TARGETING THE MARCKS-FAMILY PROTEINS TO INHIBIT NEUTROPHIL MIGRATION

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DESCRIPTION (provided by applicant): Inflammation is necessary to the maintenance of health and life and occurs most commonly in response to injury or infection. White blood cells involved in the inflammatory response, such as neutrophils, are essential both to host defense from invading microorganisms and wound healing and repair. However, inflammation that is recurrent or fails to resolve can severely damage host tissue; causing both acute and chronic diseases in children and adults. Despite their inherent benefit, neutrophils are often involved in the pathophysiology of inflammatory diseases. In order to cause tissue damage, neutrophils must migrate through the blood vessel wall and into the interstitial tissue. This process requires that neutrophils coordinate chemoattractant signals in their external environment with reorganization of their actin cytoskeleton. The Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) family of proteins, MARCKS and MARCKS-like 1 (MARCKSL1), are known cell membrane and actin binding proteins regulated by protein kinase C (PKC) phosphorylation and calcium/calmodulin binding. MARCKS and/or MARCKSL1 are essential to several events requiring dynamic reorganization of the actin cytoskeleton including: polarization of T-cell microtubule-organizing center, b2 integrin diffusion and clustering in RAW 264.7 cells, maintenance of PIP2 cell membrane microdomains and dynamic actin structure formation in the neuron-like cell line PC12 and membrane ruffling and lamellae formation in Ltk fibroblasts. This proposal will investigate the hypothesis that both MARCKS and MARCKSL1 have essential and non-redundant roles in the regulation of human neutrophil migration in vitro and in vivo. The three related and complimentary specific aims are directed at understanding: (1) the requirement for MARCKS-family proteins in neutrophil chemotaxis toward intermediate (IL-8, LTB4) and end-stage (fMLP, C5a) chemoattractants; (2) the mechanism by which MARCKS-family proteins regulate neutrophil migration; and (3) the effect of knock-down or inhibition of MARCKS or MARCKSL1 on neutrophil trafficking and recruitment in vivo. To accomplish specific aims 1 and 2, experiments will be performed using primary human neutrophils isolated from healthy volunteers as well as a promyelocitic leukemia cell line (HL60s) that can be induced to look and behave like primary neutrophils. In these experiments, MARCKS or MARCKSL1 inhibition will be accomplished using: cell permeant peptides identical to the 24 amino acids of the N-terminus of each protein, transgenic expression of a truncated fluorescently-tagged N-terminus MARCKS or MARCKSL1 transgenic protein or siRNA mediated knockdown. Specific Aim 3 will be accomplished using three different animal models of inflammation: tail-wounding in zebrafish and TNFa induced cremaster injury and LPS-mediated acute lung injury in mice. The successful outcome of this project will have direct application to therapeutic strategies targeting neutrophil migration for the treatment and prevention of numerous diseases caused or exacerbated by a dysregulation in neutrophil recruitment and activation. PUBLIC HEALTH RELEVANCE (provided by applicant): The goal of this research is to investigate promising cellular targets for the treatment and prevention of acute and chronic inflammatory airway diseases. These diseases (i.e. asthma, chronic bronchitis, acute lung injury, chronic obstructive pulmonary disease) are caused or exacerbated by white blood cells known as neutrophils, which migrate from the blood stream into the lungs. By identifying key cellular regulators of neutrophil migration, therapeutics can be developed that decrease neutrophil accumulation in the airways, diminishing and even preventing these devastating diseases.