Molecular and Cellular Pharmacology Student Seminars 2003-2004

Lina Kwong
Keely Lab
Pharmacology 901 Seminar
Monday, October 6, 2003
Biotechnology Center Auditorium

R-Ras in Integrin Signaling

R-Ras regulates integrin function, but its effects on integrin signaling pathways are not well understood. We have explored how the activation of R-Ras regulates integrin mediated events
leading to cell migration. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130Cas through a novel mechanism that differs from, but
synergizes with, the a2b1 integrin. We have also begun to examine how integrin subunits, especially a subunits, are associated with the activation of specific signaling pathways. Our
previous studies have shown that, unlike K-Ras which shows no substratum specificity for cell migration, R-Ras enhances epithelial cell migration via the a2b1, but not through the a5b1
integrin. We find that R-Ras induced focal adhesion formation was noticeably different between a2 and a5 integrins. In addition, constitutively activated R-Ras(38V) enhanced p130Cas
phosphorylation when cell were plated on collagen but not on fibronectin. These results begin to elucidate the mechanisms by which signaling pathways can regulate integrin a subunit
specificity.

Paul Nuzzi
Huttenlocher Loab
Pharmacology 901 Student Seminar
Tuesday, October 7, 2003
Biotechnology Center Auditorium
12:00 Noon

Neutrophil migration and chemotaxis are critical steps involved in innate immunity and the development of inflammation. We have examined the role that calcium-dependent cysteine protease, calpain plays during neutrophil migration. In the absence of exogenous stimulators neutrophils maintain high levels of calpain activity, display a spherical morphology and fail to adhere or migrate on extracellular matrix components such as fibrinogen (Fbg). Our results demonstrate that in their resting state, neutrophils display high levels of active calpain, with u-calpain as the predominant isoform. Using both exogenous chemical inhibitors, as well as an HIV-TAT fusion protein construct, we have been able to modulate calpain activity in primary neutrophils. Contrary to previously examined cell types, calpain inhibition leads to enhanced random migration in the absence of exogenous activators, while disrupting neutrophil chemotaxis.

These findings were further supported using the chemotactic organism Dictyostellium Discoideum, in which both the calpain inhibitor (ALLN) and a calpain knockout line using both pertussis toxin and LY294002, suggest that calpain my function downstream of GPCRs and the PI3K pathway. Interestingly, calpain inhibition also leads to an increase in the cellular levels of active Cdc42 and Rac, suggesting a possible mechanism of action. These data suggest calpain is important in neutrophil migrataion and function in neutrophils as a negative regulator. We propose, that constitutively high calpain activity in resting neutrophils normally supresses cell protrusion and migration. Calpain may function downstream of GPCR and PI3K to regulate migration by inhibiting Cdc2 and Rac. Together, these data reveal a novel function for calpain and suggest that neutrophils can regulate polarization and protrusion during chemotactic migration by spatially regulating calpain activity.

Hogune Im
Bresnick Lab
Pharmacology 901 Student Seminar
Monday, October 20, 2003
Biotech Center - 1111 Auditorium
Noon

Dynamic Regulation of Histone H3 Methylation at Lysine 79 Within A Tissue-Specific Chromatin Domain

Posttranslational modifications of individual lysine residues of core histones can exert unique functional consequences. For example, methylation of histone H3 at lysine 79 (H3-meK79)
has recently been implicated in gene silencing in Saccharomyces cerevisiae. However, the distribution and function of H3-meK79 in mammalian chromatin is not known. We found that
H3-meK79 has a variable distribution within the murine b-globin locus in adult erythroid cells, being preferentially enriched at the active bmajor gene. By contrast, acetylated H3 and
H4, and H3 methylated at lysine 4 were enriched both at bmajor and at the upstream locus control region. H3-meK79 was also enriched at the active cad gene, while the
transcriptionally inactive loci necdin and MyoD1 contained very little H3-meK79. As the pattern of H3-meK79 at the b-globin locus differed between adult and embryonic erythroid
cells, establishment and/or maintenance of H3-meK79 was developmentally dynamic. Genetic complementation analysis in null cells lacking the erythroid and megakaryocyte-specific
transcription factor p45/NF-E2 showed that p45/NF-E2 preferentially establishes H3-meK79 at the bmajor promoter. These results support a model in which H3-meK79 is strongly
enriched in mammalian chromatin at active genes, but not uniformly throughout active chromatin domains. As H3-meK79 is highly regulated at the b-globin locus, we propose that the
murine ortholog of Disruptor of Telomeric Silencing-Like (mDOT1L) methyltransferase, which synthesizes H3-meK79, regulates b-globin transcription.

References:
1. Im, H., Park, C., Feng, Q., Johnson, K. D., Kiekhaefer, C. M., Choi, K., Zhang, Y., and Bresnick, E. H. (2003) J Biol Chem 278, 18346-18352
2. van Leeuwen, F., Gafken, P. R., and Gottschling, D. E. (2002) Cell 109, 745-756

Yukiko Muroi
Meyer Jackson Lab
Pharmacology 901 Student Seminar
Monday, November 3, 2003
Biotechnology Center Auditorium
12:00 Noon

Investigation of Conformational Changes in Ligand-Gated Ion Channels

The GABAA (g-aminobutyric acid type A) receptor is a member of a superfamily of ligand-gated ion channels which includes glycine (Gly), serotonin (5-HT3), GABAC, and nicotinic
acetylcholine receptors (nACh). Neurotransmitter binding causes an allosteric transition in these receptors in which the channel opens. This is a key step in fast synaptic transmission
between neurons. Although work with radioactive ligands, site-directed mutagenesis, and electrophysiology has clarified receptor structure-function relations, information about the structural
dynamics underlying receptor activation is very limited. Using the in vivo nonsense-suppression method of fluorescent unnatural amino acid incorporation, site-specific fluorescent labeling,
and simultaneous electrophysiological analysis, the conformational changes and intermediate states of the GABAA receptor will be investigated. When the structural transitions in the
receptor involve transferring domains/amino acids residues from a polar to nonpolar environment or vice versa, an environment sensitive fluorophore can report this movement with a change
in fluorescence intensity.

We will examine membrane penetration of membrane spanning segments. Environment sensitive fluorophore will be incorporated into the membrane spanning segments as well as flanking
segments. Changes in fluorescence associated with the transitions among the resting, open, and desensitized states will be used to assess changes in depth of membrane penetration.
Moreover, we will determine whether the allosteric transitions in the GABAA receptors are symmetrical. Fluorophore will be incorporated into the ligand binding sites, and changes in
fluorescence following ligand presentation will be monitored. Changes in fluorescence will be compared for ligand binding sites and the homologous sites in different subunits. These results
will be compared with the predictions of structural models of the conformational coupling between the channel gating and the membrane spanning segments/ ligand binding sites. Real time
measurement of molecular rearrangements in the GABAA receptor will enhance our understanding of ligand-gated ion channel function.

Bernice Lin
Bradfield Lab
Pharmacology 901 Student Seminar
Monday, November 10, 2003
Biotechnology Center Auditorium
12:00 Noon

The Role of Chaperones in the Aryl Hydrocarbon Receptor Signaling Pathway

The aryl hydrocarbon receptor (AHR) is a prototype member of the PAS (Per-Arnt-Sim) superfamily of proteins. In addition to the signature PAS domain of 200 amino acids, members of this superfamily commonly have a bHLH domain that recognizes and binds specific DNA sequences in cognate enhancer elements. The Aryl Hydrocarbon Receptor (AHR) is known for its ability to mediate toxic effects of industrial byproducts such as halogenated and polycyclic aromatics (i.e. Dioxin). The outcomes of exposure to AHR agonists include, induction of xenobiotic metabolizing enzymes, teratogenicity, immune suppression, tumor promotion, and cancer. Despite the recognized involvement of AHR in such effects, the molecular pathway to toxicity still remains unclear. What also remains unresolved is the true physiological role of the AHR.

In the unliganded state, the AHR exists in the cytoplasm, bound to chaperone proteins Ara9 and a dimer of hsp90. Previous work has shown hsp90's involvement with AHR to be important in
maintaining the correct conformational state for ligand binding. Although the role of Ara9 in the AHR pathway is unclear, we propose that It may be acting to mask the nuclear localization sequence
(NLS) of the AHR; thereby inhibiting nuclear transport. Previous work in our lab gives evidence for a masked nuclear localization signal that can be unmasked by insertions within the a-helix at the basic region of the bHLH domain of AHR. As previously stated, Ara9 may be responsible for masking of this nuclear localization signal. We have constructed several DNA-binding mutants of the AHR by inserting alanines at this bHLH site to rotate the helix and uncover a masking/unmasking phenomenon.

Developmentally, Ara9 is expressed prior to, as well as in tissues distinct from AHR expression. Therefore, Ara9 may play a role outside of the AHR pathway. One ongoing project is the creation of a conditional Ara9 knockout mouse. We hope that a knockout will shed light on the importance of Ara9 in the AHR pathway, as well as provide insight into other roles for the Ara9 protein in development.

1. Carver, L.A., LaPres, J.J., Jain, S., Dunham, E.E., and Bradfield, C.A. (1998) J. Biol. Chem. 273:33580-87.

2. Gu, Y-Z, Hogenesch, J.B., and Bradfield, C.A. (2000) Annu. Rev. Pharmacol. Toxicol. 40:519-61.

3. Petrulis, J.R., Hord, N.G., and Perdew, G.H. (2000) J. Biol. Chem. 275:37448-53.

Mike Gonzales
Richard Anderson Lab
Pharmacology 901 Student Seminar
Monday, November 17, 2003
Biotechnology Center Auditorium
12:00 Noon

Nuclear Phosphoinositide Signaling and mRNA Metabolism

Phosphatidylinositol (4,5)-bisphosphate (PI4,5P2) and its derived second messengers play a pivotal role in many physiological processes at cytoplasmic membranes, ranging from regulation of membrane trafficking and the homeostasis of intracellular compartments by PI4,5P21 to signaling via the generation of the second messengers inositol (1,4,5)-trisphosphate (InsP3) and diacylglycerol (DAG). However, phosphoinositides and their biosynthetic machinery are also present in the nucleus. The regulation of the nuclear and cytosolic phosphoinositide pools appears to be largely independent, suggesting that the nucleus constitutes a functionally distinct compartment for inositol phospholipid signaling and metabolism. Several roles have been attributed to nuclear phosphoinositides. First, PI4,5P2 has been implicated in chromatin remodeling pathways. Secondly, nuclear generation of InsP3 and DAG by phospholipase C (PLC) has been implicated in regulation of DNA replication, mRNA transcription, mRNA export, apoptosis and chromatin condensation. We wish to understand the processes regulated by nuclear phosphoinositide signaling. Preliminary data from our lab indicates that a phosphoinositide signaling pathway involving both lipid and soluble phosphoinositides may be regulating various aspects of mRNA transcription, processing and metabolism.

Jaehyung (Gus) Cho
Deane Mosher's Lab
Pharmacology 901 Student Seminar
Monday, December 1, 2003
Biotechnology Center Auditorium
12:00 Noon

Matrix assembly of fibronectin (FN) is associated with diverse processes including inflammation, wound repair, malignant metastasis, microorganism attachment and thrombosis. The N-terminal 70 kDa region of FN (70K) plays an important role in initial binding of FN to cell surfaces and in matrix assembly of FN on many types of cells. The cell surface binding site for 70K, however, remains to be characterized.

A role of FN in platelet functions has long been suspected. Our group firstly demonstrated that adherent platelets have the capacity to assemble FN (1). Recently, a role of FN in platelet aggregation in injured arterioles was uncovered in mice lacking both von Willebrand factor and fibrinogen (FG) (2). Plasma FN was shown to promote thrombus growth and stability in injured arterioles (3). It has been suggested that internalized plasma FN might cause loosely packed aggregation of platelets. We hypothesize that the effect of FN on platelets be related to FN matrix assembly.

As one strategy to identify a platelet receptor for 70K, ligand blotting with FITC-70K was performed in gel-filtered platelet fractions in 1- and 2-D gel electrophoresis. FITC-70K specifically bound to several proteins (40-, 65-, 80-, and 140-kDa) in the platelet fractions. The 140-kDa region contained intracellular molecules such as filamin A and talin as well as membrane proteins such as GPIba when analyzed by mass spectrometry. The 65- and 80-kDa proteins were identified as FG Bb and Aa polypeptides, respectively. Although recombinant or purified talin head domain and filamin, and FG bind to 70K by ligand blotting in vitro, immunochemical staining of these proteins did not reveal colocalization with the linear arrays of 70K. Currently, I am experimenting with additional strategies to characterize the platelet receptor for 70K by detergent extraction. My preliminary results suggest that 0.1% Triton with 6 M urea after stabilization of bound 70K with the cell-impermeable cross-linker, BS3 allows extraction but leaves behind 70K covalently linked to molecular of 3-MDa in SDS-PAGE gels. This extraordinary complex will be further characterized by proteolysis and mass spectrometry.

1. Olorundare O.E. et al. Blood 2001;98:117-24
2. Ni H. et al. J Clin Invest 2000;106:385-92
3. Ni H et al. PNAS 2003;100:2415-9

Jianlin Chu
Bresnick Lab
Pharmacology 901 Seminar
Monday, November 24, 2003
Biotechnology Center Auditorium
12:00 Noon

Evidence that CBF1 Binding is Required for Notch-1-Mediated Repression of Activator Protein-1

Cell fate determination in invertebrate and vertebrate systems is regulated by the Notch signaling pathway. Four mammalian Notch genes, Notch1-4, encode differentially expressed transmembrane receptors, which play crucial roles in diverse biological processes including hematopoiesis and vasculogenesis. Activation of the Notch receptor results in release and subsequent nuclear translocation of the intracellular domain of Notch (NIC). Nuclear NIC regulates target gene transcription through interactions with numerous factors including CSL (CBF1/RBP-Jk in mammals, Su(H) in Drosophila, and Lag-1 in c. elegans) protein, mastermind, and ski-interacting protein (SKIP). Previously, we demonstrated that the human Notch1 intracellular domain (NIC-1) not only activates CSL-dependent transcription, but also represses activator protein-1 (AP-1) mediated transactivation [Chu et. al. (2002) J. Biol. Chem.]. AP-1 target genes play important roles in hematopoiesis, vasculogenesis, and immune cell function. We also discovered that the RAM (RBP- Jk associated molecule) domain is required for both NIC-1-mediated transactivation and repression. To investigate the sequence requirements for NIC-1-mediated repression, we systematically mutagenized the RAM domain. Analysis of the activities of the mutants by transient transfections revealed that the D1759-1778 mutant, lacking the first twenty amino acids of the N terminus of the RAM domain, phenocopied the RAM domain deletion mutant, which is impaired for both CSL-dependent transcription and AP-1 repression. The twenty-amino acid deletion mutant was incapable of binding CSL. Analysis of alanine substitution mutants revealed an essential role of eight amino acids in conferring repression, transactivation, and CSL binding. In addition, the studies revealed a correlation between AP-1 repression, CSL-dependent activation, and CSL binding, indicating the involvement of CSL in NIC-1-mediated repression of AP-1. Stably transfected pools of K562 cells were generated to analyze the activity of NIC-1 mutants in a chromatin context. We found that the cells could only tolerate very low-level expression of NIC-1 and NIC-1 mutants that retain repression/activation activities. Transcriptionally compromised NIC-1 mutants were expressed at high levels, whereas transcriptionally competent NIC-1 proteins were nearly undetectable. This result revealed a powerful cell sensing mechanism that suppresses the levels of transcriptionally competent NIC-1. Studies are underway to elucidate mechanisms underlying NIC 1-mediated repression of AP-1 and the control of NIC-1 levels. Loss-of-function analyses have revealed the crucial roles of Notch in hematopoiesis, vascular morphogenesis, and vascular remodeling. Similar in vivo analyses have shown critical roles of AP-1 in hematopoiesis, vascularization, and heart organogenesis. Based on these profound phenotypes, understanding the molecular mechanism of Notch-AP-1 signaling crosstalk is likely to have important physiological and pathophysiological implications

Kate Modzelewska
Patricia Keely Lab
Pharmacology 901 Seminar
Monday, December 8, 2003
Biotechnology Center Auditorium
12:00 Noon

Ack-1 signaling downstream of the a2b1 integrin

We have found that the Cdc42 effector - Ack-1 (Cdc42 associated kinase) enhances cell migration on collagen. Our goal is to elucidate the signaling pathway that mediates this effect. We established that Ack-1 phosphorylation was increased upon collagen stimulation.  Surprisingly, this event was not regulated by the Cdc42 GTPase.  We have found, however, that Ack-1 phosphorylation was regulated by both Src and FAK kinases.  Src kinase inhibitor PP2 decreased Ack-1 phosphorylation.  Ack-1 phosphorylation was also diminished in the SYF cells (Src, Yes, Fyn -/-) as compared to SYF cells over-expressing c-Src.  Phosphorylation of Ack-1 was minimal in the FAK -/- cells, which suggests a role for the kinase in Ack-1 activation.

To determine the role of Ack-1 in signaling downstream of the integrin receptor, we looked at Ack-1 effects on p130Cas mediated events downstream of the integrin receptor that lead to cell migration.  It has been established that phosphorylation of p130Cas is a key event in integrin signaling leading to cell migration.  We have found a role for Ack-1 in this event, since both wild type and kinase dead Ack-1 enhance p130Cas phosphorylation.  We determined that Ack-1 enhances the association of p130Cas with its effector Crk.  Furthermore, we established that Ack-1 enhances phosphorylation and binding to Crk of C3G, an exchange factor for the small GTPases Rap1 and R-Ras. Both Rap1 and R-Ras activation are implicated in cell migration. 

To further investigate the role of Ack-1 in integrin signaling, we looked at its association with focal adhesion kinase (FAK), Src kinase, Crk associated substrate (p130Cas) and the adaptor Crk.  Ack-1 could be co-immunoprecipitated with both FAK and Src, as well as with p130Cas and Crk. FAK, p130Cas and Crk association with Ack-1 is minimal in the absence of collagen and increases significantly upon a2b1 integrin stimulation.  This is in contrast to the association of Ack-1 with Src, which seems not to be regulated by collagen.  Interestingly, Ack-1 association with Src, FAK, p130Cas and Crk appears to be independent of Cdc42 activation state.  These interactions remain in place in both Src and FAK knockout cells, which suggests direct binding.  More specifically, in GST pulldown assays, Ack-1 SH3 domain associated with both FAK and p130Cas.  In addition, the SH3 domains of Src, p130Cas and the N-terminal domain of the adaptor molecule Crk associated with Ack-1. 

Our results suggest that Src and FAK regulate Ack-1 phosphorylation but not Ack-1 interactions with its signaling partners downstream of the _2_1 integrin and that Ack-1 contributes to the regulation p130Cas phosphorylation leading to enhanced complex with Crk and C3G as well as C3G phosphorylation.  We are now investigating whether this enhancement results in activation of Rap1 and/or R-Ras.--

Akhil Bhalla
Chapman Lab
Pharmacology 901 Student Seminar
Monday, December 15, 2003
Biotechnology Center Auditorium
12:00 Noon

Analysis of the divalent cation sensors that regulate exocytosis.

Synaptotagmin (syt) is a putative Ca2+ sensor proposed to trigger exocytosis in response to ca influx in nerve terminals. It has been proposed that syt triggers fusion via its interaction with membranes and SNAREs. This study focuses on syts shown to be expressed at significant levels in PC12 cells i.e. syts I and IX, as well as those present in trace amounts; syts IV and VII. I have determined the metal requirements for secretion from Pc12 cells and then asked if the metals that trigger release also activate the syts that are expressed. Finally a more defined system (reconstitution) is used as a functional read-out to ask if syt-membrane and syt-SNARE interactions important for membrane fusion? In this system two types of vesicles are used, v SNARES, which contain the synaptic vesicle protein VAMP, and t-SNARE vesicles that contain a complex of SNAP-25 and syntaxin. When these proteins assemble into a SNARE complex they catalyze membrane fusion in vitro. Preliminary data suggests that syt 1 triggers membrane fusion with a Ca2+ Ec50 resembling the Ca2+ requirement for a synapse that might harbor a low affinity sensor. Predictions made for syt 9 on the basis of biochemical evidence are similar to that of syt I. Other data suggests that syt VII on the other hand, might be a high affinity Ca2+ sensor that triggers release. The reconstituted system will be used to directly address this issue. Finally, preliminary data suggests that the ability of metals such as Sr2+ and Ba2+ to trigger membrane fusion varies with the syt isoforms. Further studies are needed to determine why such differences exists between syt isoforms.

Mechanism of Protein Kinase C anchoring in living cardiac myocytes and effect on contractile function

About 70% of the entire volume of the heart is composed of myocytes, which are the main players in the generation of myocardial contractility. Membrane receptors on cardiac myocytes receive signals from the outside and in turn, modulate cellular function. Protein Kinase C (PKC) is considered a major signal transducer in the regulation of cardiac excitation-contraction coupling by vasoactive peptides like endothelin-1 (ET-1). However, the precise identity of PKC isoforms that mediate changes in contractile function remains uncertain. In this study, the major two PKC isoforms, PKC-d and PKC-e, were tested to investigate the mechanism of PKC anchoring in myocytes and effect on cardiac muscle function. Translocation patterns of GFP fused PKCs (PKC GFP) upon stimulation with various PKC activators such as phorbol ester, arachidonic acid, diacylglycerol and ET-1 were studied in HEK 293 cells and living myocytes. It is important to better understand how different PKC isoforms respond to different signals and translocate to different cellular fractions.

Reference:
1. Localization of functional endothelin receptor signaling complexes in cardiac transverse tubules. Robu VG, Pfeiffer ES, Robia SL, Balijepalli RC, Pi Y, Kamp TJ, Walker JW. J Biol Chem. 2003 Nov 28;278(48):48154-61. Epub 2003 Sep 12.
2. Arachidonic acid stimulate protein kinase C-epsilon redistribution in heart cells.
Huang XP, Pi Y, Lokuta AJ, Greaser ML, Walker JW. J Cell Sci. 1997 Jul;110 ( Pt 14):1625-34.

Melissa Martowicz
Bresnick Lab
Pharmacology 901 Student Seminar
Monday, February 9, 2004
140 Bardeen
12:00 Noon

How does GATA Factor Interplay Control Hematopoiesis?

The GATA family of zinc finger transcription factors (GATA-1, -2, and -3) are critical regulators of hematopoiesis, whereas, GATA-4, -5, and -6 control the development of the heart and other organs. GATA-2 is more widely expressed than GATA-1, GATA-2 being expressed in hematopoietic progenitors, mast cells, megakaryocytes, endothelial cells, as well as in the CNS. GATA-2 knockout mice display an embryonic lethal phenotype with severe anemia, and the lethality occurs earlier than mice lacking GATA-1 (1). These genetic findings indicate that GATA-2 is critical at an earlier stage of hematopoiesis. Our goal is to understand the regulation of GATA-2 synthesis and the interplay between GATA factors, which controls hematopoiesis. Despite greater than eighty consensus GATA motifs in the GATA-2 locus, GATA-2 was shown by quantitative chromatin immunoprecipitation (ChIP) analysis to bind only the -2.8 kb and -1.8 kb regions, whereas GATA 1 binds only the -2.8 kb region in committed erythroid cells. Our laboratory developed a detailed molecular model for how the GATA-2 locus is controlled. This model involves positive autoregulation by GATA-2 binding to the -2.8 kb and -1.8 kb regions of its locus, and GATA-1 mediated displacement of GATA-2, resulting in repression(2,3). These studies were conducted in a GATA-1-null proerythroblast cell line, which contains a conditionally active estrogen receptor GATA-1 fusion protein (G1E-ER-GATA). During the GATA switch, histone modifications of the domain that are hallmarks of active chromatin are strongly reduced, and the levels of GATA-2 mRNA and protein decrease rapidly. Current work is aimed at further dissecting the mechanism of repression and activation of the locus, to provide a molecular explanation for how GATA factors control the differentiation of hematopoietic cells into diverse blood cells.

1. Tsai, F.Y., et al., An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature, 1994. 371(6494): p. 221-6.

2. Grass, J.A., et al., 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.

3. Pal, S. P., et al., Coregulator-dependent facilitation of chromatin occupancy by GATA-1. Proc Natl Acad Sci U S A, 2003 101(4): p.980-5.

Aude S. Ada Nguema and Patricia J. Keely
Molecular and Cellular Pharmcaology Program and the Department of Pharmacology
University of Wisconsin-Madison, Madison, Wisconsin 53706

The Small GTPase R-Ras Increases Integrin Adhesiveness by Modulating
Intracellular Calcium and the Actin Cytoskeleton.

R-ras, an atypical member of the Ras subfamily of small GTPases, has been shown to strengthen integrin-mediated adhesion through an unknown mechanism. Our primary research goal is to decipher the signal transduction pathways that connect R-ras activation to integrins. MCF10A cells were stably transfected with a constitutively active R-ras (R-Ras38V) or control vector, and the dynamics of the actin cytoskeleton were monitored during cell spreading. R-ras38V-expressing cells exhibited abundant lamellopodia formation, in contrast to control cells which exhibit both lamellopodia and filopodia. Because actin-capping protein activity must be reduced for filopodia formation, the absence of filopodia in R-ras38V cells suggests a primary defect in actin capping activity. We then used a range of pharmacological actin inhibitors to demonstrate an increase in actin-capping protein activity in R-ras 38V cells. Since many actin-capping proteins, including gelsolin, are regulated by calcium, we determined whether R-ras signaling to the actin cytoskeleton is calcium-dependent. R-ras signaling was found to be inhibited by chelating intracellular calcium. Furthermore calcium imaging revealed that R-ras38V reduced the amount of releasable calcium from the endoplasmic reticulum, suggesting that intracellular calcium levels are misregulated. Overall, our data indicate that R-ras affects integrin indirectly by regulating intracellular calcium concentrations and the changes in cytosolic calcium regulate actin dynamics and integrin activation directly.

Reema Jasuja
Greenspan Lab
Pharmacology 901 Seminar
Monday, February 23, 2004
Biotechnology Center Auditorium
12:00 Noon

Prodomains of BMP-1/mTolloid like proteases in extracellular matrix deposition and growth factor signaling

When bone morphogenetic activity in bone extracts was highly purified, it was found to correspond to peptides whose sequences led to cloning of cDNAs for the Bone Morphogenetic Proteins (BMPs). BMP-1 was found to be an extracellular zinc dependent metalloprotease, a prototype of a family of proteases implicated in pattern formation during development, while all other BMPs (which now number over 20) are TGFbeta-like molecules. BMP-1 biosynthetically processes precursors into the mature functional forms of various proteins important for the formation of extracellular matrix. Such proteins include the major and minor fibrillar collagens, laminin 5, lysyl oxidase, and biglycan. It also proteolytically processes chordin, a TGFbeta like BMP antagonist and thus enhances 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 fo rm of BMP-1 that co-purify with TGFbeta-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 but is proteolytically inactive. Moreover, ProBMP-1SQQ was found to act as a dominant negative version of BMP-1, suggesting tight binding of substrates in the absence of proteolytic activity. We have shown that ProBMP-1SQQ binds to TGFbeta-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 culture d cells. Thus the prodomain of BMP-1 may play an important regulatory role in BMP signaling.

Ashley Doan
Huttenlocher Lab
Pharmacology 901 Student Seminar
Monday, March 1, 2004
140 Bardeen
12:00 Noon

Characterization of RACK1, a regulator of adhesion, protrusion and cell migration

RACK1 is an adaptor protein that binds to the integrin cytoplasmic domain and to activated PKC. RACK1 has also been shown to bind and inhibit Src kinases (1). We have found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We have also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site (2). To further characterize the role of RACK1 in focal adhesion and cell migration, we used siRNA approaches to knockdown endogenous RACK1. Cells transfected with RACK1 siRNA oligo, but not control siRNA oligo showed reduced cell spreading. In addition, these knockdown cells display enhanced transient protrusions, a loss of lamellipodia, and are less motile. We believe that MARCKS may be a protein downstream of RACK1 that regulates cell spreading and migration. MARCKS is a major PKC substrate that has been shown to play a role in cell spreading (3). We have preliminary data that suggest that RACK1 plays a role in MARCKs phosphorylation and cellular function. Together our findings support an important role for RACK1 in cell spreading and migration.

1.Chang, B.Y. et al. Mol. Cell. Biol. 18, 3245-3256 (1998).
2. Cox, E. et al. Mol. Bio. Cell 14, 658-669 (2003).
3. Disatnik, Marie-Helene, et al. Journal of Cell Science 115,
2151-2163 (2002).

StAR: regulation and function in steroidogenesis

Steroidogenesis is the process through which cholesterol is converted to steroid hormones by the action of a series of enzymes in specialized steroidogenic cells. The rate-limiting step in steroidogenesis is the transport of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) where the enzyme for the first committed step to steroidogenesis, cytochrome p450scc, is located. cAMP acutely stimulates steroidogenesis by greatly enhancing cholesterol uptake into IMM in a very short time frame. The steroidogenic acute regulatory protein (StAR) is a vital player in the process and most extensively studied in the literature, but other proteins have also been implicated to mediate cAMP-stimulated cholesterol uptake. We have found that rodent StAR gene is mainly expressed as two mRNA isoforms, 1.6kb and 3.4kb respectively, which only differ in 3'UTR region. cAMP stimulates these two mRNA species through different kinetics, and we hypothesize they are regulated differently through mRNA stability mechanisms. Preliminary data from luciferase-StAR3'UTR chimeric constructs suggest the 3'UTR of 3.4kb StAR might confer instability to luciferase gene. Two further approaches are currently undertaken to study whether mRNA stability plays a role in their regulation, the gene gun method and IPTG-inducible stable transfection cell line. In addition, we have recently found that overexpression of StAR in sterodogenic cells does not lead to increased steroidogenesis. On the contrary, overexpressed StAR act as a dominant negative factor to suppress cAMP-stimulated steroidogenesis in these cells. This is inconsistent with the view that StAR is the solely rate-limiting factor in steroidogenesis, and may suggest that StAR is in complex with other rate-limiting factors to mediate cAMP-stimulated steroidogenesis.

DeannaLee M. Beauvais
Rapraeger Lab
Pharmacology 901 Student Seminar
Monday, March 29, 2004
140 Bardeen
12:00 Noon

The Syndecan-1 Ectodomain is Required for avb3 Integrin-Dependent Signaling in Human Mammary Carcinoma Cell Spreading

Syndecans engage the extracellular matrix (ECM) via their heparan sulfate chains. However, their core proteins likely mediate syndecans’ assembly into adhesion signaling complexes. We find that MDA-MB-231 and MDA-MB-435 mammary carcinoma cells spread when plated on syndecan-1 (S1)-specific antibody, B-B4. This syndecan-mediated spreading occurs without integrin ligation, yet requires activated avb3 integrins since spreading is inhibited by avb3-blocking antibody, LM609. Addition of recombinant murine S1 ectodomain (mS1ED), which is not recognized by mAb B-B4, has no effect on cell adhesion but blocks cell spreading suggesting that mS1ED disrupts a functional signaling complex involving syndecan-1 and avb3 integrins. Competition with other syndecan ectodomains is without effect. Interestingly, avb3-dependent spreading and migration of MDA-MB-231 and –435 cells in response to vitronectin (VN), an ECM ligand that engages both syndecan and integrin, is also blocked by soluble mS1ED or polyclonal anti-S1ED antibodies. In contrast, a5b1-dependent spreading and migration on fibronectin (FN) is unaffected. This suggests that mS1ED and anti-S1ED antibodies specifically perturb avb3-dependent signaling. Indeed, MCF-7 cells, which display no avb3 integrin activity and instead use avb1 to spread on VN, are unaffected by treatment with mS1ED or anti-S1ED antibodies. These cells also fail to spread when bound to mAb B-B4, indicating that these cells lack a functional syndecan-avb3 integrin complex. Downregulation of human syndecan-1 expression by siRNA blocks MDA-MB-231 cell spreading and migration on VN, but not on FN. Spreading and migration on VN is rescued in cells expressing a murine syndecan-1 construct that consists of the mS1ED alone fused to the GPI tail of rat glypican-1 indicating that the S1ED alone is sufficient for signaling. Indeed, mutational analysis traces syndecan-1’s signaling activity to 35 amino acids unique to the central region of the S1ED. These data suggest that syndecan-1 and avb3 integrins are functionally coupled via the S1ED and this coupling is required for both syndecan-1 and avb3 integrin signaling. As such, in response to an organized ECM, syndecan-1’s association into a signaling complex with avb3 integrins is likely to be a critical regulator of cell migration and invasion in development and in cancer.

Matt Marengo
Wassarman Lab
Pharmacology 901 Student Seminar
Monday, April 5, 2004
140 Bardeen
12:00 Noon

TAF1 Isoforms in the Context of a Dynamic TFIID Complex

We are interested in understanding how the TAF1 subunit of the general transcription factor TFIID contributes to transcriptional activation. TFIID serves three major roles during transcriptional activation. It acts as a transcriptional coactivator by binding transcription factors, it binds promoter sequences, and it post-translationally modifies histones to modify chromatin structure. Ten years ago, TFIID was largely thought to be a static, unregulated complex. However, recent studies have made it clear that TFIID composition is dynamic, and different TFIID complexes regulate the transcription of different genes. TAF1 is an evolutionarily conserved protein with two kinase domains, a ubac domain, two bromodomains, and a histone acetyltransferase domain. In Drosophila melanogaster, there is a single TAF1 gene, but the TAF1 mRNA is alternatively spliced to encode four TAF1 protein isoforms. Preliminary data indicate that the isoforms are differentially expressed in Drosophila tissues and in response to cell stress events. We are testing the hypothesis that the inclusion of alternate TAF1 isoforms alters the function of TFIID at specific genes

Bryan Glaser
Burgess Lab
Pharmacology 901 Student Seminar
Monday, April 12, 2004
140 Bardeen
12:00 Noon

Binding Factors and Drug Discovery:

Interaction of E. coli Sigma Factors with Core RNA Polymerase 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. 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 information we have started to determine the strength of interaction of each sigma factor with core RNAP and can begin to examine how small molecules (ppGpp, small RNAs, etc) alter the binding affinities. A similar assay has also been used to screen a library of 16,000 small molecules to find inhibitors of the interaction of core RNAP with sigma 70, the sigma factor responsible for regulation of housekeeping genes, as a possible new class of antibiotics.

Scott LeBlanc
Svaren Lab
Pharmacology 901 Student Seminar
Monday, April 12, 2004
140 Bardeen
12:00 Noon

Control of lipid biosynthetic gene expression by Egr2 and SREBP transactivators during peripheral nerve myelination

Myelination of peripheral nerves by Schwann cells is coordinated by transcriptional regulatory proteins that trigger various genetic events in the synthesis and compaction of myelin. One central aspect of myelin formation is the requirement for a large amount of lipid and cholesterol biosynthesis, and recent experiments have suggested that Egr2 may trigger induction of cholesterol and lipid biosynthetic enzymes at the transcriptional level. To elucidate the control of cholesterol and lipid biosynthetic genes during peripheral nerve myelination, we have investigated the developmental expression of several components of the Sterol Regulatory Element Binding Protein (SREBP) pathway, which controls expression of cholesterol and lipid biosynthetic genes. We observed a very significant induction of both SREBP1 and SREBP2 mRNA, as well as their target genes, over several timepoints of myelination in sciatic nerve, suggesting that the SREBP transactivators are important regulators in the myelination process. In addition, we investigated how expression of the SREBP pathway components is affected by the absence of Egr2. Although the expression levels of many lipid/cholesterol biosynthetic genes are dramatically reduced in sciatic nerves from mice with a targeted disruption of Egr2, mRNA levels of the SREBP transactivators do not change significantly. Instead, we found that Egr2 and SREBP2 can synergistically activate the HMG-CoA reductase, Lanosterol-14-alpha demethylase and Stearoyl-CoA desaturase 2 promoters. Therefore, our data indicate that Egr2 can interact with SREBP2 to directly regulate cholesterol/lipid biosynthetic genes as part of the myelination program.

Gennifer Mager
Svaren Lab
Pharmacology 901 Student Seminar
Monday, April 19th, 2004
140 Bardeen
12:00 Noon

Repression Egr2 Target Genes: A Nab2-NuRD Connection

The Early Growth Response (EGR) gene family is a set of immediate-early genes that respond rapidly to a wide variety of extracellular stimuli. All family members possess a three C2H2 zinc finger DNA-binding domain homology that recognizes a GC-rich sequence in the promoters of target genes. Targeted knockouts have been made for all four EGR family members, and the most dramatic phenotype is displayed by EGR2 null mice, which exhibit defects in hindbrain patterning, bone formation, and peripheral nerve myelination. Interestingly, several mutations in EGR2 have been linked to human peripheral myelinopathies, such as Charcot-Marie Tooth (CMT), Dejerene Sottas (DSS), and Congenital Hypomyelinating Neuropathy (CHN). One such EGR2 mutation was found in the R1 inhibitory domain of EGR2, and this mutation prevents EGR2 from being bound by a family of corepressors, the NGFI-A/Egr1 Binding (NAB) proteins. Nab1 and Nab2 comprise a family of nuclear cofactors, which repress the ability of EGR2 to activate its target genes. However, the function and mechanism by which Nab proteins repress EGR2 is poorly defined. Preliminary data obtained by our lab suggest that Nab2 interacts with a protein component of the Nucleosome Remodeling histone Deacetylase (NuRD) complex. The NuRD complex is one of several such complexes that utilize ATP to reorganize chromatin structure. We are investigating this postulated association of Nab2 and NuRD, and how this complex would influence the repressive function of Nab2.

Angie Mabb
Miyamoto Lab
Pharmacology 901 Student Seminar
Monday, May 3, 2004
140 Bardeen
12:00 Noon

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 at the cell surface such as tumor necrosis factor alpha (TNFa), interleukin-1 (IL-1), and lipopolysacharride (LPS). NF-kB has also been shown to be activated in response to various DNA damaging agents, such as ionizing radiation as well as chemotherapeutic drugs, such as camptothecin and etoposide. NF-kB activation in response to DNA damaging agents generally results in activation of anti-apoptotic genes such as the c-IAP and Bcl-2 family members, which promote cell survival. Although the early key molecular events involved in NF-kB activation with various stimuli are diverse, they all appear to converge at a common signaling pathway. The key component in this signaling pathway is the IKK complex. One essential component of the IKK complex needed to activate NF-kB in response to all known stimuli is NEMO. However, how NEMO is modified to permit activation of these various pathways is not well understood. Our lab has recently identified that SUMO modification of NEMO is crucial for selective NF-kB activation in response to DNA damaging agents. The mechanism responsible for SUMOylation on a protein substrate requires an activating enzyme (E1), SUMO conjugating enzyme (E2), and a SUMO ligase (E3). Currently, only 1 E1 and E2 protein have been identified. However, there are multiple E3's which are the determining factors for substrate target/s specificity. Using an siRNA screen, we have identified a SUMO E3 that is essential for DNA damage-induced activation but is dispensable for NF-kB activation with TNFa stimulation. Overexpression of this ligase enhances both basal and DNA damage-induced NF-kB activation as well as enhances NEMO SUMOylation. This thus provides strong evidence that this SUMO E3 is the SUMO E3 responsible for NEMO SUMOylation. As a result, we propose that this SUMO E3 may provide a unique target for various anticancer agents designed to treat a variety of human cancers.
1.) Karin et al. Ann Rev. Immunology 2000. 18:621-663.
2.) Melchior et al. Trends in Biochem. Sci. 2003. 28 (11): 612-618.
3.) Huang et al. Cell 2003. 115(5): 565-76.

Subu Ramachandran
Ruoho Lab
Pharmacology 901 Student Seminar
Monday, May 10, 2004
140 Bardeen
12:00 Noon

Purification and preliminary characterization of the Sigma receptor

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), immunomodulatory effects by upregulation of
IL10 (3) and antitumour effects both in vitro and in vivo (4). 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 (5). Recently sigma 1 receptor knockout mice have been generated and do not display any overt phenotype (6).

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. After purifying the fusion protein on an amylose column it is cleaved with Factor Xa, to obtain MBP and sigma receptor. The sigma receptor
along with the uncleaved material is then purified on a Ni affinity column. The material eluted from the column is further purified using an anti-MBP antibody linked to sepharose to remove the
uncleaved MBP-sigma fusion. The sigma receptor purified by this method is a 26 kDa polypeptide as assessed by SDS-PAGE and can be specifically photoaffinity labeled with sigma receptor
photoprobe (125I) iodo azido cocaine. To determine the proportion of the receptor in natively folded state, we are currently performing radioligand binding studies with a sigma receptor ligand
[3H] (+) pentazocine and affinity chromatography.

References:

1. Hayashi, T and Su, T. P. (2001). Regulating ankyrin dynamics: roles of sigma 1 receptors. Proc. Natl. Acad. Sci. USA. 98, 491-496.
2. Wilke, R. A., Mehta, R. P., Chen, Y., Ruoho, A. E., and Jackson, M. B. (1999a). K+ channels modulation in rodent neurohypophysial nerve terminals by sigma receptors and not by
dopamine receptors. J. Physiol. 517, 391-406.
3. Zhu, Li., X. et al. (2003). IL-10 mediates sigma 1 receptor dependent suppression of antitumour immunity. J. Immunology. 170, 3585-3591.
4. Berthois, Y., Bourrie, B., Gliegue, S., Vidal, H., Carayon, P., Martin, P. M. and Casellas, P. (2003). SR31747A is a sigma receptor ligand exhibiting antitumoural activity both in vitro and in
vivo. B. J. Cancer. 88, 438-446.
5. Aydar, E., Palmer, C. P., Klyachko, V. A. and Jackson, M. B. (2002). The sigma receptor as a ligand regulated auxiliary potassium channel subunit. Neuron, 34, 399-410.
6. Langa, F., Codony, X., Tokvar, V., Lovado A., Gimenez, E., Cozar, P., Cantero, M., Dordal, A., Herdanadez, E., Perez, R., Monroy, X., Zamanillo, D., Guitart, X. and Montoliu, L. (2003).
Generation and phenotypic analysis of sigma receptor type I (s1) knockout mice. Eur. J. Neuroscience, 18, 2188-2196.

Louis Lurie
Bresnick Lab
Pharmacology 901 Student Seminar
Monday, May 17, 2004
12:00 Noon
140 Bardeen
Toward an Understanding of Histone Deacetylase Specificities

The eleven class I and class II histone deacetylases (HDACs) catalyze the same enzymatic reaction. However, evidence suggests that structurally related HDACs have very different biological roles. For example, HDAC4 has been implicated in the control of neurogenesis and myogenesis. Since the determinants of HDAC specificities are largely unknown, I have initiated studies to define and mechanistically analyze such specificities in the context of erythropoiesis. I discovered that, as the transcription factor GATA-1 induces the differentiation of proerythroblasts, GATA-1 specifically upregulates expression of HDAC5 and HDAC11. Multiple other HDACs were constitutively expressed under these conditions. My current studies are designed to determine how GATA-1 coordinately induces HDAC5 and HDAC11 expression and to unravel the functional consequences of elevating HDAC5 and HDAC11 levels. Elucidating the mechanism underlying the dynamic expression of HDAC5 and HDAC11 and identifying specific target genes for these HDACs should provide important insights into how HDAC5 and HDAC11 are targeted to specific genetic loci and how they function.

References:

Yang, X.-J. and Seto, E. Collaborative spirit of histone deacetylases in regulating chromatin structure and gene expression Curr. Opin. Gen. Dev. 13, 143-153 (2003).

Emily Vaughan
Keely Lab
Pharmacology 901 Student Seminar
Monday, May 24, 2004
12:00 Noon
140 Bardeen

Proliferation of Mammary Epithelial Cells in 3D Collagen Gels

In vivo, cells respond to cues from the extracellular matrix (ECM) in order to regulate morphological behavior. In vitro, mammary epithelial cells that are cultured in rigid 3- dimensional
(3D) collagen gels continue to proliferate, while those cultured in a more flexible environment differentiate to form tubules. It is known that adhesion-mediated signaling can regulate the
G1 phase of the cell cycle. Specifically, cyclin D1, a G1 phase cyclin, can be regulated by Rho and FAK. Because of the downregulation of Rho and the increased pFAKY397
localization to focal adhesions in cells in rigid gels, it is possible that FAK and Rho regulate the cell cycle through Cyclin D1. We are now beginning to investigate the effects of matrix
rigidity on the cell cycle, and whether cyclin D1, FAK, and Rho are involved in this regulation.

References:

1. Wozniak, M., Desai, R., Solski, P., Der, C., Keely, P. ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix. The Journal of Cell Biology. 163, 583-595 (2003).
2. Zhao, J., Reiske, H., Guan, J. Regulation of the cell cycle by focal adhesion kinase. The Journal of Cell Biology. 143, 1997-2008 (1998).
3. Welsh, C., Roovers, K., Villanueva, J., Liu, Y., Schwartz, M., Assoian, R. Timing of cyclin D1 expressoin within G1 phase is controlled by Rho. Nature Cell Biology. 3, 950-957 (2001).

David Mellman
Anderson Lab
Pharmacology 901 Student Seminar
Monday, May 24, 2004
12:00 Noon
140 Bardeen

IalphaIP9: A Potential Role for this Novel Molecule in the Phosphoinositide Signaling Pathway at Nuclear Speckles

There are three types of Phosphoinositide Phosphate Kinases (PIP Kinases), Type I, II, and III. The Type I PIP Kinases are PI(4)P -5 Kinases, which generates PI(4,5)P2 utilizing PI(4)P as a
substrate. PI(4,5)P2 is ubiquitously expressed in mammalian cells. There are three separate isoforms of the Type I PIPK, PIPKI alpha, beta, and gamma, which have three distinct localization
patterns. PIPKIalpha localizes to nuclei in a speckled pattern, as all as at membrane ruffles. PIPKIbeta is mainly localized at perinuclear compartments such as the Golgi Apparatus, and the PIPKIgamma targets to focal adhesions. Interestingly, a phosphoinositide signaling pathway exists in the nucleus which is distinct from that in the cytosol. It appears that the PIPKIalpha localizes to nuclear compartments called nuclear speckles. Nuclear Speckles are enriched in pre-mRNA splicing proteins, though other proteins have been found to localize to nuclear speckles as well.

Using the C-terminal region of PIPKIalpha as a bait in a yeast two-hybrid screen, an interacting protein containing domains which suggests that it acts in the capacity of processing pre-mRNA was isolated, named IalphaIP9 (PIPKIalpha Interacting Protein #9). It was subsequently found that epitope-tagged versions of IalphaIP9 localize in nuclei, at nuclear speckles. Moreover, epitope tagged IalphaIP9 colocalizes at nuclear speckles with endogenous PIPKIalpha. It may be that IalphaIP9 is an isoform specific interacting protein partner with PIPKIalpha, which targets PIPKIalpha to nuclear speckles where the PIPKIalpha can generate PI(4,5)P2 in a temporal and spatial fashion, regulating the processing of pre-mRNA.

Ka Young Chung
Walker Lab
Pharmacology 901 Student Seminar
Monday, June 7, 2004
12:00 Noon
140 Bardeen

Induction of Purkinge Fibers from Chick Embryonic Cardiomyocytes by Endothelin

A regular heart beat is dependent on a specialized network of pacemaking and conductive cells. In the ventricles, the electrical impulses is transmitted from Purkinje fibers. During cardiac development, Purkinje fibers differentiate from cardiac myocytes in direct association with the developing endocardium and coronary arteries, but not with the venous system. This conversion of myocytes into Purkinje fibers was induced in vitro by exposing embryonic myocytes to endothelin 1 (ET-1), an endothelial cell-associated paracrine factor. In the embryonic chick heart, ET receptors are expressed throughout the whole heart, but endothelin-converting enzyme 1 (ECE1) is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber differentiation takes place. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. Endocardium and coronary arteries receive more hemodynamic forces than cardiac veins. The unique expression pattern of ECE1 in the embryonic heart suggests hemodynamic force may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. An antagonist for stretch-activated cation channels, induced delayed maturation of a ventricular conduction pathway with decreased the expression of ECE1 and Cx40. Conversely, pressure-overload results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers showing accelerated maturation of a ventricular conduction pathway.

References:

1. Gourdie RG et al., PNAS, (1998) 95 : 6815-6818
2. Takebayashi-Suzuki K et al., Development, (2000) 127 : 3523-3532
3. Hall CE et al., Development, (2004) 131 : 581-592

Amanda Branam
Greenspan Lab
Pharmacology 901 Student Seminar
Monday, June 7, 2004
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 extra cellular 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.

Cynthia Koziol
Bertics Lab
Pharmacology 901 Student Seminar
Monday, June 14, 2004
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 chemo attractant stimulation functions to activate Rac to promote eosinophil migration/chemotaxis and cause degranulation to occur.

Monica Gavala
Bertics Lab
Pharmacology 901 Student Seminar
Monday, June 14, 2004
12:00 Noon
140 Bardeen

P2X7 Nucleotide Receptor Regulation of LPS-induced Inflammatory Responses

Sepsis is defined as a spectrum of clinical conditions triggered by the immune response of a host to infection or trauma and characterized by systemic inflammation and coagulation. Septic shock is fatal in approximately 45% of those afflicted, making it the most common cause of death in intensive care units (ICUs). Recent estimates on septic shock indicate over 50% of inflicted patients were infected with gram-negative bacteria. Macrophage activation by Gram-negative bacterial endotoxin (lipopolysaccharide; LPS) is central to the mammalian inflammatory response that leads to the symptoms of septic shock. 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. Upon LPS stimulation, P2X7 expression level is enhanced. Although the physiological role of nucleotide receptors in the function of macrophages has not yet been fully established, recent research using pharmacological and molecular approaches have supported a role for P2X7 in multiple signaling events in immune cells.

The effect of P2X7 activation on LPS-induced inflammatory endpoints, such as the production of prostaglandins, in macrophages and monocytes (macrophage precursor cells) is currently under investigation. LPS-mediated cyclooxygenase-2 (COX-2) expression is responsible for increased prostaglandin synthesis at the sites of inflammation. These data have led to the hypothesis that P2X7 agonists upregulate LPS-stimulated COX-2 expression.

Delana Hopkins
Greenspan Lab
Pharmacology 901 Student Seminar
Monday, June 21, 2004
12:00 Noon
140 Bardeen

BMP-1 Cleaves and Activates GDF-11, a Negative Regulator of Neurogenesis

The metalloproteinase BMP-1 has been shown to cleave a variety of extracellular substrates including procollagen, laminin 5, lysyl oxidase and small leucine-rich proteoglycans. Recent work has shown that BMP-1 can cleave GDF-8 as well. GDF-8, also known as myostatin, is a TGF-b like molecule that acts as a negative regulator of muscle mass. The mature GDF-8 protein forms a non-covalent latent complex with its propeptide, inhibiting the action of mature GDF-8. Recently, it has been shown that BMP-1 and a group of similar proteinases are capable of cleaving pro-GDF-8 in the latent complex, liberating and thus activating the mature protein. We have evidence to show that GDF-11, a protein highly homologous to GDF-8, also forms a latent complex with its propeptide which can be cleaved by BMP-1. Furthermore, this cleavage activates the mature GDF 11 protein, which acts negative regulator of neurogenesis.

Dominique Fontanilla
Ruoho Lab
Pharmacology 901 Student Seminar
Monday, June 21, 2004
12:00 Noon
140 Bardeen

Mapping the Sigma1 Receptor Binding Site

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. Structurally, 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). These regions have thus bee n 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. Currently, the SBD1 region of the sigma1 receptor is under investigation. We propose that SBD1 and SBD2 are in close proximity to one another and that, together, they may form the binding site of the sigma receptor.