The long-term goal of this research is to determine the physiological and pathological roles of the different MLCK isoforms, encoded by the mylk1 gene, in the vasculature. The mylk1 gene encodes a 220kDa myosin light chain kinase (MLCK), a 130kDa MLCK, as well as the non-catalytic gene product telokin. Experiments described in this proposal are designed to test the hypothesis that the 130kDa MLCK plays a specific non- redundant role in regulating smooth muscle contractility and endothelial cell function in the vascular system. We will generate mice harboring a smooth muscle or endothelial cell-specific knockout of the 130kDa MLCK isoform, while maintaining expression of the other mylk1 gene products. To do this we will use a loxP/cre system to delete key regulatory elements within the 130kDa MLCK promoter. Analysis of the properties of the smooth muscle and endothelial cells in these animals under normal and pathological conditions will allow us determine the specific functions of the 130kDa MLCK in vivo.
Two specific aims are proposed to achieve these goals.
In aim 1 we will generate mice in which LoxP sites flank the core of the 130kDa MLCK promoter or a conserved element within intron 1. The LoxP mice will be crossed with smooth muscle MHC- cre/eGFP mice to facilitate deletion of the regulatory elements, and subsequent ablation of 130kDda MLCK expression, specifically in differentiated smooth muscle cells. Telemetric measurements of blood pressure will be made in live animals to assess changes in vascular tone in 130kDa MLCK knockout mice. Smooth muscle tissues will be isolated from these mice and their contractile properties determined in detail, ex vivo. MLCK has been implicated in regulating cell migration and proliferation of many cells, including smooth muscle cells. We will, therefore, also examine the effects of the ablation of the 130kDa MLCK on pathological models that induce smooth muscle cell migration and proliferation. A wire induced injury of the mouse femoral artery and collateral artery remodeling following hind limb ischemia will be used. In our second aim we will cross the 130kDa MLCK LoxP mice with Tie-2 cre mice to result in the knockout of the 130kDa MLCK specifically in endothelial cells. The subsequent effects on endothelial barrier function in vivo will be assayed by fluorescein permeability assays. Changes in angiogenesis will be assayed in a matrigel plug assay and in a mouse hind limb ischemia model. Together these data will provide a comprehensive analysis of the roles played by the 130kDa MLCK in the vasculature under physiological and pathophysiological conditions.
| Chen, Meng; Zhang, Wenwu; Lu, Xiao et al. (2013) Regulation of 130-kDa smooth muscle myosin light chain kinase expression by an intronic CArG element. J Biol Chem 288:34647-57 |
