The Na/K-ATPase, or Na+ pump, first discovered by Skou, belongs to the P-type ATPase superfamily. It transports Na+ and K+ across the plasma membrane by hydrolyzing ATP. Besides its canonic pumping function, we and others have demonstrated that a1 Na/K-ATPase could serve as a signal receptor. Binding of its specific ligands, cardiotonic steroid (CTS), could induce many signaling pathways through protein and lipid kinase cascades. Mechanistically, the receptor function of a1 Na/K-ATPase relies on the interaction with Src to form a functional receptor complex. Furthermore, we and others have demonstrated that a1 Na/K-ATPase receptor function requires its highly-exposed N and A domains to interact with other proteins to constitute a signaling microdomain mainly located in caveolae.
Caveolins are structure proteins of caveolae. It has been proposed that the interaction between caveolin-1 and other signaling proteins is important for targeting these components to caveolae. We have demonstrated that a1 Na/K-ATPase could directly interact with caveolin-1 through its highly conserved caveolin-binding motif (CBM) on the N-terminus. This interaction is not only important for CTS-induced Src-dependent signaling pathways, but also regulates caveolin-1 trafficking, and ultimately the formation of caveolae in cell culture. Disruption of a1 Na/K-ATPase/caveolin-1 interaction not only re-distributes caveolin-1 away from the plasma membrane, but also affects the signaling functions of receptors other than a1 Na/K-ATPase (e.g., IP3 receptor). Thus, we believe that a1 Na/K-ATPase is not only a pump, a signaling receptor, but also a signal integrator. We further speculate that the CBM-mediated interaction with caveolin-1 is essential for a1 Na/K-ATPase to work as a signal integrator.
On this scientific premise, we first made a CBM mutant (F97A and F100A) a1 Na/K-ATPase (mCBM), generated a stable cell line expressing mCBM we call LW-mCBM, and then characterized how such mutations affect the function of a1 Na/K-ATPase. Unlike other mammalian-expression systems reported in the literature, we used a knock-down and rescue protocol, which allows the expression of mutant a1 Na/K-ATPase in the essential absence of endogenous Na/K-ATPase. Functional studies indicate that CBM mutations did not affect the expression and membrane location of a1 Na/K-ATPase. The expressed mCBM showed comparable enzymatic activity as the wild type a1 Na/K-ATPase. On the other hand, mCBM disrupted the interaction between the a1 Na/K-ATPase, caveolin-1 and Src, resulting in a redistribution of these proteins from cholesterol-rich low density into low cholesterol high density membrane fractions. Consequently, ouabain failed to activate a1 Na/K-ATPase-mediated signal transduction. Most interestingly, LW-mCBM cells proliferated much slower than that of cells expressing wild type a1 Na/K-ATPase. Taken together, we have not only confirmed the importance of the CBM-mediated caveolin-1 interaction in the assembly of multiple protein signaling complex, but also identified a mutant a1 Na/K-ATPase that pumps normally but is defective in this assembly.
Although several lines of evidence have indicated that a1 Na/K-ATPase functions as an important signal receptor for CTS to regulate physiological processes in animals, there is no direct genetic evidence to support such a role. To seek the genetic proof of the concept that a1 Na/K-ATPase signaling function plays an important role in animal physiology, we generated a mCBM a1 Na/K-ATPase knock-in mouse line (mCBM). Two allele mutations resulted in homozygous embryonic lethality between 9.5-12.5 days post coitum (dpc.) Further analysis found that mCBM homozygous embryos exhibited an arrest of brain and somite development along with the down-regulation of transcriptional factors important for neurogenesis and muscle genesis. Moreover, these inhibitory effects appeared to be gene-dose dependent, in which the expression of these transcriptional factors were reduced in mCBM heterozygous embryos, but the degree of reduction was much less than that of homozygous embryos. Additionally, Wnt/ß-catenin signaling, known as being critical in embryonic development, appeared to be defective since the expression of multiple components of this signaling pathway was significantly altered. These data provide the first genetic evidence that the signaling function of a1 Na/K-ATPase is important for animal physiology, specifically for embryonic development. They have also suggested that a1 Na/K-ATPase is indeed a signal integrator, because neutralization of endogenous CTS only produced a mild phenotype in embryonic development.
Na/K-ATPase/Src signaling complex has been ascribed as an important regulator of pathological amplification of ROS stress, and as such it plays an important role in the progression of many chronic diseases including chronic kidney disease, obesity and nonalcoholic steatohepatitis (NASH). However, no genetic evidence has been generated to support such claim. Following up with the observations that mCBM heterozygous mice are lean and exhibit reduced caveolae number in the adipocytes, we employ mCBM heterozygous mice to a murine model of NASH. In response to Western diet, mCBM mice accumulated less fat along with a decrease in total cholesterol and LDL in the plasma in comparison to the wild type littermate control. Liver damage in terms of fat accumulation, inflammation, hepatocellular ballooning as well as plasma ALT level was significantly attenuated in mCBM heterozygous mice. Moreover, when pNaKtide, a specific peptide inhibitor of a1 Na/K-ATPase/Src complex, was administrated to wild type animals fed Western diet, we found that pNaKtide, like mCBM mice, significantly reduced body fat contents and liver damage. Thus, we conclude that a1 Na/K-ATPase plays an important role in the development of obesity and Non-alcoholic Fatty Liver Disease (NAFLD)/NASH. On this scientific premise, we further suggest that a1 Na/K-ATPase represents a novel target for developing new and effective therapeutics for NASH and other chronic conditions where ROS/inflammation is important for the disease progression.
Finally, caveolin-1, the main partner of a1 Na/K-ATPase, has been found to play a significant role in the development of pulmonary arterial hypertension through its regulation of eNOS pathway. Because we have found a significant alteration in caveolae formation in adipocytes of mCBM heterozygous mice, we were prompted to explore whether mCBM animals develop pulmonary arterial hypertension. Similar to caveolin-1 null mice, mCBM heterozygous mice showed elevated pulmonary artery systolic pressure, most likely due to an increase in the muscularization of pulmonary arteries, a hall-marker of pulmonary arterial hypertension. Consistently, we also detected significant right ventricular hypertrophy as evidenced by an increase in Fulton index and increased expression of hypertrophic marker genes. Unlike caveolin-1 knockout mice, we failed to detect any structural and functional changes in the left ventricles of mCBM mice. Taken together, these findings provide further support to our hypothesis that a1 Na/K-ATPase is not only a signaling receptor but also a signal integrator. Moreover, they suggest that mCBM mice may be used as a unique pulmonary arterial hypertension model to probe for signaling pathways important for this clinical condition as well as to aid the development of new therapeutics of this disease.