Molecular and Cellular Pharmacology Student Seminars 2004-2005

Xiao-Fan Wang, PhD
Professor, Department of Pharmacology and Cancer Biology, Duke University Medical Center
Tuesday, September 14, 2004
12:00 Noon
Biotechnology Center Auditorium
425 Henry Mall

Novel Players in Tumor Metastasis

For additional information, please contact Student Host Sujatha Venkataraman, svenkatarama@wisc.edu. Note to Graduate Students: There will be a Graduate Student Lunch with Dr. Wang following his seminar in the Pharmacology Conference Room, 3765 MSC.

Beth Chang
Miyamoto Lab
Monday, September 20, 2004
12:00 Noon
140 Bardeen

NF-kB Super-Induction: An NF-kB Memory Pathway Mediated via the Exchange of NF-kB Dimer Subunits

The NF-kB family of transcription factors regulates various physiological and developmental processes. The transcriptional activity of NF-kB can self-regulate via controlling the expression of its family members. With the exception of p65, all of the NF-kB family members (c-Rel, RelB, p52 and p50) contain at least one NF-kB binding site (kB site) within their promoter regions. Thus, previous activation of NF-kB could lead to changes in the levels of different NF-kB protein expression and associated changes in biological readouts. We have identified a phenomenon termed "NF-kB super-induction" where an initial NF-kB activating event results in increased NF-kB DNA binding activity to selective kB sites when a second stimulus is applied. My studies to determine the mechanism of super-induction indicate that super-induction occurs through an NF-kB and Elf-1 dependent transcription of p50. The increased levels of p50 in turn exchange the pre-existing NF kB dimers into p50-containing dimers. A second NF-kB stimulus applied at this point activates these p50-containing dimers. Moreover, in the context of certain leukemia cell lines exposed to anti cancer agents, p50 containing dimers caused heightened expression of certain NF-kB regulated genes, such as cell cycle regulators (e.g. p21) resulting in super-survival of these malignant cells. My studies thus suggest that the super-induction phenomenon represents an NF-kB memory pathway in which an initial NF-kB activating event results in augmented response to second NF-kB stimuli in a gene-selective fashion through the increased expression of the p50 NF-kB subunit. These findings further suggest that auto-regulatory induction of specific NF-kB subunits provides a mechanism for diversification and/or amplification of NF-kB regulated biological responses.

Ashley Doan
Huttenlocher Lab
Monday, October 4, 2004
12:00 Noon
140 Bardeen

Characterization of RACK1, a Regulator of Cell Adhesion and Migration

RACK1 is an adaptor protein that binds to and regulates signaling via Src and PKC-dependent pathways. Elevated levels of rack1 mRNA has been demonstrated in non-small cell lung, colon, and breast carcinoma cells compared to normal tissue. Thus identification of the function of RACK1 and its regulation in normal and tumor cells appears to be essential to evaluate oncogenic or tumor-suppressor potential. I have evaluated the roles of paxillin as a potential downstream effector of RACK1 in the regulation of adhesions and cell migration. Subsequently, I would also like to elucidate the role of RACK1 in modulating cell-cell contact and metastasis in breast epithelial cells. Our previous studies demonstrated that RACK1 modulates cell migration and adhesion complex organization through its interaction with Src. We now show that down-regulation of RACK1 using siRNA increases the random and haptotactic migration of fibroblast cell lines, suggesting that endogenous RACK1 acts as a negative regulator of cell migration. We have evidence to suggest that paxillin is an important target downstream of RACK1 that regulates adhesion complex dynamics and cell migration. Together, our findings suggest that RACK1 may be playing a role in adhesion complex dynamics and cell migration by modulating Src-mediated phosphorylation of the focal adhesion protein, paxillin.

Hogune Im
Bresnick Lab
Monday, October 25, 2004
12:00 Noon
140 Bardeen

Dissecting the Activation of Gata-1-Dependent Endogenous Murine b-Globin Locus

Defining factors and signals that establish and maintain the native nucleoprotein structure of endogenous chromatin domains represents a powerful approach for elucidating transcriptional mechanisms.  In adult erythroid cells, the locus control region (LCR) and the adult b-globin genes of the murine b-globin locus are highly enriched in acetylated histones H3 and H4 and H3 methylated at lysine 4 (H3-meK4).  By contrast, the embryonic b-globin genes reside in a broad region of reduced acetylation.  Histone H3 methylated at lysine 79 (H3-meK79) is highly enriched at the adult b-globin genes, but not at the LCR.  To identify molecular steps in b-globin transcriptional activation, genetic complementation experiments were conducted in GATA-1-null, G1E cells containing an estrogen receptor hormone binding domain-GATA-1 fusion protein (ER-GATA-1).   Kinetic analysis of ER-GATA-1 occupancy of chromatin and establishment of the histone modification pattern by chromatin immunoprecipitation (ChIP) revealed that GATA-1 occupies multiple regions within the LCR prior to the bmajor promoter.  Chromatin accessibility at the promoter was low until ER-GATA-1 assembled into regulatory complexes at the LCR.  Subsequently, ER-GATA-1 accessed the bmajor promoter, induced histone H3 acetylation, RNA polymerase II recruitment, and elevated H3-meK79 levels.  Blocking transcriptional elongation did not erase H3-meK79, indicating that maintenance of H3-meK79 does not require ongoing elongation.  Analysis of N-terminal GATA-1 deletion mutants resembling truncated GATA-1 polypeptides expressed in megakaryoblastic leukemia, which retain Friend of GATA-1 (FOG-1) binding and DNA binding activities, revealed that FOG-1 binding and DNA binding activities are defective in polymerase recruitment. These results support a model in which acquisition of transcriptional competence requires establishment of H3-meK4, which is GATA-1-independent.  ER-GATA-1 binding to the LCR increases histone H3 acetylation at the promoter as an early event in transcriptional activation, which is tightly coupled to increased promoter accessibility and polymerase recruitment. Increased promoter accessibility permits ER-GATA-1 access to the promoter, which precedes maximal induction of H3-meK79, a late event in activation.  Given the dynamic regulation of H3-meK79 by GATA-1 and NF-E2 and the modulation of H3-meK79 levels during erythropoiesis, we propose that H3-meK79 is a crucial signal that controls the rate of b-globin transcription. Studies are underway to dissect mechanisms underlying the requirement of N-terminal sequences of GATA-1 for chromatin modification.  As the N-termini of GATA factors differ considerably, one might expect these divergent sequences to establish GATA factor-specific functions.

Katarzyna Modzelewska
Keely Lab
Week of November 1, 2004

Ack-1 regulates Cdc42 induces integrin-mediated cell migration through a signaling complex with p130Cas, FAK and Src

Activation of Cdc42, a member of the Rho family of small GTPases, promotes integrin-mediated migration and invasion of breast epithelial cells across collagen matrices (Keely et al, 1997. Nature 390: 632). To elucidate the molecular basis for the effects of Cdc42, we tested whether Cdc42 regulates integrin signaling pathways that lead to cell migration.  We found that Cdc42 is a necessary component of the a2b1 integrin signaling pathway leading to p130Cas phosphorylation, since activated Cdcd42(61L) lead to enhanced and dominant negative Cdc42 (17N) blocked p130Cas phosphorylation.  Additionally, Cdc42 activation occurred downstream of the a2b1 integrin, indicating it is a direct component of this signaling pathway.  Signaling from Cdc42 to p130Cas and FAK was functionally relevant, since the SH3 domain of p130Cas and FRNK, which function as a dominant negatives of the corresponding molecules, blocked Cdc42-induced cell migration.  In addition, both FAK and p130Cas can be immunoprecipitated with Cdc42 in a collagen dependent manner.  Moreover, we find a role for the Cdc42 effector, the tyrosine kinase Ack-1.  Ack-1 interacts with the beta1 integrin and becomes tyrosine phosphorylated following collagen stimulation. This phosphorylation is dependent on both Src and FAK kinases. Overexpression of either wild-type or kinase dead Ack-1 constructs enhanced Cdc42-induced migration, suggesting a role for Ack-1 in regulating cell migration.  Both wild type and kinase dead Ack-1 enhanced p130Cas phosphorylation, with wild type being more potent. Finally, we find evidence for a signaling complex between Ack-1, p130Cas, FAK and Cdc42.  Importantly, the formation of this complex was collagen-dependent, even in Cdc42(12V)-expressing cells.  Our results suggest a signaling pathway downstream of the a2b1 integrin by which collagen stimulates the activation of Cdc42, the phosphorylation of Ack-1, and their association with p130Cas and FAK.  These signaling events ultimately lead to cell migration.

Misuk Kang
Walker Lab
Department of Physiology and Molecular and Cellular Pharmacology Training Program
Monday, November 8, 2004
12:00 Noon
140 Bardeen

Correlation of cardiac contractile function with translocation of novel PKC isoforms in adult ventricular myocytes

In order to examine cardiac contractile regulation and protein kinase C (PKC) translocation in parallel, the d- and e-isoforms of PKC were fused to green fluorescent protein (GFP) and expressed in adult rat ventricular myocytes maintained in short term culture.  PKC-d-GFP and PKC-e-GFP were predominantly cytosolic until phorbol dibutyrate (PDBu) was introduced.  PKC-e-GFP redistributed preferentially to the surface sarcolemma, whereas PKC-d-GFP redistributed preferentially to perinuclear structures that co-localized with a Golgi marker.  Similar translocation preferences were observed in human embryonic kidney (HEK 293) cells, a non-cardiac cell type with a distinct morphology.  Myocyte contractile function was assessed by monitoring twitch shortening with field stimulation at 0.5 Hz, 22oC.  In PKC-e-GFP expressing myocytes, PDBu caused a 40% decrease in twitch amplitude that paralleled accumulation at the surface sarcolemma, but this response did not differ from myocytes expressing GFP alone.  In PKC-d-GFP myocytes, PDBu caused a transient negative inotropic response followed by a robust, sustained and bis-indoylmaleimide (BIM)-sensitive positive inotropic response that paralleled perinuclear PKC-d accumulation.  PKC-e-GFP myocytes at higher expression levels responded more like PKC-d-GFP myocytes including perinuclear accumulation and a sustained and BIM-sensitive positive inotropic response.  Conversely, PKC-d-GFP myocytes behaved more like PKC-e-GFP myocytes when stimulated with phorbol 12-myristate-13-acetate (PMA, a more hydrophobic phorbol ester) including PKC-d-GFP accumulation at the surface sarcolemma and an attenuated positive inotropic response.  The data suggest that these novel PKC isoforms have little effect on contractile function when accumulating at the cell surface, but produce strong positive inotropic responses upon accumulation at intracellular sites.

Paul Nuzzi
Huttenlocher Lab
Monday, November 15, 2004
12:00 Noon
140 Bardeen

The Role of Calpain-II in Neutrophil Polarization and Chemotaxis

Neutrophil migration and chemotaxis are critical steps involved in innate immunity and the development of inflammation. The ability of neutrophils to migrate within a shallow gradient of chemoattractant requires a dynamic system with tight regulation and specific localization of intracellular proteins. In our previous work we demonstrated that calpain inhibition induced neutrophil adhesion, polarization and rapid chemokinesis in the absence of exogenous activators, while disrupting neutrophil chemotaxis and directional persistence in a gradient of IL-8 and fMLP. In this study we further characterize the role of calpain in regulating neutrophil migration and examined isoform specificity. Using RT-PCR and Western blot analysis we show that neutrophils express both calpain-1 and calpain-2 isoforms with calpain-1 as the more abundant isoform. Upon IL-8 or fMLP stimulation, immunofluorescence microscopy shows an asymmetric redistribution of the two isoforms with calpain-2 localization at the leading edge and calpain-1 localized to the rear. Biochemical analysis indicates that upon activation each isoform also partitions into distinct cellular fractions, with calpain-2 localized to lipid rafts. To examine the importance of this asymmetry, HL-60 cell lines over- expressing wt and proteolytically dead calpain-2 were examined for effects on polarization and chemotaxis. Over-expression of calpain-2 resulted in an increase in chemotactic response over control, while over-expression of the proteolytically dead calpain-2 abrogated chemotaxis. This suggests that the two ubiquitous isoforms of calpain, calpain-1 and 2, have distinct distribution and functions during neutrophil motility and suggests an essential role for calpain-2 in neutrophil chemotaxis.

Haichuan Duan
Jefcoate Lab
Monday, December 13, 2004
12:00 Noon
140 Bardeen

Post-transcriptional Regulation of the Steroidogenic Acute Regulatory Protein (StAR) mRNA

The steroidogenic acute regulatory protein (StAR) mediates cholesterol import from outer to inner mitochondrial membrane, a rate-limiting step for steroid hormone biosynthesis. Two major polyadenylation sites give rise to two StAR mRNA forms (1.6kb and 3.5kb) differing only within their 3' untranslated regions (3'UTR). AU-rich Elements (AURE) which mediate fast degradation and selective stabilization of many cytokine transcripts are found at the 3' end of the 3.5kb StAR mRNA. We are looking at the post-transcriptional regulation of StAR transcripts and have found that the long but not the short 3'UTR contain destabilizing sequences. At least two elements are found to be involved in the destabilization, one nonspecific tract and AURE, which is specific for steroidogenic cells. TIS11b, a member of the CCCH tandem zinc finger family of RNA binding proteins which destabilize AURE-containing mRNAs, is rapidly induced in steroidogenic cells after cAMP-PKA stimulation and precedes further downregulation of StAR transcripts. We found that TIS11b selectively targets wild-type but not mutant StAR AURE through cotransfection assays. Taken together, we hypothesize that StAR mRNA half-life is kept low by a nonspecific degradation pathway but can be quickly changed through proteins interacting with AURE during stimulation.

Rapid synaptic transmission is mediated by ligand gated channels that bind ligand and undergo conformational transitions to open and desensitized states. To investigate the structural nature of these transitions we introduced cysteine residues at selected locations in the GABAA receptor. Wild-type and mutant a1b2 receptors were expressed in Xenopus oocytes, and the introduced cysteines were labeled with the environment-sensitive fluorophores, Alexa Fluor 546 C5 maleimide (Molecular Probes), sulforhodamine methanethiosulfonate (MTSR), and tetramethylrhodamine-maleimide (TMRM). Combined voltage-clamp and fluorescence measurements revealed GABA-induced fluorescence changes occurring simultaneously with GABA-induced current. Fluorescence changes and current both increased with the concentration of GABA. In receptors labeled at a1L127C, receptor activation was accompanied by an increase in fluorescence. This suggests that the side chain of this residue moves from a hydrophilic to a hydrophobic environment when the receptor is activated. In receptors labeled at the homologous site of the b2-subunit (L125C), receptor activation was accompanied by a similar increase. In the a-subunit, this site is near the GABA binding region, but in the b-subunit this site is far away from regions directly engaged in agonist binding. Thus, a global change in receptor structure probably contributes to the fluorescence changes in these labels. Labels at residue a1E122C or b2P120C report a fluorescence decrease in response to GABA. Thus, residues 122 and 127 of the a-subunit, which reside in b strand 5` and 6 in the extracellular domain of the GABAA receptor, undergo opposite changes in environment polarity during the gating of the channel.

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Frontiers in Pharmacology Spring 2005 Schedule

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R-Ras is a member of the Ras superfamily of small GTPases, which act as molecular on/off switches for intracellular signaling pathways.  R-Ras shares ~55% identity with classical Ras proteins H-, N-, and K-Ras (1), and has a unique 26-amino acid N-terminal extension (2).  Activation of R-Ras disrupts ductal morphogenesis of mammary epithelial cells (MECs) in 3D collagen gels and enhances cell migration (3,4).  We show here that constitutive activation of R-Ras causes a decrease in adherens junction (AJ) formation and corresponding increase in focal adhesions (FAs).  R-Ras inhibits AJ formation to a greater extent on collagen than on fibronectin, suggesting that R-Ras disrupts AJs by affecting a collagen receptor.  It has been shown previously that R-Ras enhances adhesion through the a2b1 integrin, a collagen and laminin receptor (4).  The a2b1 integrin not only mediates MEC polarization and tubulogenesis in 3D collagen matrices, it also mediates haptotactic migration on collagen (5,6,7).  Because the a2b1 integrin can mediate these two opposing phenotypes, regulation of a2b1 integrin is likely important in epithelial vs. mesenchymal phenotype choice made by MECs in response to the extracellular matrix (ECM).  We find that inhibition of the a2b1 integrin through addition of a blocking antibody, P1E6, causes a partial rescue of AJ formation on collagen but not fibronectin.  In addition, E-cadherin co-immunoprecipitates with the a2b1 integrin in control cells, but not in cells overexpressing activated R-Ras.  These data suggest that R-Ras disrupts adherens junctions in mammary epithelial cells through its effects on the a2b1 integrin, likely through increasing cell-substratum adhesion.

(1) Self, A.J., H.F. Paterson, and A. Hall.  Oncogene, 1993. 8(3):655-61.
(2) Lowe, D.G., et al.  Cell, 1987.  48(1):137-46.
(3) Keely, P.J., et al.  Journal of Cell Biology, 1999.  145(5):1077-88.
(4) Kwong, L., et al.  Molecular and Cellular Biology, 2003.  23(3):933-49.
(5) Berdichevsky, F., et al.  Journal of Cell Science, 1992.  102(Pt 3):437-46.
(6) Chen, J., et al.  American Journal of Pathology, 2002.  161(1):337-44.
(7) Zutter, M.M., et al.  Molecular Biology of the Cell, 2001.  12(S):8-9a.

Seminar Title:  Membrane-Cytoskeletal Dynamics in the Metastatic Process

For additional information, please contact Chateen Carbonara, ccarbonara@wisc.edu, 262-7436.

NOTE TO MOLECULAR AND CELLULAR PHARMACOLOGY GRADUATE STUDENTS:  There will be a student lunch with Dr. McNiven following his seminar in the Pharmacology Conference Room, 3765 MSC.

There is no Frontiers in Pharmacology Seminar scheduled for Tuesday, March 1, 2005.

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Gennifer Mager
Svaren Lab
Monday, March 7, 2005
12:00 Noon
140 Bardeen

Nab2 represses Transcription by recruiting the Nucleosome Remodeling and Deacetylase Complex
 
The EGR2/Krox-20 transcriptional activator plays a critical role in vertebrate hindbrain development and in peripheral nerve myelination. Our previous studies revealed that EGR2 expression in Schwann cells regulates genes for a number of myelin proteins, as well as genes involved in the lipid/cholesterol synthesis required for myelin formation. Recently, several independent mutations have been associated with human peripheral neuropathies. One such mutation highlights the importance of regulation of EGR2 activity by the NAB1 and NAB2 (NGFI-A/EGR1-binding) corepressors, which are recruited to EGR2 target promoters by direct interaction with EGR2. Using protein interaction assays, we have found that NAB2 interacts with the CHD4 (Chromodomain Helicase DNA-binding protein 4) subunit of the NuRD (Nucleosome Remodeling and Deacetylase) chromatin remodeling complex,  and dominant negative mutants of CHD4 have revealed that NAB2 represses via interaction with the NuRD complex. In addition, we have also characterized the domains required for interaction between NAB2 and CHD4. This unexpectedly revealed that EGR2 activity is modulated by at least two repression domains within NAB2, one of which uniquely requires interaction with CHD4 in order to repress transcription.  Finally, the interaction with CHD4 is regulated by alternative splicing of the NAB2 mRNA. Overall, our studies provide the first evidence implicating a chromatin remodeling complex in the transcriptional regulation of the myelination program in Schwann cells. 

Dentin Sialophosphoprotein (DSPP) is a member of the SIBLING (Small Integrin Binding LIgand N-linked Glycoproteins) family of proteins.  Members of the SIBLING family have a variety of functions including: regulating the biomineralization of bone and dentin and activation of matrix metalloproteinases, and may be upregulated in some cancers.  DSPP is a precursor protein comprised of a N-terminal dentin sialoprotein (DSP) and C-terminal dentin phosphoprotein (DPP) and is processed /in vivo/ to generate these two proteins.  DPP is the major non-collagenous protein in dentin and is involved in the biomineralization of dentin.  The Bone Morphogentic Protein 1/Tolloid (BMP-1/TLD) family of metalloproteinases are involved in the proteolytic processing of a variety of extracellular proteins including procollagen, chordin, alpha 2 macroglobulin, myostatin, lysyl oxidase and small leucine rich proteoglycans such as osteoglycin, biglycan, and decorin.  BMP-1/TLD like proteinases usually cleave just N-terminal to an aspartate residue, although little else is known about any sort of consensus sequence for these proteins.  When isolated from tissue, DPP begins with an Asp-Asp-Pro sequence.  This observation combined with a previous paper from our lab showing that BMP-1/TLD like proteinases cleave dentin matrix protein 1, another SIBLING, led us to investigate these enzymes in the processing of DSPP.   We have shown that BMP-1/TLD like proteinases can process recombinant DSPP to generate fragments consistent in size to those observed /in vivo/.

Breast cancer is the second leading cause of death among women  in the United States.  Estrogen receptors (ER-alpha, ER-beta) are a primary focus of the etiology and therapy of breast cancers.  ER-alpha-positive breast cancers have a better prognosis and have traditionally  been treated with tamoxifen, a selective estrogen receptor modulator (SERM), or inhibitors of aromatase, an enzyme involved in estrogen synthesis.  ER-alpha-negative breast cancers, which occur in 25% of patients, do not respond to anti-estrogen treatment, are more aggressive, and function through unknown mechanisms (Rochefort et al., 2003).  Estrogen-related receptor alpha (ERR-alpha) is a protein with sequence similarity to ER-alpha, yet it does not bind estrogen.  ERR-alpha can function as either a repressor, down-modulating ER-alpha-regulated transcription, or as a constitutive activator, acting independently of the presence of estrogens and tamoxifen.  Whether ERR-alpha functions as a repressor or as a constitutive activator is likely determined in part by post-translational phosphorylation modifications occurring via the ErbB2/MAPK signaling pathway (Ariazi et al., 2004).  Thus, ERR-alpha probably plays an important role in the etiology and progression of a subset of breast cancers, especially ErbB2-positive cases that are ER-alpha-negative or resistant to tamoxifen.The long-term goal of my project is to find a new treatment for human breast cancer, particularly for aggressive breast cancers that are ER-alpha-negative or tamoxifen resistant, and resistant to currently available therapies. I hypothesize that ERR-alpha is constitutively activated in a subset of breast cancers and is regulating transcription of estrogen-response element (ERE)-containing genes by binding specific coactivators.  Therefore, I believe that the ERR-alpha-coactivator interaction is a promising target for new chemotherapeutic drug development.  The immediate goals are to purify specific coactivators of ERR-alpha by gentle immunoaffinity chromatography, to identify these associated proteins, and to study their binding properties to ERR-alpha in vitro.  By understanding these aspects of ERR-alpha regulation, I will be able to develop a high-throughput screen to identify compounds that interfere with the target interaction between ERR-alpha and the coactivator. This screen could provide a possible new chemotherapeutic candidate for treating aggressive tumors that do not respond to anti-estrogen therapies.
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Rochefort H, Glondu M, Sahla ME, Platet N, Garcia M.  How to target estrogen receptor-negative breast cancer?  Endocr Relat Cancer 10: 261-266, 2003.
Ariazi EA, Kraus RJ, Jordan VC, Mertz JE.  Estrogen-related receptor alpha's transcriptional activities are regulated in part via the EGFR
and ErbB2 (HER2) signaling pathways.  In revision, 2004.

Nuclear Factor-kappaB (NF-kB) is a transcription factor that regulates a diverse subset of genes such as those involved in immune function, growth control, and regulation of apoptosis.  NF-kB can be activated by a wide variety of stimuli such as tumor necrosis factor alpha (TNFa), interleukin-1 (IL-1), lipopolysacharride (LPS), and DNA damaging agents (ionizing radiation and chemotherapeutic drugs such as camptothecin and etoposide).  Recently, it has been proposed that enhanced NF-kB activity in cancer cells may promote their survival.  As a result, inhibitors for this pathway are being investigated for their ability to enhance cancer cell death.  The orchestration of events responsible for activation of NF-kB in response to DNA damaging agents requires sequential posttranslational modifications of a regulatory subunit essential for NF-kB activation, known as NEMO.  We are interested in the first known posttranslational modification of NEMO in this pathway, SUMOylation.  The process of SUMOylation requires the use of two enzymes, an E1 (Aos1-Uba2) and E2 (Ubc9).  However, whether there is a SUMO ligase (E3) for NEMO is unknown.  Using an siRNA screen for all known SUMO E3's, we have identified a SUMO E3 that plays a critical role in NF-kB activation in response to DNA damaging agents.  Furthermore, overexpression of this E3 is able to enhance NF-kB activity in a DNA damage specific manner and has the ability to promote SUMOylation of NEMO both in vitro and in vivo.  We found that endogenous NEMO interacted with this SUMO E3 in a DNA damage inducible manner.  Analysis using NEMO truncation mutants revealed that this SUMO E3 binds NEMO in its CC1 domain region thus suggesting that this SUMO E3 is the putative E3 for NEMO.  As a result, we propose that inhibition of the SUMO E3 for NEMO may enhance the proapoptotic effects of certain chemotherapeutic agents by preventing NF-kB activation.

Cynthia Koziol
Bertics Lab
Monday, April 18, 2005
12:00 Noon
140 Bardeen

Asthma is a disease that affects over 20 million Americans today.  To date, there is no cure for the disease, only temporary treatments.  The disease is characterized by variable airflow obstruction, airway hyper-responsiveness, and chronic airway inflammation.  These symptoms are due to cellular responses from both immune cells and the surrounding tissues.  Eosinophils, a type of immune cell associated with the inflammation in allergic disease, are commonly found to be highly activated and present in the lungs of asthmatic patients.  The molecular mechanisms of how the eosinophils migrate/chemotax to the area of inflammation, and the mechanism behind how chemoattractants modulate other processes such as degranulation, are poorly understood. The low molecular weight G protein, Rac, is known to modulate a number of functions in neutrophils, a cell closely related to eosinophils.  The role that Rac plays in eosinophil function is not well understood.  We hypothesize that chemoattractant stimulation functions to activate Rac to promote eosinophil actin reorganization, migration/chemotaxis, and cause degranulation to occur.

Amanda Branam
Greenspan Lab
Monday, April 18, 2005
12:00 Noon
140 Bardeen

Chordin is an extracellular BMP (Bone Morphogenetic Protein) signaling antagonist.  It functions by binding to the BMPs and preventing the BMPs from binding to their receptors and initiating signaling.  BMP-1 differs from the rest of the BMP family in that it does not function as a growth factor but rather as a proteinase.  BMP-1 has been shown to be involved in the proteolytic processing of several extracellular matrix proteins.  One of its substrates is chordin.  Upon BMP-1 cleavage of chordin, the bound BMP is released and free to bind to its receptor and initiate signaling.  Recently, two proteins with homology to chordin, Chordin- Like 1 (CHL1) and Chordin-Like 2 (CHL2), have been discovered.  Both of these proteins have been shown to function similarly to chordin and share similar domain structures.  From this information, we suspect that CHL1 and CHL2 may also be substrates for BMP-1.

Inflammation is a localized protective reaction of tissues to injury or infection and is mediated by substances released from leukocytes present in the inflammatory microenvironment. Usually accompanying infection or inflammation is the release of a high concentration of extracellular nucleotides, supplying a source of ligands for purinergic receptors that are present on the cell surface of many immune cells. One particular nucleotide receptor that is mainly expressed on leukocytes is the P2X7 receptor (P2X7R). Previous work with P2X7R has linked this receptor to the modulation of macrophage responsiveness to inflammatory stimuli. In a mAb-induced arthritis model, the P2X7R knock out mice were markedly less affected by the disease.

To elucidate the mechanism by which the P2X7R modulates inflammation, the effect of P2X7R activation on induced inflammatory endpoints, such as the production of prostaglandins, in macrophages and monocytes (macrophage precursor cells) is currently under investigation. Cyclooxygenase-2 (COX-2) expression has been implicated as the main route for increased prostaglandin synthesis at the sites of inflammation and data in our lab supports that its expression is up-regulated upon P2X7R stimulation.

Previous studies, along with my preliminary data, suggest that stimulation of mitogen-activated protein kinase (MAPK) cascades, and the control of transcription via cAMP response element binding protein (CREB)-linked pathways, are potentially involved in the nucleotide regulation of inflammation. Therefore, it is hypothesized that signaling cascade can provide a link between P2X7R activation and COX-2 expression.

Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, which contain binding sites for a range of various compounds including cocaine, antipsychotics such as haloperidol and steroids such as progesterone. The sigma 1 receptor has been cloned from guinea pig liver, rat brain, mouse kidney and human placenta and encodes a protein of 223 amino acids. All cloned sigma receptors share grater than 90% sequence identity. Various functions have been proposed for the sigma 1 receptor such as regulation of intracellular Ca2+ release [1], inhibition of K+ channels [2] and antitumour effects both in vitro and in vivo [3]. Association of the sigma 1 receptor with voltage gated potassium channels and inhibition of channel gating has led to the idea that the sigma 1 receptor is a subunit voltage gated potassium channels [4]. Recently sigma 1 receptor knockout mice have been generated and do not display any overt phenotype [5].

As a first step to characterizing the sigma receptor, we have developed a scheme for the purification of the guinea pig sigma receptor from E. coli. The sigma receptor is expressed in E. coli as a maltose binding protein (MBP) fusion protein. The fusion protein was purified on an amylose column, cleaved with Factor Xa and further purified on a Ni2+ affinity column. The Ni2+ column eluate was purified using an anti-MBP antibody linked to sepharose to remove the uncleaved MBP-sigma fusion protein. Radioligand binding studies using [3H] (+) pentazocine indicated that 40-45% of the fusion protein is in a state competent to bind ligand, assuming a 1:1 stoichiometry ligand to protein molecules. Surprisingly when a haloperidol affinity column was used, all the  receptor bound tightly to the column. This observation suggests that the receptor may exist in an oligomeric state perhaps binding [3H] (+) pentazocine as a homodimer.

References:

1.  Hayashi, T. and T.P. Su, Regulating ankyrin dynamics: Roles of sigma-1 receptors. Proc Natl Acad Sci U S A, 2001. 98(2): p. 491-6.

2.  Wilke, R.A., et al., K+ channel modulation in rodent neurohypophysial nerveterminals by sigma receptors and not by dopamine receptors. J Physiol, 1999. 517 ( Pt 2): p. 391-406.

3.  Spruce, B.A., et al., Small molecule antagonists of the sigma-1 receptor causeselective  release of the death program in tumor and self-reliant cells and inhibit tumor growth in vitro and in vivo. Cancer Res, 2004. 64(14): p. 4875-86.

4.  Aydar, E., et al., The sigma receptor as a ligand-regulated auxiliary potassium channel subunit. Neuron, 2002. 34(3): p. 399-410.5.  Langa, F., et al., Generation and phenotypic analysis of sigma receptor type I (sigma1) knockout mice. Eur J Neurosci, 2003. 18(8): p. 2188-96.

References:

1.   Dalpe, G., Mathieu, M., Comtois, A., Zhu, E., Wasiak, S., De Repetigny, Y., Leclerc, N., and Kothary, R. (1999) Dev.Biol. 210, 367-380

2.   Dowling, J., Yang, Y., Wollmann, R., Reichardt, L. F., and Fuchs, E. (1997) Dev.Biol. 187, 131-142

3.   Guo, L., Degenstein, L., Dowling, J., Yu, Q. C., Wollmann, R., Perman, B., and Fuchs, E. (1995) Cell 81, 233-243