Our studies have demonstrated that the gene encoding the multi-functional cytoskeletal protein, myosin light chain kinase (MLCK), contains coding polymorphisms which are highly associated with susceptibility to severe asthma. The non-muscle isoform, nmMLCK, is a critical cytoskeletal effector which regulates the participation of the EC actin cytoskeleton in vascular barrier disruption, in barrier restoration, in lung inflammatory cell trafficking and in vascular responses to mechanical stretch. Following edemagenic agents, MLCK phosphorylates MLCs on Ser19 and Thr18, producing barrier-disrupting cytoplasmic stress fibers, spatially-localized actomyosin contraction and paracelular gaps. In contrast, EC barrier-protective agonists induce the rapid translocation of MLCK to lamellipodial membrane protrusions (to close paracellular gaps and restore barrier integrity) and to cortical actin networks (to enhance linkage to junctional complexes and increase barrier properties). The mechanism by which the full length nmMLCK1 (and its alternatively spliced variant nmMLCK2) is targeted to specific cellular sites is entirely unknown. Furthermore, the influence of the asthma-associated nmMLCK coding SNPs (Pro21His, Pro147Ser, Val261Ala) on MLCK structure/function are similarly unknown. We hypothesize that site-specific nmMLCK regulation involves post-translational modifications (PTMs) and results in variant- and SNP-specific MLCK activities.
Specific Aim (SA) #1 will conduct case control studies in African Americans with asthma who reside in Harlem, NY to validate our earlier observations in asthma cohorts from Chicago and Barbados.
Specific Aim #2 studies will characterize nmMLCK (nmMLCK1, nmMLCK2, MLCK-coding SNPs) utilizing kinase and actin polymerization assays, GFP/YFP-MLCK fusion proteins and cytoskeletal binding assays. SA #3 will examine the influence of kinase-mediated PTMs (Src, Abl, and PKA) on site-specific MLCK responses (nmMLCK1, nmMLCK2, nmMLCK-SNPs) utilizing mass spectroscopy, phosphopeptide mapping, GFP-MLCK fusion proteins, and binding partner assays. SA #4 will utilize available and novel genetically-engineered mice to further define the site specific in vivo involvement of nmMLCK ( SNPs) in lung inflammatory injury. We believe these integrated translational studies will lead to mechanistic insights into asthma pathobiology and the development of novel edema-reducing therapies.

Public Health Relevance

Asthma is a disorder affecting over 20 million Americans with unacceptable morbidity and mortality, particularly in under-represented minority populations such as African Americans. Our proposal will offer clues for the involvement and importance of the cytoskeleton in the development and severity of asthma.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL091889-02
Application #
7681504
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Banks-Schlegel, Susan P
Project Start
2008-09-01
Project End
2010-01-31
Budget Start
2009-07-01
Budget End
2010-01-31
Support Year
2
Fiscal Year
2009
Total Cost
$392,207
Indirect Cost
Name
University of Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Bime, Christian; Pouladi, Nima; Sammani, Saad et al. (2018) Genome-Wide Association Study in African Americans with Acute Respiratory Distress Syndrome Identifies the Selectin P Ligand Gene as a Risk Factor. Am J Respir Crit Care Med 197:1421-1432
Liu, Pengfei; Rojo de la Vega, Montserrat; Sammani, Saad et al. (2018) RPA1 binding to NRF2 switches ARE-dependent transcriptional activation to ARE-NRE-dependent repression. Proc Natl Acad Sci U S A 115:E10352-E10361
Wang, X; Wang, L; Garcia, J G N et al. (2018) The Significant Role of c-Abl Kinase in Barrier Altering Agonists-mediated Cytoskeletal Biomechanics. Sci Rep 8:1002
Mascarenhas, Joseph B; Tchourbanov, Alex Y; Danilov, Sergei M et al. (2018) The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene. Am J Respir Cell Mol Biol 58:604-613
Szilágyi, Keely L; Liu, Cong; Zhang, Xu et al. (2017) Epigenetic contribution of the myosin light chain kinase gene to the risk for acute respiratory distress syndrome. Transl Res 180:12-21
Mascarenhas, Joseph B; Tchourbanov, Alex Y; Fan, Hanli et al. (2017) Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene. Am J Respir Cell Mol Biol 56:29-37
Wang, Ting; Shimizu, Yuka; Wu, Xiaomin et al. (2017) Particulate matter disrupts human lung endothelial cell barrier integrity via Rho-dependent pathways. Pulm Circ 7:617-623
Wang, Ting; Mathew, Biji; Wu, Xiaomin et al. (2016) Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury. Pulm Circ 6:234-9
Pouladi, Nima; Bime, Christian; Garcia, Joe G N et al. (2016) Complex genetics of pulmonary diseases: lessons from genome-wide association studies and next-generation sequencing. Transl Res 168:22-39
Tao, Shasha; Rojo de la Vega, Montserrat; Quijada, Hector et al. (2016) Bixin protects mice against ventilation-induced lung injury in an NRF2-dependent manner. Sci Rep 6:18760

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