Current Research Projects

• Mapping the signaling pathways that link integrin ligation with cytoskeleton-depdendent function in dendritic cells. This project focuses on SLP-76 and Vav1 as key regulators of integrin signaling and dendritic cell adhesion, motility, and function.
• Cloning and characterization of a novel SLP-76 homolog. We have identified a fourth member of the SLP-76 family of adaptors that may represent the only member that is expressed in non-hematopoietic tissues. We hypothesize that this protein may link integrin signaling with downstream events in a manner analogous to that described for SLP-76 in neutrophils and dendritic cells.
• Defining the molecular events controlling thymocyte selection and lineage commitment. These studies will focus on the role of Ets-1 in silencing expression of CD4 as selected thymocytes commit to the CD8 T cell lineage.

Introduction

My laboratory has two major areas of interest.  The first is to define the molecular signaling pathways that regulate the dendritic cell cytoskeleton as a means to understand how dendritic cell function is achieved.  Coordinated cell positioning and migration are central aspects of embryogenesis, a functional immune system, and metastatic spread of cancer.  Certain aspects of cell adhesion and motility are common to virtually all cell types, such as the dependence on dynamic changes in the actin and microtubule cytoskeletons.  Indeed, it has been postulated that metastatic cancer cells “hijack” one or more of these “core” mechanisms to modulate adhesive and tissue invasion potential (1).  However, it remains unknown how the signaling pathways upstream of these common or core mechanisms are regulated in different cell types (eg; cells derived from non-hematopoietic versus hematopoietic origins). 

Our previously published work (2) and preliminary data have identified two signaling intermediates that regulate the adhesive and motile properties of DCs, a potent APC of the immune system.  Expression of these proteins (SLP-76 and Vav1) is largely restricted to cells of the immune system, suggesting participation in a hematopoietic-specific pathway or pathways for regulating DC adhesion and motility.  Still, most of what we know regarding SLP-76 and Vav1 mediated signaling comes from studies of the T cell receptor or other ITAM-coupled antigen receptors.  However, it should not be assumed that the signaling events proximal and distal to SLP-76 or Vav1 are similar in T cells (following TCR ligation) and DCs (following integrin engagement), as the downstream biological effects are likely to be quite different (eg; gene induction vs. adhesion).  The challenge now is to determine a) how these important signaling intermediates couple surface receptors with cell adhesion and motility in cells of hematopoietic origin, and b) what these “hematopoietic specific” signaling pathways contribute to specialized cell functions.

Figure 1:  A polarized, motile murine dendritic cell.  The arrow indicates direction of movement.  Note the intensely stained podosomes that cluster at the leading edge of the cell (green, actin; red, vinculin).

Fully differentiated non-hematopoieitc cells rarely migrate far from one position.  While fibroblasts can migrate short distances in response to wounding, a sedentary, firmly adherent phenotype is quickly re-established upon contact with neighboring cells.  In marked contrast, antigen-loaded DCs traverse relatively long distances in a directed fashion to seek out and stimulate (or, under appropriate conditions, tolerize) naïve T cells (a polarized, motile dendritic cell can be seen in Figure 1). 

Once in the LN, DCs continue to migrate and extend long, sweeping dendrites as a means to optimize contact with as many T cells as possible (3,4).  Upon contact with an antigen-specific T cell, stable adhesion is established (following an initial period of more transient contacts) and the DC and T cell engage in two-way communication resulting in T cell activation, proliferation, and effector function.  In elegant studies performed both in vitro and in vivo, it has been demonstrated that contact between a DC dendrite and an antigen-specific T cell triggers polarized movement of the DC body to the T cell, resulting in more intimate contact (5).  This process is likely to be critically dependent on the cytoskeleton, as DCs deficient for both Rac1 and Rac2 (key enzymes that regulate cytoskeletal dynamics) fail to translocate to the T cell and establish stable, productive contact upon encountering an antigen-specific T cell (5).  As a consequence, the T cell stimulatory capacity of Rac1/2 deficient DCs is markedly impaired.  Still, the actual mechanisms upstream of Rac1/2 function and specific downstream targets of this pathway have not been identified. 

Integrin function in DCs has been shown to be important for proper localization, migration, and morphology (6-8).  While cells of the immune system are likely to use some of the same signaling mechanisms described in non-hematopoietic cells to propagate integrin signaling, including those involving FAK, it is becoming evident that integrins also engage several signaling intermediates that are unique to hematopoietic cells.  In this regard, two ITAM-bearing accessory proteins (FcR -chain and DAP12) have been found to be important for integrin signaling in neutrophils and macrophages, likely by providing a platform for the recruitment and assembly of signaling complexes in response to integrin ligation (9,10).  This new and exciting data provides a more complete framework for understanding the molecular basis of integrin signaling in cells of hematopoietic origin, and provides a mechanism for how the Syk PTK is rapidly activated in platelets and neutrophils following integrin ligation (11-14). Several genetic studies have demonstrated just how important the “hematopoietic specific” pathway is to immune cell function.  Syk deficient neutrophils are markedly impaired in their capacity to spread on surfaces pre-coated with an integrin agonist, and display defects in multiple signaling pathways following integrin ligation (15).  Phosphorylation of Pyk2, a FAK-family member that is expressed predominantly in hematopoietic and neuronal cells, is compromised in Syk-deficient neutrophils stimulated via integrins (15).  The SLP-76 adaptor is a known substrate of Syk family PTKs.  Similar to neutrophils lacking Syk, SLP-76 deficient neutrophils are impaired in the ability to spread or produce reactive oxygen intermediates following direct integrin ligation (16).  In addition, the activation of the p38 MAPK and the inducible phosphorylation of PLC2 and Vav are compromised upon integrin ligation in the absence of SLP-76.  Interestingly, murine neutrophils isolated from mice lacking Vav1 and Vav3 also display defects in spreading and adhesion following ligation of 2 integrins (17).  Biochemically, Vav1/3-deficient neutrophils demonstrate reduced phosphorylation of Pyk2, Akt, and PAK as well as impaired activation of all three Rho-family GTPases following integrin ligation (17).  These data strongly implicate Syk, SLP-76, and Vav family members in propagating multiple downstream signaling pathways following integrin ligation in neutrophils and platelets.  In contrast, very little is known regarding mechanisms of integrin signaling and downstream targets in dendritic cells.  To date, our previously published work and preliminary data provided below are the only evidence that we are aware of indicating an important role for the hematopoietic-specific pathway in supporting integrin function in DCs.  How SLP-76 and Vav integrate integrin-triggered signaling pathways and the biological relevance of SLP-76 or Vav dependent signaling to integrin-mediated cell function has not been defined in any hematopoietic cell type.  One particularly interesting area that remains to be explored is the potential interplay between the proposed Syk/SLP-76/Vav1 signaling “axis” and the more well-defined signaling pathways mediated by Src family kinases and FAK.  Clearly, FAK-dependent signaling does not compensate for loss of SLP-76 or Vav1 in DCs, as integrin signaling is almost completely abolished in the absence of these hematopoietic-specific signaling proteins. 

The existence of hematopoietic-specific signaling pathways comprised of ITAM-bearing accessory proteins (eg: FcR -chain) and proximal signaling intermediates (eg; SLP-76) beg the question of what this pathway actually contributes to immune cell function.  Cells of the immune system demonstrate great plasticity in their adhesive and motile characteristics.  Perhaps this is accounted for, at least in part, by the existence of a specialized hematopoietic-specific signaling pathway or pathways downstream of integrin ligation.  In addition, distinct cell types of the immune system manifest unique morphologies and migratory capacities that are central to function.  This raises the interesting possibility that “variations” of the hematopoietic-specific signaling pathways may exist that engage or target unique downstream effectors, depending on the cell type.  Understanding how the existence of a hematopoietic-specific signaling pathway contributes to function will require a detailed analysis of these pathways in individual cell types.  A major focus of my laboratory is defining how SLP-76 and Vav1 function within one or more hematopoeitic-specific signaling pathways to regulate dendritic cell cytoskeletal dynamics and contribute to DC function.  As the cytoskeleton is central to multiple aspects of DC function (morphology, adhesion, motility), elucidation of this pathway is expected to greatly enhance our current understanding of DC biology.   It is also anticipated that these studies will facilitate the identification of signaling mechanisms governing the adhesive and migratory properties of additional hematopoietic cell types, and will provide a valuable basis for comparing these fundamental processes between cells of hematopoietic and non-hematopoietic origin. 

A second major area of study in my laboratory focuses on defining the molecular regulators of thymocyte selection and linage commitment.  T cell development in the thymus is characterized by multiple maturational stages and tightly regulated developmental checkpoints that ultimately give rise to a MHC-restricted and self-tolerant peripheral T cell repertoire (Figure 2).  The early stages of thymocyte development (prior to expression of CD4 and CD8) are dependent upon expression and functional signaling via the pre-TCR, which is composed of a functionally rearranged TCR chain and a surrogate pre-T chain (18,19).  Once thymocytes pass this checkpoint, rearrangement at the TCR locus begins and the mature  TCR is expressed at low levels.  This coincides with expression of both CD4 and CD8 (CD4+CD8+, or “double-positive”) and the onset of an MHC-dependent selection process.  The survival and maturation of double-positive (DP) thymocytes depends on a functional interaction between the TCR and MHC/peptide complexes expressed on thymic stromal elements.  Signal strength and the nature of subsequent MAPK activation are key mediators of selection, with strong TCR dependent signals giving rise to a transient but robust activation of ERK MAP kinases and deletion (20,21).  Positively selecting signals are characterized by lower-affinity TCR interactions that promote a low but sustained level of ERK phosphorylation.  Still, it is not clear if the mechanisms governing lineage commitment and downregulation of CD4 or CD8 are distinct from those governing the selection process.  While evidence for a stochastic model of lineage commitment has been provided (22-25), it has become more generally accepted that lineage commitment is dictated by the quality of signal emanating from the TCR/co-receptor complex that is productively engaged (26-32).  Most recently, a “kinetic signaling” model of lineage commitment has been put forth suggesting that the CD4 co-receptor does not transduce unique “instructional” signals per se, but that sustained signals from the TCR/CD4 co-receptor complex appear required for commitment to the CD4 lineage (33). 

Figure 2:  Schematic depicting the major stages of thymocyte development including selection and lineage commitment.

Thus, thymocytes surviving the selection process may be pre-programmed to adopt a CD8 SP fate unless directed to the CD4 lineage by sustained signaling via the TCR and CD4 co-receptor (Figure 2).  Presumably qualitative differences in the signaling pathways engaged by differential TCR/co-receptor engagement ultimately influence transcriptional control of the CD4 and CD8 genes and lineage commitment.

 As indicated above, regulated expression of the CD4 and CD8 genes is a critical component of thymocyte development and selection.  The gene loci encoding the CD4 and CD8 co-receptors contains multiple cis-regulatory sequences, which include both promoter and enhancer elements (34,35).  These elements are likely sites of convergence for upstream signaling pathways influenced by the nature of the TCR/co-receptor signals initiated at the cell surface.  To date, several transcription factors have emerged as central regulators of CD4 or CD8 gene expression during thymic development.  These include Th-POK and TOX, which promote CD4 or CD8 lineage commitment, respectively (36,37).  The CD4 locus also contains an intronic silencer element that functions to actively quench CD4 expression in DN thymocytes as well as CD8 SP thymocytes (38,39).  Members of the RUNX family of transcription factors have been shown to regulate CD4 silencer activity in both DN and CD8 SP thymocytes (40,41).  The CD4 silencer contains two RUNX binding sites, and the absence of RUNX3 results in impaired generation of CD8 SP thymocytes and the presence of DP T cells in the periphery (42,43), consistent with a role for RUNX3 in silencing CD4 gene expression as thymocytes commit to the CD8 lineage.  Indeed, RUNX3 expression increases substantially in CD8 SP thymocytes (41,43), providing a potential mechanism for specific silencing of the CD4 gene in thymocytes selected by TCR interactions with MHC class I.  Still, the upstream mediators and signaling pathways regulating RUNX3 expression during this transition remain to be identified.

Members of the Ets family of transcription factors are important regulators of hematopoietic development and immune cell function, and typically bind to DNA as monomers via an ~85 amino acid winged-helix-turn-helix DNA binding motif (Ets domain) (44).  Ets-1 is expressed in multiple hematopoietic tissues, including the thymus, suggesting that this protein may play an important role in the development or function of lymphoid cells (45-47).  In support of this idea, the numbers of thymocytes and lymph node (LN) resident T cells is markedly decreased in the absence of Ets-1, and Ets-1 deficient T cells undergo apoptosis more readily than wild type counterparts (48,49).  More recently, Ets-1 has been shown to be important for optimal transition through early pre-TCR-dependent stages of thymocyte development and allelic exclusion at the TCR locus (50).  In the B cell compartment, Ets-1 deficiency results in impaired B cell development, an elevated frequency of IgM+ plasma cells in vivo and enhanced in vitro differentiation responses to CpG-containing oligonucleotide (a Toll-like receptor 9 ligand) (48,49,51,52).  Ets-1 deficient mice produce both IgM and IgG autoantibodies leading to immune complex deposition in the kidney (48,51,52).  The numbers of splenic NK cells is also substantially reduced in Ets-1 deficient mice (53).  Collectively, these studies demonstrate that Ets-1 plays an important role in both early stages of lymphocyte development and lymphocyte homeostasis in the periphery.  Our recently published work provides the first evidence that Ets-1 is also important for later stages of thymocyte development (54), see preliminary studies).  Specifically, the generation of CD8 SP thymocytes is markedly impaired in mice lacking Ets-1 while the development of CD4 SP thymocytes is not visibly effected.  We hypothesize that this is due to aberrant silencing of the CD4 gene in Ets-1 deficient thymocytes that commit to the CD8 lineage.  We need to test this hypothesis by assessing occupancy of the CD4 silencer by Ets-1 and the ability of Ets-1 to cooperate with Runx3 in silencing the CD4 gene.