this family of phosphatases that has been well documented to negatively regulate the innate immune response by dephosphorylation p38, ERK, and JNK. Previous work has shown that mice lacking this protein show enhanced levels of pro-inflammatory cytokines in response to periodontal pathogenic endotoxin with accelerated bone loss and increased levels of osteoclasts in vivo. Thus, we explored the hypothesis that MKP-1 may attenuate osteoclast differentiation. Bone marrow macrophages (BMMφ) from femurs of MKP-1-/- and MKP-1+/+ mice were collected and allowed to adhere to a 100mm plate in the presence of mM-CSF (10ng/ml). The non-adherent cells were removed 24 hrs post initial culture and treated with mM-CSF (10 ug/ml) and mRANKL (100ug/ml). Primary BMMφ will be cultured at 2 × 104 cells/well in α-MEM containing 10% serum for 8 days with the medium replaced every 3 days. Following cell culture, cells are cytochemically stained for tartrate-resistant acid phosphatase (TRAP), using a commercially available kit (Sigma). TRAP-positive multinucleated cells containing ≥ 3 nuclei were used to define and identify osteoclasts and enumerated by light microscopy. All experiments have been conducted to date in triplicate on 2 separate experiments. Images of cultured cells are digitally captured using a Nikon TS100 inverted scope and Nikon 5.1 megapixel camera. Preliminary data show that the BMMφ from MKP-1-/- mice differentiate into more numerous osteoclasts per well after 8 days than do MKP-1+/+ mice. In addition, it was observed that these osteoclasts had almost 2 more nuclei/osteoclast, suggesting that they may exhibit accelerated function (bone resorption). Future experiments will look into the mechanistic involvement of p38 and MKP-1 in macrophage fusion and steoclast maturation.
MAGNETIC RESONANCE SPECTROSCOPY REVEALS THAT NAA LEVELS MAY BE AN EARLY INDICATOR OF NEURODEVELOPMENTAL OUTCOME IN NEONATES WITH CHORIOAMNIONITIS
C. Bryce Johnson,1 Denise M. Mulvihill,2,3 Karen C. Lee,3 Paul S. Morgan,1,2 Lakshmi D. Katikaneni,3 and Dorothea D. Jenkins3
1.
Department of Neuroscience’s Center for Advanced Imaging Research, MUSC, Charleston, SC; 2. Department of Radiology, MUSC, Charleston, SC; 3. Department of Pediatrics, MUSC, Charleston, SC
Development of novel therapeutics for chorioamnionitis is hindered by the length of time required to assess efficacy of treatment since predictive neurological exams cannot be administered until 1 y of age. Therefore, we tested whether a correlation exists between neonatal brain metabolites, measured using magnetic resonance spectroscopy (MRS), and 1 y neurodevelopmental outcomes, determined by the Bayley Scales of Infant Development (BSID). For this study, 10 neonates with chorioamnionitis underwent MRS, which was performed in both the basal ganglia (BG) and watershed areas (WS) with echo times (TE) of 32 and 270 ms. Using the LC Model data analysis software, mI, NAA, Cho, and Cr were reliably quantified (%SD < 20) and ratios obtained (mI/Cho, mI/Cr, mI/NAA, NAA/Cho, NAA/Cr, and Cho/Cr). The correlation between metabolite ratio in each brain area at both TE's and average BSID composite scores (cognitive, language, and motor) was determined using the Pearson product-moment correlation coefficient (r). The analysis revealed that the BG mI/NAA ratio obtained at TE = 32 ms and the WS NAA/Cr ratio at TE = 270 were most strongly correlated to BSID (r = -0.904, n=6 and r=0.785, n=10, respectively; both p<0.01). All other ratios containing NAA obtained at TE = 270 ms showed a significant correlation , regardless of brain area (p<0.05). These results reveal that NAA levels may prognosticate poor neurodevelopmental outcome in eonates afflicted with chorioamnionitis.
KSHV modulation of xCT and RNS affects murine macrophage susceptibility to infection and survival
Patricia Kearney, Zhiqiang Qin, Chris Parsons
The gammaherpesvirus known as the Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS)—the most common cancer arising in the setting of HIV infection and a common cancer arising in patients receiving organ transplants. Existing cytotoxic chemotherapies for KS are relatively ineffective and difficult to tolerate for these patients, necessitating the development of safer and more effective therapies. KSHV-infected macrophages and their less differentiated monocyte counterparts are found in KS tumors and within the peripheral circulation of patients with KS, but whether KSHV regulates monocyte/macrophage function to promote KS pathogenesis is unknown. Macrophages generate reactive nitrogen species (RNS) which have been implicated in KS pathogenesis, and preliminary in vitro experiments in our laboratory indicate that KSHV or KSHV-encoded microRNA (KSHV miRNA) in isolation induce macrophage secretion of RNS. Interestingly, inhibition of inducible nitric oxide synthase (iNOS), an important mediator of RNS production, significantly reduces macrophage permissivity to KSHV infection. Moreover, both KSHV and KSHV miRNA protect macrophages from exogenous RNS-mediated cell death. Finally, we have found that KSHV miRNA up-regulate the expression of xCT, a membrane-bound amino acid transporter that serves as a fusion/entry receptor for KSHV and maintains intracellular glutathione homeostasis in an environment of RNS. Based on these data, we hypothesize that KSHV miRNA regulate interdependent pathways associated with viral entry, RNS secretion, and cell survival through their up-regulation of xCT.
ROLE OF REACTIVE INTERMEDIATES IN LUPUS NEPHRITIS
Ahmad K Mashmoushi1, Ann F Hofbauer1, 2, Peter Komlosi1, Phillip Darwin Bell1, Jim C Oates1, 2
1Medicine, Medical University of South Carolina, Charleston, SC
2Medicine, Ralph H. Johnson VA Medical Center, Charleston, SC
Purpose of Study: Pharmacological inhibition of inducible nitric oxide synthase (iNOS) in murine lupus nephritis (LN) decreased disease activity and markers of reactive nitrogen and oxygen intermediates (RNI and ROI), yet genetic modulation of iNOS had little effect on murine LN. These results suggest that additional enzyme targets for NOS inhibitors may be important for disease progression. Little is known about enzyme source and cellular origin of reactive intermediates (RIs) in LN glomeruli. Il-6 is associated with proliferative LN. The purpose of this study is to determine the cellular origin of iNOS-mediated RI production in LN glomeruli and the role of RI in mesangial cell IL6 production.
Methods Used: Glomeruli were isolated from 3 groups of mice at 16 weeks of age: MRL-MpJFASlpr(MRL/lpr) mice either lacking functional NOS2 gene or with a functional NOS2 gene and MRL/MPJ controls. Glomeruli were then examined by 3-color confocal microscopy after staining with fluorescent probes for NO (DAF-FM DA) and reactive oxygen (RO)(CM-H2DCF-DA). Immortalized MRL/lpr mesangial cells were stimulated with LPS(100ng/ml)/IFγ(10U/ml) in the presence of increasing concentrations of S-nitroso-N-acetylpenicillamine (SNAP 50,100,250,500 uM). After overnight culture, supernatants were analyzed for IL6 by ELISA.
Summary of Results: Levels of fluorescence indicating NO and ROIs production were greatest in glomeruli from wt mice, and significantly lower levels were observed in NOS2 knockout and control glomeruli. Fluorescence intensity in wt glomeruli was localized to the capillary loops region. Stimulated mesangial cell IL6 production was inhibited more by low (50 mM) than high (500 mM) concentrations of the SNAP(see table).
Conclusions: Glomerular iNOS activity is a major source of RI production in this model of murine LN. Podocytes and/or endothelial cells appear to be a source of RI production rather than mesangial cells in murine LN. Given that lower concentrations of NO had a greater effect on IL6 production, paracrine NO production may act through multiple mechanisms to modify IL6 production in mesangial cells.
Effect of NO donor
(SNAP) on IL6 production in MRL/lpr mesangial cells stimulated with LPS/IFγ
| SNAP(uM) | 0 | 50 | 100 | 250 | 500 |
| IL6(pg/ml) | 116.387 | 47.27 | 56.51 | 65.61 | 79.12 |
KNOCKDOWN OF CERAMIDE SYNTHASES REVEALS COMPLEX INTER-REGULATION OF SPHINGOLIPID METABOLISM
Thomas D. Mullen1 and Lina M. Obeid 1,2
1Departments of Medicine and Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425; 2Division of General Internal Medicine, Ralph H. Johnson Veterans Affairs Hospital, Charleston, South Carolina 29403
De novo ceramide synthesis is necessary for sphingolipid homeostasis in metazoans and has been demonstrated to regulate a wide array of biological phenomena. Ceramide synthases (CerSes) are crucial enzymes in the de novo synthetic pathway; these enzymes catalyze the N -acylation of free dihydrosphingosine to form dihydroceramide which is subsequently converted to ceramide by dihydroceramide desaturase. Alternatively, free sphingosine produced from the breakdown of complex sphingolipids can be re-acylated by these same enzymes to form ceramide. Little is known about the biochemistry and biology of these enzymes, but it is known that each mammalian CerS has a unique preference for its acyl-CoA substrate. However, it is not known how each CerS contributes to the overall sphingolipid homeostasis of the cell. In this study, we have investigated the contribution of each CerS to the regulation of sphingolipid levels including dihydroceramides, ceramides, sphingomyelins, and simple glycosphingolipids. Using the breast adenocarcinoma cell line MCF-7, we targeted siRNA to each human CerS (CerS1-6) and analyzed sphingolipid mass changes by liquid chromatography coupled with mass spectrometry (LC/MS). According to our data, knockdown of individual CerS has profound effects on cellular sphingolipid levels. Some of these changes are predicted by the acyl-chain length specificities that have been previously described: knockdown of CerS2 resulted in decreases in very long-chain ceramides and sphingomyelins and knockdown of CerS6 resulted in decreases in C16-ceramide. However, other changes were not expected. For example, loss of CerS2 results in an up-regulation of CerS4, CerS5, and CerS6 with a consequent up-regulation in long-chain ceramide production. Intriguingly, the increased ceramide appears to be preferentially converted into hexosylceramides and lactosylceramides. Overall, this study demonstrates the complex inter-regulation of CerS and their ability to control not only the acyl chain lengths of ceramide but also the cellular levels of multiple sphingolipid species.
Support: NIH/NIEHS 5T32ES012878, NIH/NIEHS 1F30 ES016975-01, NIH R01AG016583, and NIH C06 RR018823.
CERAMIDE ACTIVATION OF PROTEIN PHOSPHATASES: IN VITRO RESULTS AND A PHYSIOLOGICAL ROLE IN MEDIATING INHIBITION OF p38 SIGNALING IN RESPONSE TO INFLAMMATORY CYTOKINES
David Perry, Patrick Roddy, Vinodh Rajagopalan, Kazuyuki Kitatani, Russell Jenkins, Mohamad El-Osta, Yusuf Hannun
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC
Ceramide has been implicated in many biological processes but elucidating an exact mechanism has not been as clear as other second messengers (DAG, cAMP). Previous studies have shown that ceramide can activate protein phosphatases 1 and 2A. In the search to further understand this activation an Okadaic Acid-insensitive protein phosphatase was found to be responsive to ceramide. This protein phosphatase was identified by MS/MS to be protein phosphatase 2Cγ, which is in a different phosphatase family than PP1 or 2A. It shares little sequence homology and exists in the cell predominantly as a monomer unlike PP1 and PP2A. In an effort to apply these in vitro findings to a relevant biological pathway the SAPK pathway has appeared to be a potential candidate for the model of ceramide activated protein phosphatases. The family of SAPK includes JNK and p38 of the MAPKs. A variety of phosphatases are known to regulate SAPKs including MKP, PP2A, PP1, and PP2C. Once the kinase signal is stimulated, one or more of these phosphatases are responsible for turning off the kinase cascade through a currently unknown mechanism. Since PP1, PP2A, and PP2C are activated by ceramide, a logical hypothesis is that coordinated and transient ceramide production binds and activates one of these phosphatases to turn off the kinase cascade in a manner to regulate the intensity and duration of the signal. Previous studies have shown that fumonisin B1, an inhibitor of ceramide synthases, augments the phosphorylation of p38 upon PMA treatment but not of ERK or JNK. Conversely the addition of exogenous ceramide was able to decrease the phosphorylation of p38 to PMA and TNFα. More specifically, inhibiting glucosylceramidase, augmented the phosphorylation of p38 in response to PMA. These findings are noteworthy for a few reasons: p38 signaling is known to be involved in inflammation and cancer, and loss of glucosylceramidase is the etiology of Gaucher disease (a lysosomal storage disease associated with increased inflammation, neurodegeneration, and increased rates of multiple myeloma.) Therefore understanding the mechanism of ceramide regulation of p38 signaling holds large potential for future therapeutic benefit.
A NEW POTENTIAL TREATMENT AGAINST CRYPTOCOCCOSIS
Ryan Rhome and Maurizio Del Poeta
Cryptococcus neoformans (Cn) is a significant fungal pathogen that can cause lethal meningoencephalitis in humans. Current treatments are limited by toxic side effects and drug resistance. In previous studies, we found that a Cn strain (∆gcs1) lacking the glycosphingolipid glucosylceramide (GlcCer) does not cause meningoencephalitis and, as a result, mice infected with ∆gcs1 do not die. These studies indicate that GlcCer is critical for infection. Therefore, we hypothesized that decreasing GlcCer levels in Cn will result in a defect in pathogenicity. To assess this hypothesis, we used Cerezyme (Cz), a recombinant enzyme that metabolizes GlcCer which is used as a treatment for Gaucher’s disease. Cz treatment was shown to reduce fungal GlcCer in vitro, in live Cn, and in Cn growing inside host lung. Interestingly, Cz treatment reduced in vitro growth of Cn and, in mouse models of infection, increased the lifespan of he infected mice. These studies suggest that targeting novel factors like GlcCer ay represent a new avenue of antifungal treatment.
ROLES OF SPHINGOLIPID SIGNALING IN THE REGULATION OF CHEMOTHERAPY-INDUCED CELL DEATH IN HUMAN HEAD AND NECK SQUAMOUS CELL CARCINOMAS (HNSCC): FROM BENCH TO BED SIDE
David Sentelle1,2, Can E. Senkal1,2, Sarah Sprout1,2, Uzair Chaudhary2, Paul O'Brien2, Larry Afrin2,
Keisuke Shirai2, Terry A. Day2,3, Boyd Gillespie2,3, Joshua Hornig2,3,Anand Sharma4, Christina Wilhoit2,
Sahar Saddoughi1,2, Yusuf A. Hannun1,2, and Besim Ogretmen1,2
1Department of Biochemistry and Molecular Biology, 2Hollings Cancer Center , 3Department of Otolaryngology and Head and Neck Surgery, and 4Department of Radiation Oncology
In this study, endogenous long chain ceramides were measured in 45 human head and neck squamous cell carcinoma (HNSCC) and 10 non-squamous head and neck carcinoma (NSHNC) tumor tissues. These groups were compared to their paired adjacent non-cancerous tissue counterparts, by liquid chromatography/mass spectroscopy (LC/MS). Interestingly, only C18-ceramide, the main product of ceramide synthase 1 (CerS1), was down regulated significantly in the majority of the patients. Importantly, decreased C18-ceramide in HNSCC tumor tissues significantly associated with increased incidences of lymphovascular invasion and nodal metastasis. In another line of investigation, quantitative isobologram studies showed that treatment with gemcitabine (GMZ) and doxorubicin (DOX), known inducers of ceramide generation, in combination, supra-additively inhibited the growth of human UM-SCC-22A cells in situ . Furthermore, treatment with GMZ/DOX also resulted in a significant suppression of HNSCC tumor growth in SCID mice bearing the UM-SCC-22A xenografts. Interestingly, LC/MS analysis showed that only the levels of C18-ceramide, were elevated significantly (about 7-fold) in these HNSCC tumors after GMZ/DOX treatment as compared to controls. In functional studies, knock-down of CerS1 using siRNA prevented, while tetracycline-inducible expression of wt-CerS1 enhanced GMZ/DOX-mediated apoptosis in HNSCC cells. Based on these data, a clinical phase II trial was initiated at the Hollings Cancer Center to develop GMZ/DOX combination therapy against recurrent HNSCC, which has not been tested previously. Our initial data showed promising partial and complete responses in some patients with interesting changes in ceramide and sphingolipid profiles in their serum samples, collected after each cycle of treatment. In conclusion, these data suggest an important role for CerS1/C18-ceramide signaling in HNSCC tumor growth and therapy. In future studies, the mechanistic link between CerS1/C18-ceramide and drug-induced apoptosis will be defined both in situ and in vivo models of HNSCC.
The neuroprotective efficacy of calpain inhibition in acute and chronic-progressive optic neuritis
Amena W. Smith, Mary Kelly Guyton, Supriti Samanta Ray, Arabinda Das, Narendra L. Banik
Department of Neurosciences, Medical University of South Carolina , Charleston
Optic neuritis (ON), inflammation of the optic nerve, is due to influx of auto-reactive T cells and other immune cells, and is manifested by visual dysfunction. ON is strongly associated with multiple sclerosis (MS), and is the first sign in the diagnosis of 15-20% of MS cases. Decreased amplitude or increased latency of visual evoked potential (VEP) and electroretinogram (ERG) responses in patients indicate degeneration of the optic nerve and retinal damage, respectively. Findings of increased activity and expression of calpain, a Ca2+-activated protease, in the optic nerves and retinas of experimental autoimmune encephalomyelitis (EAE) animals suggests a role for calpain in the pathogenic events of ON. The roles of calpain in inflammation (T cell activation, immune cell migration), demyelination and neurodegeneration (apoptosis and axonal degeneration) are well established. We thus hypothesize that Ca2+ influx and calpain activation will lead to inflammation and oligodendrocyte damage in the optic nerve and RGC death in the retina of animals with ON, and that calpain inhibition will provide neuroprotection. An acute EAE model was utilized in which rats were immunized subcutaneously with an emulsion of complete Freund's adjuvant containing Mycobacterium tuberculosis , guinea pig spinal cord homogenate and myelin basic protein. Rats were treated b.i.d. by intraperitoneal injection of vehicle (DMSO) or calpeptin on days 1-9 post-EAE induction. A chronic-progressive EAE model was induced in B10.PL mice by a similar protocol. Mice received b.i.d. oral dosing of calpain inhibitor from day 7 post-induction until day of sacrifice. All rodents were enucleated and frozen sections of optic nerves and retinas were prepared for immunohistochemistry and TUNEL staining. Minimal calpain staining was present in control optic nerve, but calpain expression was upregulated in TUNEL+ oligodendrocytes (Olig-2) at acute EAE disease onset. This implies a role for calpain in death of mature myelin-producing cells. Staining of EAE optic nerve sections at disease peak demonstrated high levels of OX-42 (microgliosis) and GFAP (astrogliosis) relative to control. Increases in de-NFP (axonal damage marker) also occurred in EAE versus control optic nerves. In chronic-progressive EAE optic nerves, calpain inhibition prevents the loss of oligodendrocytes, astrocytosis, and axonal degeneration in the optic nerves relative to EAE-vehicle treated mice. Findings in the retinas of these animals are pending.
KNOCKDOWN OF THE NMDAR SUBUNIT NR3A ALTERS VIABILITY, GROWTH RATES, AND INTRACELLULAR CALCIUM CONCENTRATIONS IN INNER MEDULLARY COLLECTING DUCT CELLS
Adrian D. Sproul, Stacy Steele, P. Darwin Bell
N-Methyl-D-Aspartate Receptors (NMDARs) are non-selective cation channels which are expressed both in the CNS and in peripheral tissues. NMDARs are composed of NR1 and either NR2 or NR3 subunits, with the NR3 subunits (NR3a and NR3b) conferring reduced Ca2+ permeability. We have previously shown that the NR3 subunits are expressed in vivo and in vitro in the collecting duct of the kidney and that NR3a is upregulated and trafficked to the cell surface under hypoxic conditions. We have hypothesized that this increase in surface expression may may protect collecting duct cells by reducing influx of extracellular Ca2+ and preventing subsequent activation of downstream Ca2+ -dependent cell death pathways. To test this hypothesis we stably transfected IMCDs with shRNA directed against the NR3a subunit or with control constructs. This reduced levels of NR3a mRNA by approximately 90% and reduced NR3a protein levels by 75-80%. IMCDs with reduced NR3a were found to differ in several respects from control transfected cells: knockdown of NR3a produced significant increases in Fluo-4 fluorescence intensity, and in basal rates of LDH release, but resulted in significant decreases in cell growth rates and cell viability during combined oxygen and glucose deprivation (OGD) as assessed by cyquant and exclusion of trypan blue, respectively. Future studies will verify the specificity of the observed effects of shRNA knockdown by reintroducing NR3a via transient transfection with shRNA-insensitive NR3a constructs.
SPARC knock out mice have Irregular Collagen Morphology within the Periodontal Ligament
Jessica Trombetta, Amy Bradshaw*
College of Dental Medicine, *Department of Medicine, Medical University of South Carolina, and the Ralph H. Johnson Medical Center, Department of Veteran's Affairs, Charleston, SC
The periodontal ligament (PDL) is vital to maintaining oral health because the PDL fibers anchor each tooth into the surrounding bone. The PDL also serves to absorb shock from the forces of mastication to prevent bone resorption. Cell types within the PDL include fibroblasts, endothelial cells, and neuronal cells. However, fibroblasts are the main cell type and function to produce collagen and other components that comprise the extracellular matrix (ECM). The ECM of the PDL, composed of fibrillar collagens I and III, undergoes high rates of collagen turnover mediated by resident fibroblasts. SPARC, a matricellular protein, associates with collagens I and III and is implicated as a mediator of collagen turnover and phagocytosis. We have studied the role of SPARC in the PDL using a SPARC knock out murine model and human PDL fibroblast cells. Preliminary results suggest the SPARC knock out mice have decreased numbers of fibroblasts and increased disorganization of existing fibers within the PDL. The phenotype of the SPARC knock out model is more severe as the mice age. Our model has shown that SPARC is essential for the organization of collagen within the PDL.
Increased risk of second primary cancers after non-melanoma skin cancer: a meta-analysis
Lee Wheless, Joshua Black, Anthony J Alberg
BACKGROUND: Based on empirical evidence, the hypothesis has been set forth that a personal history of non-melanoma skin cancer may be a marker of a high cancer-risk phenotype. Numerous studies have reported on this potential association, with varying interpretations of the relative benefits and harms, but this evidence has yet to be systematically reviewed and summarized. To address this gap in the evidence, we carried out a systematic review to determine the overall association between non-melanoma skin cancer and subsequent risk of other cancers.
METHODS: We conducted a search of the Ovid/MEDLINE and PubMed databases to identify articles. Our search was supplemented by crosschecking the reference list of the selected papers, plus the related articles feature of PubMed. We screened all eligible articles, and for those included we abstracted in duplicate all measures of association between non-melanoma skin cancer and second primary cancers.
RESULTS: We identified 22 articles that met our criteria (19 cancer registry-linked, 3 prospective cohorts), 16 of which presented data on total risk of cancer following a history of non-melanoma skin cancer. A random-effects model including measures abstracted from these studies found a significant increase in total risk of cancer following non-melanoma skin cancer: random effects risk ratio (RR) 1.20, 95% confidence interval (CI) 1.07-1.35. The risk of second primary cancers was significantly elevated for both squamous cell carcinoma (RR 1.28, 95% CI 1.18-1.39) and basal cell carcinoma (RR 1.19, 95% CI 1.01-1.41) in random effects models. The cancer risk associated with prior non-melanoma skin cancer was observed in both males (RR 1.15, 95% CI 1.09-1.20) and females (RR 1.10, 95% CI 1.04-1.15). This increased burden of cancers appears not to be associated with an increase in breast (RR 1.06, 95% CI 0.98 - 1.15), prostate (RR 1.03, 95% 0.96 - 1.11), or colorectal cancer (RR 1.05, 95% CI 0.97 - 1.14), but rather may be due to site-specific increases in cancers of the lung (RR 1.27, 95% CI 1.11 - 1.45), as well as both lymphohematopoietic (Non-Hodgkin lymphoma RR 1.55, 95% CI 1.40 - 1.71; Hodgkin lymphoma RR 1.38, 95% CI 1.09 - 1.75; Leukemia RR 1.25, 95% CI 1.06 - 1.48) and head and neck cancers (Salivary gland RR 4.83, 95% CI 3.35 - 6.95, Lip RR 3.07, 95% CI 2.38 - 3.98; Oropharynx RR 2.88, 95% CI 1.90 - 4.36).
CONCLUSIONS: There is strong, consistent evidence that a personal history of non-melanoma skin cancer is associated with an increased risk of developing second primary cancers other than skin cancer. Non-melanoma skin cancer may be a marker of a high cancer-risk phenotype, but further research is needed to understand the mechanistic underpinnings of this association to translate these findings to improved cancer prevention and control.
ROLE OF CALPAIN 10 IN MITOCHONDRIAL DYSFUNCTION AND KIDNEY FUNCTION
Ryan M. Whitaker, Marisa D. Covington, Rick G.Schnellmann
Department of Pharmaceutical and Biomedical Sciences, MUSC
Aging of the kidney is associated with both morphological and physiological changes. Nephron number and kidney mass are decreased, and the functional capacity of the kidney for both absorption and secretion becomes significantly reduced. Because cells tend to accumulate abnormal or modified proteins over time, it has been postulated that cell death in aged renal tissue and associated kidney dysfunction may be a result of decreased proteolytic activity due to loss of certain proteases. Our laboratory recently identified calpain 10, a Ca2+ activated cysteine protease, as a resident in kidney mitochondria of rabbits, rats and mice. Calpain 10 was shown to cleave complex I proteins ND6, NDUFV2 and ATP synthase b, and inhibit state 3 respiration. Over-expression of calpain 10 led to mitochondrial dysfunction. Finally, levels of calpain 10, but not other common calpains, were found to be reduced in rabbit, mouse, rat and human kidneys with increasing age. To begin to elucidate the role and mechanisms of action of calpain 10 in kidney aging, we created a shRNA adenoviral construct to knockdown calpain 10. Rabbit renal proximal tubule cells (RPTCs) were treated with the calpain 10 shRNA or control vector and harvested each day over 5 days. Nuclear, mitochondrial and cytosolic fractions were prepared via differential centrifugation. Expression of calpain 10, ND6, NDUFV2 and ATP synthase ß were examined via immunoblotting. Cell death was assessed by DAPI staining and immunoblotting for the cleavage of caspase 3. Levels of calpain 10 decreased at differential rates in the mitochondrial and cytosolic fractions with significant knockdown observed after day 1 (40%) and day 3 (45%) in the mitochondrial and cytosolic fractions, respectively. Knockdown of calpain 10 led to the accumulation of its substrates ND6, NDUFV2, and ATP synthase ß. Examination of nuclear condensation via DAPI staining showed condensed nuclei (15%) as early as day 1 post-infection and continuing throughout the 5 day time course. Additionally, cleavage of caspase 3 was also observed beginning on day 1. The 48 hour gap between knockdown in the mitochondrial and cytosolic fractions indicates that cell death that occurred during this period was attributable to loss of calpain 10 activity in the mitochondria. We suggest that calpain 10 is playing a pro-survival role in the kidney by preventing the toxic buildup of calpain 10 substrates. We believe calpain 10 may play a universal role as a mediator of both mitochondrial function and cell survival. Loss of calpain 10 protein expression during aging or certain disease processes can lead to kidney cell death, functional loss and decreased tolerance to ischemic or toxicant induced injury.