Molecular and Cellular Pharmacology Student Seminars 2005-2006

Gennifer Mager
Monday September 12, 2005
12:00 Noon
140 Bardeen

Repression of Egr2 target genes by the NuRD complex

Gennifer Mager and John Svaren (Comparative Biosciences)
4th Year Molecular and Cellular Pharmacology Graduate Students

Abstract:

The EGR2/Krox-20 transcriptional activator plays a critical role in vertebrate hindbrain development and 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.

Haichuan Duan
Colin Jefcoate Lab
October 10, 2005
12:00 Noon
140 Bardeen
 
The steroidogenic acute regulatory protein (StAR) is a key mediator of steroid hormone biosynthesis. StAR transfers cholesterol from the outer to the inner mitochondrial membrane, a rate limiting step in steroidogenesis. StAR expression is stimulated by the cAMP-PKA pathway in many steroid producing tissues. Two StAR transcripts are present in rodent steroidogenic cells, 1.6kb and 3.5kb in length respectively. They arise from alternative use of polyadenylation signals in the last exon and differ only in their 3' untranslated regions (3'UTR). An AU-rich element (AURE), which is the most common mRNA stability regulatory element, is found only in the 3.5kb mRNA. StAR promoter constructs only accounted partly for several features of regulation by cAMP, so we investigated the role of mRNA stability in regulating StAR expression. We examined the effects of 3'UTR on the stability of both StAR mRNA and luciferase chimeric constructs. We compared 3 methods: measurement of steady state luciferase expression from luciferase-StAR 3'UTR chimeric constructs; degradation of mRNA after transcription inhibition by actinomycin D; and direct transfection of in vitro transcribed StAR mRNA. Although there are some discrepancies among these data, conclusions can be drawn that (a) the long 3'UTR destabilizes StAR message 2 fold under basal condition; (b) prolonged cAMP stimulation selectively destabilizes 3.5kb message 2 fold. We hypothesized that AURE renders cAMP-PKA destabilization to StAR message, which will be tested in future experiments. TIS11b is a candidate protein that destabilizes StAR message through AURE. TIS11b is rapidly stimulated by cAMP-PKA in steroidogenic cells, prior to downregulation of StAR message. Cotransfection of TIS11b with various luciferase-StAR 3'UTR chimeric constructs suggested that TIS11b selectively targets StAR AURE. Future experiments will aim to define the role of TIS11b in StAR message stability regulation. In short, our studies uncover a novel regulatory mechanism of StAR expression in steroidogenic cells.

Signaling by bone morphogenetic proteins plays a central role in early embryonic patterning, organogenesis and homeostasis in a broad range of species. This signaling is regulated by BMP-1/TLD like metalloproteases which were initially isolated from bovine bone along with bone inducing BMPs but found to be astacin like metalloproteases. The BMP-1 family of enzymes proteolytically processes the extracellular BMP antagonist chordin and thereby promotes BMP signaling. BMP-1 is synthesized with an N-terminal prodomain that is removed by furin-like proprotein convertases, presumably activating the protease. Interestingly, all characterized peptide sequences for the form of BMP-1 that co-purify with TGFb-like BMPs in osteogenic fractions are from the prodomain region. To gain more insight into possible functional roles for the BMP-1 prodomain in vertebrates, we have evaluated the proteolytic activity and the binding properties of a furin site-mutated form of ProBMP-1 (ProBMP-1SQQ) that retains its prodomain sequence. We found that ProBMP-1SQQ is efficiently expressed and secreted. We have shown that ProBMP-1SQQ binds to TGFb-like BMPs while the mature BMP-1 does not. This interaction is direct and specific as it cannot be competed away with excess of unrelated growth factors. BMP-2, which is structurally and functionally similar to BMP-4 can compete with BMP-4 for binding to ProBMP-1SQQ . ProBMP-1SQQ can also alter the BMP-4 signaling in cultured cells. We have also shown that this interaction with BMP-4 occurs via the prodomain region of BMP-1 and prodomain by itself is also capable of inhibiting BMP signaling in cultured cells. We have shown that prodomain can also exist in complex with BMP-4 in vivo and can affect BMP signaling by dorsalization of zebrafish embryos while it is tethered to the membrane. In conclusion, the prodomain of BMP-1 may act as a negative regulator of BMP signaling.

In another project, we show that mammalian Chordin, an extracellular antagonist of BMP-2/4 binds heparin with an affinity similar to that of factors known to functionally interact with heparan sulfate proteoglycans (HSPGs) in tissues. We further demonstrate that Chordin binding in mouse embryonic tissues is dependent upon its interaction with cell surface HSPGs and that Chordin binds to cell surface HSPGs (e.g. syndecans). Also, Chordin-HSPG interactions strongly potentiate the antagonism of BMP signaling by Chordin and are necessary to the retention and uptake of Chordin by cells.

Signal-Dependent Transcriptional Control of Notch4 Signaling in Vascular Endothelium: A Key Angiogenic Mechanism        

Notch signaling is an evolutionarily conserved pathway that modulates cell fate determination. The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenesis, is controlled by multiple signals, including the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. The endothelial cell-specific expression of Notch4 suggests that dynamic changes in Notch4 expression are crucial for the regulation of angiogenesis. As mechanisms that
confer Notch4 expression were unknown, we investigated how Notch4 transcription is established and regulated in endothelial cells and in transgenic mice. The Notch4 promoter assembles into an endothelial cell-specific histone modification pattern and confers endothelial-specific activity both in transfection assays and in the vasculature of transgenic mouse embryos. Activator Protein-1 (AP-1) complexes occupy Notch4 chromatin in a cell-type-specific manner and conferred endothelial cell-specific transcription. Vascular angiogenic factors synergize with cortisol to activate and maintain Notch4 transcription in endothelial cell lines. Both AP-1 and glucocorticoid receptor (GR) occupy a single AP-1 motif at the Notch4 promoter. Ectopic signals activate AP-1 and glucocorticoid receptor and reprogram the endogenous Notch4 gene from a repressed to a transcriptionally active state in non-endothelial cells. As the reprogramming involves signal-dependent histone modifications, we predict that AP-1 and glucocorticoid receptor integrate the growth factor and glucocorticoid signals and establish the active chromatin domain. Base on these results the Model we propose is that, unlike the previously described activity of the GR to antagonize AP-1-mediated transcription activation, growth factor activated AP-1 bind to the AP-1 motif at the Notch4 promoter and recruits GR as a coactivator to activate Notch4 transcription. These results reveal a growth factor/glucocorticoid-AP-1/GR-Notch4 axis that we propose is crucial for transducing angiogenic signals during vascular development/remodeling and is deregulated upon aberrant signal transduction in cancer.

Reference:

• Wu J., Iwata F., Grass J.A., Osborne C.S., Elnitski L., Fraser P., Ohneda O., Yamamoto M. and Bresnick E.H. 2005 Molecular determinants of NOTCH4 transcription in vascular endothelium. Mol Cell Biol. 25(4):1458-74.

Jill L. Humston
Ervasti Lab
Monday, November 28, 2005
12:00 Noon

Probing utrophin function at the neuromuscular junction.

Duchenne Muscular Dystrophy (DMD) is an X-linked progressive muscle wasting disorder that affects 1/3500 boys. This disease is caused by mutations in the DMD gene resulting in absent or abnormal expression of the protein, dystrophin. A compensatory upregulation of the dystrophin homolog, utrophin, is observed in the mdx mouse, a dystrophin-deficient animal model for DMD. Utrophin is ubiquitously expressed in all tissues and is highly expressed at the sarcolemma in skeletal muscle during development. However, in normal, adult skeletal muscle, utrophin is localized to the neuromuscular and myotendinous junctions. Despite their homology and ability to perform important equivalent functions, there are differences between them. Work in our lab and others suggests that novel interacting proteins exist and we hypothesize that these could have a preference to one or the other and define their specific functions. Here we investigate utrophin-interacting proteins through af finity chromatography and mass spectrometry and identified proteins localized and important for clustering of acetylcholine receptors at the neuromuscular junction.

Gerry Dodson
Monday, December 19, 2005
12:00 Noon
140 Bardeen

DNA replication stress-induced phosphorylation of CREB mediated by ATM

The DNA damage response-regulators ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) are structurally and functionally related protein kinases that exhibit nearly identical substrate specificities in vitro. Current paradigms hold that the relative contributions of ATM and ATR to nuclear substrate phosphorylation are dictated by the type of initiating DNA lesion; ATM-dependent substrate phosphorylation is principally activated by DNA double-strand breaks (DSBs), whereas ATR-dependent substrate phosphorylation is induced by UV light and other forms of DNA replication stress. We employed the cyclic AMP response element-binding protein (CREB) to provide evidence for substrate discrimination by ATM and ATR in cellulo. ATM and ATR phosphorylate CREB in vitro and CREB is phosphorylated on Ser-121 in intact cells in response to ionizing radiation (IR), UV light, and hydroxyurea (HU). The UV- and HU-induced phosphorylation of CREB was delayed in comparison to the canonical ATR substrate, CHK1, suggesting potentially different mechanisms of phosphorylation. UV-induced CREB phosphorylation temporally correlated with ATM autophosphorylation on Ser-1981 and an ATM-specific siRNA suppressed CREB phosphorylation in response to this stimulus. UV-induced CREB phosphorylation was absent in ATM-deficient cells, confirming that ATM is required for CREB phosphorylation in UV-damaged cells. Interestingly, RNAi-mediated suppression of ATR partially inhibited CREB phosphorylation in response to UV, which correlated with reduced phosphorylation of ATM on Ser-1981. These findings suggest that ATM is the major genotoxin-induced CREB kinase in mammalian cells and that ATR lies upstream of ATM in a UV-induced signaling pathway. Future studies will be focused on determining the mechanism of replication-stress induced activation of ATM.

Reference:

Dodson, GE and Tibbetts, RS 2005 DNA replication stress-induced phosphorylation of cyclic AMP response element-binding protein mediated by ATM. J Biol Chem. 2005 Nov 17; [Epub ahead of print]

There is no Frontiers in Pharmacology Seminar scheduled for Tuesday, December 20, 2005.

The Frontiers in Pharmacology Seminar Series will resume in January, 2006.

Seminar Title: "Guided by COMPASS on an Expedition Defining the Role of MLL and its Histone Methylase Function in Leukemogenesis"

NOTE TO GRADUATE STUDENTS:  There will be a graduate student lunch with Dr. Shilatifard on Tuesday, January 17, 2006 following the seminar in the Pharmacology Conference Room, 3765 MSC.

Dynamic GATA Factor Interplay at a Multi-Component Regulatory Region of the GATA-2 Locus

Melissa L. Martowicz, University of Wisconsin Medical School, Molecular and Cellular Pharmacology Program, 383 Medical Sciences Center, Madison, WI 53706

Previous studies indicated that GATA-2 selectively occupies the -2.8 kb region of the GATA-2 locus in the active state, despite there being numerous GATA motifs throughout the locus (1, 2).  The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription.  We demonstrated that GATA-1 and GATA-2 occupy two additional regions, the -3.9 kb and -1.8 kb regions of the GATA-2 locus (3).  Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and GATA-2 displacement at the sites, suggesting that ER-GATA-1 occupies the regions simultaneously.  In the transcriptionally-active state, DNaseI hypersensitive sites (HSs) were detected at the -3.9 kb and -1.8 kb regions, and a weak HS was detected at the -2.8 kb region.  Whereas ER-GATA-1-instigated repression abolished the -1.8 kb HS, the -3.9 kb HS persisted in the inactive state.   As a first step to test whether the HSs function differentially, transient transfection analysis was conducted which evidence that the -3.9 kb region functions distinctly from the -2.8 kb and -1.8 kb regions.  The -3.9 kb region had no activity in a GATA-1-null cell line (G1E) which expresses GATA-2, whereas, the -2.8 and -1.8 kb regions activated transcription.  In mouse erythroleukemia cells (MEL) which express GATA-1 but not GATA-2, the -3.9 kb region activated transcription, whereas, the -2.8 and -1.8 kb regions had no activity.  GATA motifs were required for the differential activities. We hypothesized that the accessible DNaseI HSs are sites of RNA polymerase II (Pol II) recruitment.  ChIP analysis demonstrated that Pol II localizes to the HSs and the -77 kb region, which was recently shown by the Bresnick group to be an additional GATA switch site.  Pol II was phosphorylated at Ser-5, a hallmark of elongating polymerase, and colocalized with TFIIB, a component of the basal transcription machinery. These results provide evidence that the intergenic Pol II is transcriptionally competent.  Furthermore, Pol II dissociates from the -3.9, -2.8, and -1.8 kb regions upon repression, whereas it is retained at the -77 kb region.   Acetylation is also maintained at the -77 kb region upon repression, whereas it is lost at the HSs.  Our results support a model in which GATA switches at multiple intergenic sites repress GATA-2 via dynamic changes in GATA factor occupancy, chromatin structure, and Pol II occupancy.  We hypothesize that intergenic Pol II exerts an important function required for activation of the GATA-2 chromatin domain.

1.      Grass, J.A., M.E. Boyer, S. Pal, J. Wu, M.J. Weiss and E.H. Bresnick, GATA-1-dependent transcriptional repression of GATA-2 via disruption of positive autoregulation and domain-wide chromatin remodeling. Proc Natl Acad Sci U S A, 2003. 100(15): p. 8811-6.

2.       Pal, S., A.B. Cantor, K.D. Johnson, T.B. Moran, M.E. Boyer, S.H. Orkin and E.H. Bresnick, Coregulator-dependent facilitation of chromatin occupancy by GATA-1. Proc Natl Acad Sci U S A, 2004. 101(4): p. 980-5.

3.      Martowicz, M.L., J.A. Grass, M.E. Boyer, H. Guend and E.H. Bresnick, Dynamic GATA factor interplay at a multicomponent regulatory region of the GATA-2 locus. J Biol Chem, 2005. 280(3): p. 1724-32.

The Escherichia coli transcription machinery is comprised of core RNA polymerase (a2bb'w) along with one of seven sigma factors.  Core, which is able to bind DNA nonspecifically, is only able to recognize specific promoters once it has bound a sigma factor, forming the holoenzyme.  Each sigma factor is responsible for driving transcription of a different set of genes.  Sigma70, which regulates the "house keeping" genes, is thought to be the most abundant sigma factor while also having one of the highest affinities for core.  If core is truly limited in the cells, the question remains as to how the competition among the seven sigma factors is regulated.  We have constructed a homogeneous assay based on Luminescence Resonance Energy Transfer (LRET) to measure the binding affinity of each sigma factor for RNAP in vitro.  With this assay we have started to determine the strength of interaction of each sigma factor with core RNAP.  We are also examining how other proteins, DNA, small molecules, or the environment can alter the binding affinities.

Characterization of the Sigma receptor - a modulator of voltage gated K+ channels

Sigma-1 receptor once considered as an opioid receptor can be grouped under the family of sterol isomerases and isomerase like proteins. Sterol isomerases catalyze the isomerization of the D8 bond in the steroid nucleus, an important step in cholesterol synthesis in mammals and ergosterol synthesis in yeast. The sigma-1 receptor does not have any enzymatic activity but has affinity for the same range of pharmacological agents that bind to the isomerases. Antipsychotics such as haloperidol and steroids such as progesterone are known to bind to the sigma receptor although its endogenous ligand is not known. The sigma-1 receptor has been cloned from different species and encodes a protein of 223 amino acids. All cloned sigma-1 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).  It has been shown that sigma-1 receptors specifically target to galactoceramide and cholesterol containing lipid microdomains and this was proposed to play an important role in oligodendrocyte differentiation (4). We have expressed the guinea pig sigma-1 receptor in E. coli. as a maltose binding protein (MBP) fusion protein and developed a purification scheme to obtain pure sigma-1 receptor. The purified sigma-1 receptor showed specific binding with sigma ligand [3H] (+) pentazocine. The detergent LDAO was found to act like a sigma-1 receptor ligand in that it inhibited [3H] pentazocine binding activity in guinea pig liver membranes with a Ki of 6 nM. Endogenous sphingolipids sphingosine and NN dimethyl sphingosine were also shown to inhibit [3H] pentazocine binding on the purified sigma receptor preparation with a Ki of 55 and 59 nM respectively. The sigma-1 receptor inhibits voltage gated potassium channel activity when co-expressed with the potassium channels (Kv1.4 and Kv1.5) in Xenopus oocytes (5). We have expressed C-terminal truncations of the sigma-1 receptor along with the Kv 1.4 in Xenopus oocytes to determine the region on the sigma receptor responsible for inhibition of the K+ channel. The C- terminal truncation mutant lacking 110 amino acids from the C-terminus of the sigma receptor is still seen to inhibit the K+ channel. This suggests that the interaction occurs through the transmembrane domains or the N terminus of the sigma-1 receptor.
 
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., P.J. Lupardus, D.K. Grandy, M. Rubinstein, M.J. Low, and M.B. Jackson, K+ channel modulation in rodent neurohypophysial nerve terminals by sigma receptors and not by dopamine receptors. J Physiol, 1999. 517 ( Pt 2): p. 391-406.

3. Spruce, B.A., L.A. Campbell, N. McTavish, M.A. Cooper, M.V. Appleyard, M. O'Neill, J. Howie, J. Samson, S. Watt, K. Murray, D. McLean, N.R. Leslie, S.T. Safrany, M.J. Ferguson, J.A. Peters, A.R. Prescott, G. Box, A. Hayes, B. Nutley, F. Raynaud, C.P. Downes, J.J. Lambert, A.M. Thompson, and S. Eccles, Small molecule antagonists of the sigma-1 receptor cause selective 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. Hayashi, T. and T.P. Su, Sigma-1 receptors at galactosylceramide-enriched lipid microdomains regulate oligodendrocyte differentiation. Proc Natl Acad Sci U S A, 2004. 101(41): p. 14949-54.

5. Aydar, E., C.P. Palmer, V.A. Klyachko, and M.B. Jackson, The sigma receptor as a ligand-regulated auxiliary potassium channel subunit. Neuron, 2002. 34(3): p. 399-410.

Matt Marengo
Wassarman Lab
Monday, March 6, 2006
12:00 Noon
140 Bardeen


Developmental and stress signals regulate the splicing of TAF1, a central regulator of eukaryotic transcription.

Understanding the control of transcription is a key to understanding the molecular basis of development and disease.  TFIID is a protein complex composed of the TATA box binding protein (TBP) and 10-14 TBP associated factors (TAFs).  TFIID binds the core promoter of most eukaryotic genes.  Previous studies of TFIID indicate that it has a broad role in transcription.  We propose that TFIID complexes containing different TAF1 isoforms regulate specific genetic programs.Previously, our laboratory has observed that TAF1 is alternatively spliced in Drosophila melanogaster and that the ratio of TAF1 isoform mRNAs is regulated by tissue-specific and cell stress signals.  Recombinant fragments of TAF1 isoforms differentially bind core promoter DNA and differential binding correlates with transcriptional activation.  By Western blot, I found different TAF1 proteins in testis, head, and embryonic protein lysates.  By coimmunoprecipitation, I demonstrated that TAF1 isoforms associate in TFIID complexes. Using a TAF1 minigene system, I identified an intronic element that is necessary for the camptothecin-induced upregulation of the TAF1-4 isoform. These results contribute to a model in which cells use signal-dependent alternative splicing to regulate the function of a central regulator of eukaryotic transcription.

Examining Functions of BMP-1/TLD-like Proteinases in the Nervous System

Bone Morphogenetic Protein 1 (BMP-1) is a metalloproteinase that serves as the prototype of a family of astacin-like metalloproteinases (BMP-1/TLD-like Proteinases).  These proteinases cleave a variety of extracellular substrates including components of the extracellular matrix, some TGF-beta-like molecules and Chordin, an antagonist of the TGF-beta-like BMPs.  Our lab is investigating the roles of these proteinases within the nervous system using two approaches.  First, we are generating a brain-specific conditional gene ablation of mTLL1, a member of the BMP1/TLD-like proteinases which is expressed in the hippocampus and cerebellum.   Secondly, we are using a candidate based approach to identify novel substrates for the BMP-1/TLD-like proteinases in the nervous system.  Recently, we've begun to investigate gliomedin, a Type II transmembrane protein shown to mediate Node of Ranvier formation in the peripheral nervous system, as a possible substrate for the BMP-1/TLD-like proteinases.

Eosinophil, a type of leukocyte, is thought to contribute to the pathology of allergic disorders including asthma. Interleukin-5 (IL-5) is a primary eosinophil-active cytokine and it regulates many aspects of eosinophil biology. In vivo and in vitro studies have shown that IL-5 can potentiate cell responsiveness to subsequent chemokine stimulation in human eosinophils, a process known as "priming". Eosinophils isolated from asthmatic patients show increased release of cytotoxic granule proteins and enhanced production of proinflammatory mediators in response to chemokine stimulation. The increased cellular responses contribute to the chronic inflammation in asthma.  Although priming has been known for years, the intracellular mechanism is not yet clear.  My study suggests that IL-5 not only potentiates chemokine receptor mediated pathways but also pathways of other G-Protein Coupled Receptors (GPCRs) in human eosinophils. Preliminary data also support my hypothesis that Src kinase family members and b-arrestin are important intracellular players for priming process.

Isolation and Characterization of Ventricular Cardiomyocytes (CMs) derived from human Embryonic Stem Cells (hESCs)

Human embryonic stem cells are pluripotent, and can differentiate into CMs in vitro.  These CMs are spontaneously beating, displaying characteristics of embryonic cardiac muscle. It has been shown that hESC-derived CMs can have action potentials resembling embryonic atrial, ventricular and sinus-nodal action potentials. Traditionally, myocytes are isolated by microdissection of the beating area. This method cannot ensure the isolation of cardiac tissue exclusively. It is practically impossible to isolate the beating area exclusively, and some surrounding tissue (that would be non-cardiac) gets excised as well with the beating area. Another consideration is the purity of the cells in the beating area. It is unknown whether these beating areas are made up exclusively of cardiac tissue. There might be some non-cardiac cells present in a beating area, which cannot be distinguished from the majority of the beating cells. Various groups have tried to use the expression of a selective cassette under a cardiac-specific marker to isolate CMs from differentiating murine ESCs. MYL2 and IRX4 are both expressed exclusively in cardaic ventricles. These might be used as cardiac-ventricular specific markers to isolate ventricular myocytes from hESCs.

Macrophage activation is an essential part of normal immune function that is often regulated by select products from activated Type 1 or Type 2 T helper lymphocytes at sites of inflammation. Classical macrophage activation involves priming of na=EFve monocytic cells with interferon-gamma (IFN-_) that is released from T helper-1 (TH1) cells in response to invasive bacterial, protozoal and viral infections, but can sometimes lead to pathologies such as sepsis, autoimmunity, or graft rejection. When the host is invaded by extracellular parasites or helminths, or if the host is asthmatic or atopic, T helper-2 (TH2) cells readily release interleukin (IL)-4 which primes macrophages to release different products than their classically primed counterparts and is often referred to as "alternative" activation. 

Infection and inflammation are often accompanied by the release of a high concentration of extracellular nucleotides, thereby supplying a source of ligands for nucleotide receptors that are present on the cell surface of many immune cells, such as monocytes and macrophages. One nucleotide receptor that is expressed on leukocytes is the P2X7 purinoreceptor (P2X7R). Interestingly, activity of the P2X7R has been demonstrated to enhance macrophage responsiveness to pro-inflammatory stimuli, which is consistent with classical TH1 macrophage activation. Intriguingly, when the P2X7R activity is attenuated, either through genetic polymorphisms, pharmacological inhibitors, or generated knockouts, hallmarks of TH1-induced macrophage activation are also attenuated (e.g.,  decrease in TNF-_ production) and a more TH2-induced macrophage phenotype develops (e.g.,   enhanced IL-10 release). Therefore it has been proposed that activation of the P2X7R is a marker of classical TH1-driven macrophage activation and attenuation of P2X7R activity leads to a more TH2-driven activated phenotype. Accordingly, I propose to test the hypothesis that the P2X7R signaling is a principal component of classical macrophage activation.

To address this hypothesis, I am first examining the potential role for the transcription factor cyclic AMP response element binding protein (CREB) in P2X7R-induced inflammatory mediator production, thereby providing a potential regulatory signaling mechanism for classically activated macrophages. In addition, it is of interest to know how P2X7R signaling is affected by different cytokines that would be present in different disease states (a.k.a. TH1-mediated versus TH2-mediated microenvironments). Accordingly,  I will examine the effect of a classical, TH1-predominant activator (e.g., IFN-_) and an alternative, TH2-predominant activator (e.g., IL-4) on the activation of select proteins found to be downstream of P2X7R activation using pharmacological and molecular approaches on various macrophage model systems. These studies will help provide a better understanding of the signaling capacity of differentially-primed macrophages as well as better understanding of what impact the P2X7R has in different disease states.

Cynthia Koziol
Bertics Lab
Monday, April 3, 2006
12:00 Noon
140 Bardeen

Eosinophils are terminally differentiated innate immune cells that have both advantageous and detrimental functions within the host.  It has been reported that eosinophils kill parasites and alert the immune system to presence of a foreign invaders, but eosinophils are also thought to contribute to disordered airway function that is characteristic of asthma.   Interleukin-5 (IL-5) strongly affects many aspects of eosinophil biology.  Expression of the IL-5 receptor has been detected on eosinophils and basophils only, giving IL-5 functions that are specific to only a few cell types.  Interleukin-5 mediates survival of peripheral blood eosinophils /in vitro/, suggesting that IL-5 potentiates the lifespan of airway eosinophils.  The potentiation of lifespan thereby contributes to the pathology of the disease through prolonging the presence of eosinophils, exacerbating the inflammation already found in the airways.  However, the mechanisms by which IL-5 mediates eosinophil survival have not been elucidated.  Expression of the survival promoting gene Pim-1 has been observed following IL-5 stimulation.  Our lab has demonstrated that STAT5 phosphorylation and activation occurs following IL-5 stimulation of peripheral blood eosinophils, and expression of Pim-1 is thought to be STAT5-dependent.

Accordingly, the overall hypothesis of my studies is that IL-5-dependent eosinophil survival is mediated through STAT5-dependent expression of the anti-apoptotic proteins, including Pim-1.

Bone morphogenetic protein 1 (BMP1) is an astacin-like metalloproteinase.  It is the founding member of a small group of metalloproteinases with similar domain structure and function.  These proteinases can be found in a wide variety of organisms, ranging from sea urchins to mammals.  Mammals contain four BMP1-like metalloproteinases, designated as BMP1, mammalian tolloid (TLD), mammalian tolloid-like 1 (TLL1), and mammalian tolloid-like 2 (TLL2).   These proteinases have been shown to be involved in the processing of several extracellular matrix proteins along with proteins involved in TGF-beta-like BMP signaling, such as chordin.  Chordin is an extracellular antagonist of BMP signaling.  This antagonism is accomplished by binding to the BMP in the extracellular space and preventing the BMP from binding to its cognative receptor and initiating signaling.  BMP1/TLL1 have been shown to play very important roles in this mechanism in that they are able to process chordin, releasing the BMP from its inactive complex and allowing signaling to commence.  It has been shown in both xenopus and zebrafish models that BMPs and chordin are involved in dorsal-ventral patterning of these organisms.  Chordin-like 1 (CHL1) and chordin-like 2 (CHL2) are two proteins with homology to chordin, which have been shown to bind a variety of BMP proteins and are also able to induce dorsalization in xenopus embryos upon injection of their mRNA.  This effect is very similar to that of chordin.  For these reasons, our lab has decided to further study these proteins and determine if they are substrates for BMP1/TLD-like proteinases.

The sigma receptor represents a ubiquitously expressed unique binding site in the CNS and is a member of the orphan receptor class for which no endogenous ligand is known. It is known, however, that the sigma receptor binds with high affinity to several classes of chemically unrelated ligands such as neurosteroids, neuroleptics, dextrobenzomorphans,and cocaine. Consequently, it is thought that the sigma receptor may mediate the immunosuppressant, antipsychotic, and neuroprotective effects of drugs. Functionally, sigma-1 receptor ligands have been shown to modulate voltage-gated K+ channels independently of G-proteins or kinases (1). They have also been shown to mediate calcium release from intracellular stores (2), regulate compartmentalization of lipids on ER (3), and have antitumour activity in vitro and in vivo (4).The sigma1 receptor has been shown to have three hydrophobic regions, two of which, have highly conserved residues between the sigma receptor and the yeast C8-C7 sterol isomerase (ERG2) (5). These regions have thus been termed steroid binding domains 1 and 2 (SBD1 and SBD2). Previous photoaffinity labeling work in our lab has implicated Asp 188 in SBD2 as a ligand-binding target using a 3-iodo-4-azidococaine photoprobe. Currently, we aim to probe the SBD1 region by synthesizing photoprobes that have their photoreactive group on moieties that we hypothesize are interacting with this domain. Therefore, N-alkyl-N'-aralkyl derivatives and trace amine derivatives, which both mimic the pharmacophore found in many sigma1 ligands have been synthesized in addition to an N-substituted cocaine derivative.  The binding affinities of these compounds have been characterized by competitive displacement against [3H] (+) pentazocine, which is selective for sigma1. These compounds have also been characterized functionally at the cellular level through whole cell voltage clamp methods.  At the whole animal level, preliminary data indicates these compounds can be tested in the open-field mouse assay.  Biochemically, preliminary data for one N-alkyl-N'-aralkyl derivative as a radioactive photoaffinity label shows sigma1-specific binding that can be protected by haloperidol, a neuroleptic with high affinity for sigma1 receptors.

References:

1) Wilke, R.A. et al. (1999). J. PHysiol. 517, 391-406.
2) Hayashi, T. and Su. T.P. (2001). PNAS. USA. 98, 491-496.
3) Hayashi, T. and Su, T.P.(2003). JPET. 306, 718-725.
4) Berthois, Y. et al. (2003). B.J. Cancer. 88, 438-446.
5) Jbilo, O. et al. (1997) JBC. 272(43):27107