As such, it is likely that only one of the two proteins is the important interactor with Rock1 in determining the native airway hyperresponsiveness seen in A/J mice. Since Myl7 encodes the cardiac atrial isoform of myosin regulatory light chain, it seems more likely that Limk2 interacts with Rock1 to determine the constrictor responsiveness of pulmonary airways to methacholine. Rock1 encodes rho kinase, an enzyme expressed in airway smooth muscle that promotes actin filament polymerization by phosphorylating and activating Lim kinase, which in turn phosphorylates and thereby inactivates cofilin, whose function promotes actin filament depolymerization. Actin polymerization plays critical roles in airway smooth muscle contraction. As such, we think it plausible that A/J mice express genetic variants or abundances of Rock1 and Limk2 proteins that uniquely interact to accentuate actin polymerization during contraction. Thus, having only the A/J variant of each gene might result in enhanced contractile function or in reduced force fluctuation-induced relengthening of contracting airway smooth muscle. These functional consequences might result in the native airway constrictor hyperresponsiveness so characteristic of A/J mice. It is also conceivable that the A/J variant of Limk2 potentiates other functions of A/J rho kinase. Importantly, rho kinase also promotes smooth muscle contraction by phosphorylating the myosin targeting subunit of myosin light chain phosphatase thereby promoting the phosphorylated state of myosin regulatory light chain, and perhaps also by promoting focal adhesion assembly. In the Npc1, Npc1l1 network, each gene individually and the genes’ interaction were prioritized with false discovery rate less than 0.0001. Npc1 encodes a protein located in the membranes of lysosomes and endosomes, and is thought to regulate intracellular trafficking of cholesterol. Mutations of Npc1 are responsible for most cases of Niemann-Pick disease type C, which is associated with abnormal accumulation of cholesterol and other lipids within cells. Niemann-Pick disease can involve the lung with foam cell infiltration, but airway hyperresponsiveness has not been reported. Npc1l1 is a separate gene expressed in gut epithelium whose protein product regulates cholesterol absorption, thereby influencing whole body cholesterol homeostasis, and is the target of ezetimibe. A growing body of literature implicates cholesterol or modulators of metabolism in the regulation native airway responsiveness and/or allergic airway inflammation. Depletion of cholesterol from membrane caveoli disrupts contractile activation of airway smooth muscle during muscarinic, serotonin, or KCl-stimulation, and further inhibits rho kinase activation. As such, strainrelated variations in two genes, Npc1 and Npc1l1, which each influence cholesterol homeostasis could plausibly interact to regulate AR in A/J vs C57BL/6J mice. There are potential limitations to our study. We tested only male mice, and so might have missed the opportunity to detect other interacting gene pairs that might be important in females. There were other pairs of interacting loci that were suggested by our two-QTL analysis as potential regulators of AR, but these did not reach Z-VAD-FMK statistical significance.