Hallmarks of the inflammatory response are the rapid recruitment of leukocytes and vascular leakage. We defined a role for the Rap GTPases and its guanine exchange factors (GEFs) in both these processes. In human T cells, RaplGTase and its calcium and diacylglycerol responsive GEF CalDAG-GEF1 promoted chemokine induced LFA-1-integrin mediated adhesion, while cAMP induced PKA activation downregulated this process. In human endothelial cells, activation of the cAMP inducible GEF Epad enhanced barrier properties: Epad activation of Rap GTPases led to enhanced cortical actin, while Epad adaptor functions promoted microtubule (MT) growth. There is emerging evidence that compartmentalization of cAMP signaling to distinct subcellular sites by the multivalent scaffold proteins, A-kinase anchoring proteins (AKAPs), controls its diverse intracellular functions. Our hvpothesis is that a specific microtubule associated AKAP, AKAP9 selectively relays cAMP signals that regulate endothelial cell barrier function and leukocyte CD18-inteqrin mediated adhesion. Our preliminary data show that AKAP9 co-localizes with Epad at the Golgi and the centrosome of human endothelial cells. Its silencing selectively abrogates the Epad induced enhancement of barrier properties. This was associated with reduced MT growth but intact Rap activation, cortical actin and junctional VE-cadherin. In a published report, delocalization of AKAP9 at the centrosome interfered with LFA-1 mediated polarization of a T lymphoma cell line. Here we show that in primary human T cells, AKAP9 colocalized with LFA-1 in SDF-1a stimulated cells migrating on ICAM-1. Furthermore, a peptide that disrupts PKA interaction with AKAPs enhanced SDF-induced, LFA-1 integrin-mediated cell adhesion. The proposed specific aims will define a molecular framework for AKAP9 function in endothelial cells and T cells and delineate its role in regulating vascular permeability and T cell migration in vivo.
In SPECIFIC AIM 1, studies will examine the molecular mechanisms underlying the role of AKAP9 in Epad-mediated reduction in permeability by exploiting AKAP9 silencing, structure-function analysis and proteomic approaches in primary human endothelial cells. We will also explore how Epac-AKAP9-microtubule growth and the Epac-RapGTPase-cortical actin pathway integrate to modulate endothelial barrier properties.
In SPECIFIC AIM 2, we will examine the contribution of AKAP9 to chemokine induced LFA-1 integrin function in human T cells. For this, a detailed analysis of the spatiotemporal distribution of AKAP9, relative to MTs, LFA-1 and cAMP gradients in SDF stimulated adherent T cells will be investigated. The approach will include live cell, FRET and confocal microscopic analyses. The effect of AKAP9 silencing, or AKAP anchored PKA in T cell integrin function, GTPase activation, cytoskeletal reorganization and cAMP generation will be assessed using imaging and biochemical approaches coupled with in vitro adhesion assays.
In SPECIFIC AIM 3, conditional gene targeting approaches will be used to define the function of AKAP9 in the endothelium and T cells in vascular permeability and T cell recruitment observed in a dermal model of vascular permeability (Miles assay) and intravital microscopy respectively. The pathophysiological relevance of AKAP9 will be assessed by examining its contribution to T cell migration and permeability in a model of contact hypersensitivity and in experimental autoimmune encephalomyelitis. We anticipate that completion of these aims will provide a greater understanding of cAMP dependent mechanisms that stabilize endothelial junctions and reduce T cell migration. This could provide a rationale for enhancing these mechanisms to counteract inflammation induced edema and leukocyte diapedesis in inflammatory diseases.
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|Venkatesh, Deepak; Mruk, Dolores; Herter, Jan M et al. (2016) AKAP9, a Regulator of Microtubule Dynamics, Contributes to Blood-Testis Barrier Function. Am J Pathol 186:270-84|
|Milstone, David S; Ilyama, Motoi; Chen, Mian et al. (2015) Differential role of an NF-?B transcriptional response element in endothelial versus intimal cell VCAM-1 expression. Circ Res 117:166-77|
|Cullere, Xavier; Plovie, Eva; Bennett, Paul M et al. (2015) The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3. Proc Natl Acad Sci U S A 112:14284-9|
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|Mayadas, Tanya N; Cullere, Xavier; Lowell, Clifford A (2014) The multifaceted functions of neutrophils. Annu Rev Pathol 9:181-218|
|Massaad, Michel J; Oyoshi, Michiko K; Kane, Jennifer et al. (2014) Binding of WIP to actin is essential for T cell actin cytoskeleton integrity and tissue homing. Mol Cell Biol 34:4343-54|
|Leick, Marion; Azcutia, Veronica; Newton, Gail et al. (2014) Leukocyte recruitment in inflammation: basic concepts and new mechanistic insights based on new models and microscopic imaging technologies. Cell Tissue Res 355:647-56|
|Azcutia, Veronica; Routledge, Matthew; Williams, Marcie R et al. (2013) CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions. Mol Biol Cell 24:3358-68|
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