Molecular and Cellular Pharmacology Student Seminars 2001-2002

Understanding the Role of Cdc42 and Calpain in Neutrophil Chemotaxis and Chemokinesis

Neutrophils are a class of terminally differentiated leukocytes that comprise 60-80% of the total white blood cells circulating in the human blood. Neutrophil chemotaxis and activation are the first critical steps involved in innate immunity and are essential for fighting human infection. Defects in neutrophil response as well as chronic activation, plays a central role in illnesses such as: ischemia-reperfusion, heart disease, arthritis and asthma. In addition the absence of effective technology and hyper-reactivity of neutrophils make them difficult to work with and hard to manipulate. These obstacles have hampered our understanding of the mechanisms that regulate neutrophil chemotaxis and directional migration. Our lab has sought overcome these problems by developing new techniques that will allow us to study neutrophil chemotaxis. In order to utilize primary neutrophils, we have established a Tat mediated "Transit Peptide System" which allows for the introduction of full-length protein into neutrophils. Using several purification techniques and the process of protein conjugation we where able to demonstrate both internalization and in vivo function of V12Cdc42. This technology in combination with chemokinetic and chemotactic assays, allow us for the first time to manipulate and examine morphologic and signaling changes that occur during chemotactic migration in primary neutrophils This allows for manipulation of chemotactic cells using an established cell line. Recent evidence for our lab suggests that the Rho family GTPase Cdc42 and the calcium dependent cystine protease Calpain may play a central role in chemotactic migration. Using these new approaches we stand poised to finally begin to decipher and elucidate the underlying mechanisms of neutrophil chemotaxis.

References:
Allan et al. (1998) The Journal of Cell Science, Vol.141, 1147-1157
Servant et al., (1999) MBC, Vol. 10, 1163-1178
Glading et al., (2002) TCB, Vol 12, 46-54

Siobhan Wilson
Laboratory of Patricia J. Keely
Pharmacoloy 901 Student Seminar
Monday, March 11, 2002
12:00 Noon
140 Bardeen

Integrin-associated protein (IAP/CD47) augments the function of 2ß1 integrin in smooth muscle cells (SMC), resulting in enhanced chemotaxis toward soluble collagen (Wang, X-Q., and W.A. Frazier. 1998. Mol. Biol. Cell. 9:865). IAP-deficient SMC derived from IAP-/- animals did not migrate in response to 4N1K (KRFYVVMWKK), a peptide agonist of IAP derived from the COOH-terminal domain of thrombospondin-1 (TSP1). When normal SMC were preincubated with 4N1K or an anti- 2ß1 function-stimulating antibody, cell migration to soluble collagen was significantly enhanced. 4N1K-induced chemotaxis was blocked by treatment of SMC with pertussis toxin indicating that IAP acts through Gi. In agreement with this, 4N1K evoked a rapid decrease in c! AMP levels which was intensified in the presence of collagen, and forskolin and 8-Br-cAMP both inhibited SMC migration stimulated via IAP. 4N1K strongly inhibited extracellular regulated kinase (ERK) activation in SMC attaching to collagen and reduced basal ERK activity in suspended SMC. Pertussis toxin treatment of SMC significantly activated ERK, suggesting that an inhibitory input was alleviated. Inhibition of ERK activity by (a) the MAP kinase kinase (MEK) inhibitor, PD98059, (b) antisense oligonucleotide depletion of ERK, and (c) expression of mitogen-activated protein (MAP) kinase phosphatase-1 in SMC all led to increased migration to collagen, 4N1K, or 4N1K plus collagen. Thus, IAP stimulates 2ß1 integrin-mediated SMC migration via Gi-mediated inhibition of ERK activity an! d suppression of cyclic AMP levels. Both of these signaling pathways could directly modulate the state of the integrin as well as impact downstream components of the cell motility apparatus.

Reference:
Xue-Qing Wang, Frederik P. Lindberg, and William A. Frazier
Integrin-associated Protein Stimulates 2ß1-dependent Chemotaxis via Gi-mediated Inhibition of Adenylate Cyclase and Extracellular-regulated Kinases
J. Cell Biol. 1999 147: 389-400.

Akhil Bhalla
Chapman Lab
Pharmacology 901 Student Seminar
Monday, March 4, 2002
12:00 Noon
140 Bardeen

Metal Selectivity of Synaptotagmin-Membrane andSynaptotagmin-SNARE Interactions

Physiological studies show that divalent cations other than calcium induce secretion of neurotrasmitters form cells. The degree of secretion (potency and efficacy) varies with the type of metal used and also the cell type.

Our lab studies the function of Synaptotagmin and its role in exocytosis. We believe that synaptotagmin acts as the calcium sensor for secretion. We hypothesize that inorder for fusion to occur, synaptotagmin must bind phospholipids and SNAREs as a coupling step. Also, if these various metals induce secretion they probably do it by affecting the same or similar processes (i.e. membrane binding and SNARE binding). So we would like to use these metals to ask whether there is any correlation between the metal requirements for these individual steps (membrane binding and SNARE binding) to that which is observed in physiology (secretion from intact cells). If we see a correlation it could mean that synaptotagmins are the sensors for those metals in cells. If not, it could mean that there are other sensors present, or other effector molecules that work togther with synaptotagmin. In any case, this study will help us better understand the mechanism by which synaptotagmin facilitates neurotrasmitter release.

The assays I will use to address these questions are membrane penetration assays using fluorescent probes (AEDANS) and FRET.

Reference:
Ion selectivities of the calcium sensors for exocytosis in rat phaeochromocytoma cells
Kishimoto T. et al., Journal of Physiology (2001) 533.3 pp 627-637

Steroidogenic Acute Regulatory Protein

mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.

Our lab is studying the Steroidogenic Acute Regulatory Protein (StAR), a critical mediator of de novo steroid hormone systhesis. StAR is subject to both transcriptional and post-transcriptional regulation, the latter one being more important in the acute phase of steoid hormone synthesis. We have evidence that StAR message stability is coupled to ongoing translation, and several mCRD-like elements within the StAR coding region and 3'UTR have recently been identified, suggesting a mechanism close to c-fos mRNA regulation.

Reference
Christophe Grosset, C.-Y. A. C., Nianhua Xu, Nahum Sonenberg, Helene Jacquemin-Sablon, and Ann-Bin Shyu (2000). "A Mechanism for Translationally Coupled mRNA Turnover: Interaction between the Poly(A) Tail and a c-fos RNA Coding Determinant via a Protein Complex." Cell 103: 29.

Understanding the Regulation of Spemann's Organizer by FGFR

Elucidating the molecular mechanisms that regulate vertebrate development is the primary goal of the work in the Sheets lab. In order for the normal development of a multicellular organism to occur, cell-cell signaling events must take place to instruct cells where to go and what to become. Spemann's organizer is an essential inducing center in the Xenopus functioning organizer developing embryos show reduced cell differentiation and body axis definition. The presence of an ectopic organizer can by itself induce secondary body axes in host tissues. Thus showing that the signals that emanate from the organizer are sufficient to direct development [1]. Our lab has demonstrated that a key regulator of organizer function is the fibroblast growth factor receptor (FGFR) signaling pathway [2]. The specific goal of my work is to elucidate the mechanism by which FGFR regulates signaling in the organizer, as well as to determine how the signals that emanate from the organizer are regulated during development. In order to elucidate the features of FGFR responsible for downstream signaling I am using mutant receptor constructs in the organizer and assaying organizer function. This focus of my research will allow me to understand what properties of FGFR mediate its ability to transduce specific signals. To determine how signaling down stream of FGFR regulates development I am trying to determine how the organizer specific gene chordin is regulated. Our lab has demonstrated that FGFR is a regulator of the transcription of chordin [2]. This provides a good starting point from which I can determine how organizer specific genes are regulated by FGFR.

References:
1. Gerhart, J., Evolution of the Organizer and the chordate body plan. International Journal of Developmental Biology, 2001. 45: p. 133-153
2. Mitchell, T.S. and M.D. Sheets, The FGFR Pathway Is Required for the Trunk-Inducing Properties of Spemann's Organizer. Developmental Biology, 2001. 237(2): p. 295-305.

DeannaLee Beauvais
Rapraeger Lab
Pharmacology 901 Student Seminar
Monday, February 18, 2002
12:00 Noon
140 Bardeen

Syndecan-1 Adhesion Mediated Signaling in Human Breast Carcinomas

Syndecans are a family of four distinct, but homologous, type I transmembrane cell surface heparan sulfate proteoglycans. These receptors are expressed in all adherent cells and have been attributed important regulatory roles in cellular differentiation, proliferation, cell-cell/cell-matrix adhesion, cell migration and regulation of cell morphology. Endowed by their heparan sulfate (HS) chains, syndecans interact with components of the extracellular matrix (ECM). Through these binding interactions, syndecans can act as co-receptors to modify integrin-ECM adhesions, but can also influence intracellular signaling events to elicit changes in the cortical actin cytoskeleton. Though it is clear that the syndecan HS chains are essential for matrix binding, less is known about the role the syndecan core proteins play in cell adhesion signaling. The focus of my research is to investigate how cis protein-protein interactions of the syndecan-1 core protein mediate the formation of macromolecular cell adhesion and signaling complexes that regulate organization of the actin cytoskeleton. 
MDA-MB-231, a highly invasive human mammary carcinoma cell line, endogenously express syndecan-1, -2 and -4 at their cell surface, along with a wide complement of b1 integrins and modest levels of the primary vitronectin receptor, avb3. These cells will strongly adhere to a monoclonal antibody (B-B4) directed against the extracellular domain of the syndecan-1 core protein, but fail to spread. Though the MDA-MB-231 initially fail to spread in response to syndecan-1 ligation, these cells can be induced to spread in response to 1mM Mn2+ (an exogenous activator of integrins) or with function blocking b1 integrin antibodies (P5D2 or mAb13). This cell spreading occurs in the absence of an integrin ligand, yet requires avb3 integrins activity as treatment with a soluble avb3 integrin blocking antibody (LM609) inhibits spreading. Interestingly, recent studies indicate that cell spreading is dependent on a region located within the ectodomain of the syndecan-1 core protein. These results suggest that anchorage of syndecan-1 to extracellular matrix ligands initiates formation of a syndecan-1/avb3 integrins signaling complex that mediates reorganization of the actin cytoskeleton. Further, our results suggest that the failure of these cells to spread in response to syndecan-1 ligation is due to transdominant inhibition of avb3 integrins by constitutively active b1 integrins on the surface of these cells. This phenomenon may be a critical regulator of invasiveness in mammary carcinoma cells.

Reema Jasuja
Greenspan Lab
Pharmacology 901 Student Seminar
Monday, February 11, 2002
12:00 Noon
140 Bardeen

Creation of a Sog Morphogen Gradient in the Drosophila Embryo

A variety of genetic evidence suggests that a gradient of decapentaplegic (Dpp) activity determines distinct cell fates in the dorsal region of the drosophila embryo, and that this gradient may be generated indirectly by an inverse gradient of the BMP antagonist Short gastrulation (Sog). It has been proposed that the Sog diffuses dorsally from the lateral neuroectoderm where it is produced and is cleaved and degraded dorsally by the metalloprotease Tolloid (Tld). In this paper, it has been shown directly that Sog is distributed in a graded fashion in dorsal cells and that Tld degradation limits the level of Sog dorsally. In addition, the paper discusses the dynamin dependent retrieval of Sog which acts in parallel with degradation by Tld as a dorsal sink for active Sog.

Reference:

Shaila Srinivasan, Kay E Rashka and Ethan Bier, Developmental Cell, Vol.2, 91-101, Jan 2002.

NFkB Activation Induced by DNA Damaging Agents: Mechanistic Investigation of Super-induction by TPA

The transcription factor NF-kB was originally found in mature B cells as a nuclear protein having the ability to enhance the transcription of the k light chain gene. Since then, NF-kB has been found in various cell types as a complex in the cytoplasm with its inhibitor protein, IkB. IkBa degradation can be stimulated by a variety of agents, including: pro-inflammatory molecules, pro-oxidants, and DNA damaging agents. Inducible NF-kB activity is initiated by degradation of its inhibitor protein, IkB. Induction of NF-kB activity by pro-inflammatory agents is known to induce a phosphorylation cascade. This cascade leads to a site-specific phosphorylation of IkB by IkB kinase (IKK), followed by ubiquitination, and 26S proteasome-mediated degradation. Free NFkB undergoes nuclear translocation and can activate target genes with a kB DNA binding site.
DNA damaging agents also activate PKC. A correlation between PKC activity and NFkB activity has been demonstrated, but there is no evidence that PKC might be modulating components in the NFkB cascade directly.
TPA is a phorbol ester that activates NFkB. It is a very potent tumor promoter known to activate various PKC isoforms including PKCa, bI, bII, g, d, e, h, q and m. A short-term treatment of TPA induces translocation of various PKCs from the cytoplasm to the membrane where they become activated by auto-phosphorylation.
I down-regulated PKC by prolonged TPA treatment. I pre-treated human T cells (CEMp) with TPA for 21 hours prior to the addition of VP16 (treated for 3 additional hours). NFkB activity in the TPA treated cells was five times higher than the cells treated with VP16 alone despite the fact that PKCs (a, b) have been down-regulated. This activation of NFkB is termed ìsuper-inductionî. My research goal has been focused on determining the underlying mechanism and functional consequence of this "super-induction" phenomenon.

References:
1. Silverman N. and Mandates T. Genes and Development, 15:2321-2342
2. Chapman D. Pate A. Heissmeyer V and Scheidereit C. , MCB 21 (19) 6640-6650

Differentiation of Embryonic Stem Cell-Derived Cardiomyocytes (SCDCs)

The observation that embryonic stem (ES) cells, when allowed to differentiate, produced areas of spontaneously contracting cells has opened up a new system to study mammalian cardiac development. Much work has been done with mouse embryonic stem cells. However, the isolation of primate ES cell lines allows for new studies that more closely mimic human cardiac development.
The embryoid body culture system has been widely used to produce cardiomyocytes from mouse ES cells. In this system, spontaneous contraction begins approximately one week after the cells begin to differentiate. Cardiac markers (a-MHC, b-MHC, MLC-2v, etc.) are seen to increase both from RT-PCR and immunostaining experiments. Electrophysiological studies help to confirm the presence of functional cardiomyocytes. Comparison to mouse fetal tissue suggests the system's validity as a model for cardiac development. Using a modified embryoid body system, we are currently attempting to differentiate and characterize cardiomyocytes from human and rhesus ES cells.

References:

Doevendans et al.  Differentiation of Cardiomyocytes in Floating Embryoid Bodies is Comparable to Fetal Cardiomyocytes.  J Mol Cell Cardiol 32:839- 851, 2000.

Kehat et al.  Human Embryonic Stem Cells Can Differentiate into Myocytes with Structural and Functional Properties of Cardiomyocytes.  J Clin Invest 108(3):407-414, 2001.

R-Ras Enhances Integrin-Mediated Signaling to FAK and p130Cas to Promote Migration

The a2b1 integrin mediates signaling events that determine whether an epithelial cell will polarize or migrate. Changes in a2b1 integrin expression can disrupt cell polarization, which may lead to uncontrolled cell migration and invasion. The small GTPase, R-Ras, has been shown to promote a2b1 integrin mediated cell adhesion and migration in breast epithelial cells (Keely, et al. 1999, JCB 145:1077). It is unknown how R-Ras regulates these integrin mediated signaling events. Integrin mediated cell migration and adhesion to the extracellular matrix involves forming focal adhesions and signaling to molecules such as focal adhesion kinase (FAK) and p130Cas. In human breast carcinomas, T47D cells, we found that activated R-Ras enhanced focal adhesion formation and the phosphorylation of FAK and p130Cas mediated by a2b1 integrins. I will discuss the following three possible mechanisms by which R-Ras may enhance FAK and p130Cas phosphorylation: (1) R-Ras conformationally changes the integrin receptor to induce higher affinity binding of ligand, (2) R-Ras clusters integrins to increase the avidity to induce an increase in receptor ligand binding, and (3) R-Ras activates direct signaling pathways. Our data suggests that R-Ras enhances FAK and p130Cas phosphorylation and focal adhesion formation by a novel mechanism that differs from but also synergizes with the a2b1 integrin.

References:

Keely, P.J. et al., 1999. R-Ras Signals through Specific Integrin a Cytoplasmic Domains to Promote Migration and Invasion of Breast Epithelial Cells. JCB 145(5): 1077-1088

Giancotti, F.G. and E. Ruoslahti 1999. Integrin Signaling. Science 285: 1028-1032.

Understanding the AhR 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 enhancer elements. The Aryl Hydrocarbon Receptor (AHR) is known for its ability to mediate toxic effects of industrial chemical 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 death. Despite the recognized involvement of AHR in such effects, the causal molecular pathway to toxicity still remains unclear. What also remains unresolved is the true physiological role of the AHR. However, recent work on the AHR-/- does suggest importance in hepatic vasculogenesis.
In the unliganded state, the AHR exists in the cytoplasm, bound to a dimer of hsp90, and Ara9. Previous work has shown hsp90's involvement with AHR to be important in maintaining the correct conformational state for ligand binding. However, the role of Ara9 in the AHR pathway is still unclear. It may be interacting to stabilize AHR, or function as part of a DNA binding complex; important in recruitment of cofactors, or may be acting to mask the nuclear localization sequence (NLS) of the AHR; thereby inhibiting nuclear transport. 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 creating 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. 
Previous work in our lab gives evidence for a masked nuclear localization signal which 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 are working on construction of DNA-binding mutants of the AHR by inserting alanine's at this bHLH site and thereby rotating the helix to uncover a masking/unmasking phenomenon. These DNA-binding mutants will also prove useful in studying the role of AHR DNA-binding in transactivation of genes important in either the toxic, adaptive, or endogenous AHR pathway.

References:
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.

Syndecans are a family of four distinct, but homologous, type I transmembrane cell surface heparan sulfate proteoglycans. These receptors are expressed in all adherent cells and have been attributed important regulatory roles in cellular differentiation, proliferation, cell-cell/cell-matrix adhesion, cell migration and regulation of cell morphology. Endowed by their heparan sulfate (HS) chains, syndecans interact with components of the extracellular matrix (ECM). Through these binding interactions, syndecans can act as co-receptors to modify integrin-ECM adhesions, but can also influence intracellular signaling events to elicit changes in the cortical actin cytoskeleton. Though it is clear that the syndecan HS chains are essential for matrix binding, less is known about the role the syndecan core proteins play in cell adhesion signaling. The focus of my research is to investigate how cis protein-protein interactions of the syndecan-1 core protein mediate the formation of macromolecular cell adhesion and signaling complexes that regulate organization of the actin cytoskeleton. 
MDA-MB-231, a highly invasive human mammary carcinoma cell line, endogenously express syndecan-1, -2 and -4 at their cell surface, along with a wide complement of b1 integrins and modest levels of the primary vitronectin receptor, avb3. These cells will strongly adhere to a monoclonal antibody (B-B4) directed against the extracellular domain of the syndecan-1 core protein, but fail to spread. Though the MDA-MB-231 initially fail to spread in response to syndecan-1 ligation, these cells can be induced to spread in response to 1mM Mn2+ (an exogenous activator of integrins) or with function blocking b1 integrin antibodies (P5D2 or mAb13). This cell spreading occurs in the absence of an integrin ligand, yet requires avb3 integrins activity as treatment with a soluble avb3 integrin blocking antibody (LM609) inhibits spreading. Interestingly, recent studies indicate that cell spreading is dependent on a region located within the ectodomain of the syndecan-1 core protein. These results suggest that anchorage of syndecan-1 to extracellular matrix ligands initiates formation of a syndecan-1/avb3 integrins signaling complex that mediates reorganization of the actin cytoskeleton. Further, our results suggest that the failure of these cells to spread in response to syndecan-1 ligation is due to transdominant inhibition of avb3 integrins by constitutively active b1 integrins on the surface of these cells. This phenomenon may be a critical regulator of invasiveness in mammary carcinoma cells.

MAKING SENSE OF WORM SEX

All higher organisms have evolved specialized sensory cells and receptors for perceiving selective features of their external environment to ultimately coordinate behaviors for survival and reproduction. While the cellular bases of sensory systems are well characterized, less is understood about the genes and proteins responsible for sensory detection. Our lab is using male mating behavior of C. elegans as a model system to study the genes, proteins and cells involved in sensory mediated behaviors.

To identify genes required for male mating behavior, we are undertaking a candidate gene approach and are surveying the cellular expression profiles of all 13 C. elegans TRP genes to see if they are expressed in male specific sex sensory cells. This family of genes encodes 6 TM domain ion channels, some of which have been shown to be important for sensory behaviors in C. elegans and other organisms (1, 2). In addition, available TRP mutant strains are being put through behavioral assays to see if males exhibit defects in mating. Expression data so far suggests that the TRP channels osm-9 and ocr-2 are expressed in subsets of male-specific sex sensory cells, including spicule and ray neurons. Behavioral data supports a role for these channels in these cells, as the males are unresponsive to hermaphrodite contact (a ray behavior) and are often unable to insert their copulatory spicules into their mates' vulva.

In parallel with the candidate gene approach, we are utilizing a forward genetics approach to clone new genes involved in male mating. A mutant strain sy511 was originally isolated from a mating behavior screen based on its abnormal responsiveness to contact with hermaphrodites, and we are in the process of mapping it genetically. It will be ultimately cloned by cosmid rescue, which will then make it possible to determine the molecular lesions that result in abnormal mating behavior. By using a combination of both forward and reverse genetic approaches, we hope to unravel the molecular basis of sensory behaviors.

Bibliography:
1) Harteneck, C., Plant TD, and Schultz, G. From worm to man: three subfamilies of TRP channels. TINS. 23 (4): 159-166.

2) Colbert, HA, Smith, TL, Bargmann, CI. (1997)   OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J Neurosci. 17 (21):8259-69.