Fine-tuning Of Amphiregulin Expression By Na,K-ATPase Ligands And Its Biological Significance

The Na,K-ATPase(NKA),also named as the sodium pump,is a kind of transmembrane protein generally expressed in mammals.Through the hydrolysis of adenosine triphosphate,the redundant intracellular sodium ion is extruded out of the cell with the entry of the excellular potassium ion.The sodium-potassium exchange creates the electrochemical gradient across the plasma membrane and facilitates the entry of other ions,amino acids,glucose and many other compounds into the cell.NKA is one specifical receptor of cardiotonic steroids(CTS)which are able to inhibit the activity of ion transportation of NKA by the competitive combination of the potassium ion binding site on the plasma membrane.CTS are derived from plants and interact with NKA,inhibiting the activity of NKA.However,later studies demonstrated that endogenous CTS(like ouabain)also exist in mammals.These substances have been used in clinical research to treat diseases,such as heart failure or arrhythmia based on their physiological function in myocardium and vessels.However,the functions of those endogenous CTS are still unclear.NKA was found to activate multiple growth signals and promote proliferation,differentiation or apoptosis in a cell-type-specific manner.For example,ouabain was able to modulate the process of cell proliferation,apoptosis,cell-to-cell contact and migration.It is worth noting that the pro-proliferating effect of CTS on the cell growth occured at low concentrations at which the inhibiton of the activity of cellular Na/K-ATPase was not observed.Thus,the NKA has dual functions:On one hand,NKAacts as an ion transporter;on the other hand,NKA functions as a receptor of CTS and mediates the downstream signaling independently of the activity of ion transport.Amphiregulin(AREG),a ligand of the epidermal growth factor receptor(EGFR),activates major intracellular signaling cascades governing cell survival,proliferation and motility through interacting with EGFR.AREG is always up-regulated in a wide variety of neoplasms.However,AREG production during inflammation and injury participates in defensive and repair mechanisms in different tissular backgrounds.In our experiments,we found that ouabain at low concentration was able to promote the expression of amphiregulin(AREG)significantly in some epithelial cells including A549?HK-2 and Hela cells in a dose-and a time-dependent manner.It is suggested that ouabain is highly expressed in multiple renal cyst and acute kidney injury patients,but the role of ouabain still remains unclear.It was also found in our experiment that ouabain promoted HK-2 cell growth at low concentration.And when AREG was depleted,the pro-growth effect of ouabain was significantly inhibited.Further study showed that AREG induced by ouabain also promoted HK-2 cell growth through paracrine signaling.Oxalate is a metabolic end product and forms calcium oxalate stones in the kidney.This substance is often deposited at the renal tubules and is toxic to the proximal tubule(PT)cells.We have found that the HK-2 cells growth was inhibited significantly by calcium oxalate monohydrate(COM).However,ouabain decreased its inhibition through the induction of the AREG expression.In conclusion,AREG plays a key role in the protection of the epithelial cells(especially the renal cells)by ouabain.To investigate the mechanisms in which AREG is upregulated by ouabain.The following experiments were performed.It was found that ouabain upregulated AREG mRNA at transcriptional level.The deletion analysis of promoter region revealed that multiple transacting factors,including CREB?ATF-2 and ATF-3 were involved in AREG transcriptional activation by ouabain.Besides,we also found ouabain prevented the degradation of AREG mRNA,indicating that ouabain could also regulate the expression of AREG in posttranscriptional level.Other study have reported that ultraviolet B(UVB)exposure increased a mRNA-binding protein,human antigen R(HuR)binding to AREG 3'-UTR to enhance the stabilization of AREG mRNA.However,in our study,ouabain increased the level of AREG mRNA in the absence of HuR and overexpression of exogenous HuR decreased the level of AREG mRNA.Our previous work has found that Na+,K+-ATPase ligands were capable of upregulating cytokines mRNA stability by HuR translocation,as well as promoting cytokines expression.So the main purpose of the research is to clarify the relationship between Na+/K+-ATPase-mediated signal transduction and HuR-mediated genetic expression.Src kinase is often involved in the signalling network of ouabain.Ouabain was able to stimulate the tyrosine phosphorylation of Src.Moreover,addition of a specific Src Family Kinase Inhibitor PP2 was sufficient to block ouabain-induced tyrosine phosphorylation,and subsequently blocked the effect of AREG upregulation induced by ouabain.These indicated that ouabain induces AREG expression through the activition of Src kinase.Interestingly,we found that PP2 enhanced the effect of HuR nuclear export.Thus,Src has a dual role in AREG regulation during the process of cell protection induced by ouabain:one is the activation of the transcription of AREG mRNA,the other is the inhibiton of the HuR nuclear translocation.All these results illustrated the mechanisms of AREG regulation mediated by ouabain.The novelties of this study lie in following findings:(1)AREG was first discovered as a cell protective role mediated by ouabain in epithelial cells and the underlying molecular mechanisms were well elaborated.(2)The link between the CTS/NAK/Src signaling pathway and the post-transcriptional regulation of HuR was first clarified,which helped broaden the knowledge of the biological effect of ouabain.Our further research should be focused on the precise regulation of AREG mRNA stability by the crosstalk between Src signaling and HuR signalosome.