Abstract
The functional expression of the amiloride-sensitive epithelial sodium channel (ENaC) in select epithelia is critical for maintaining electrolyte and fluid homeostasis. Although ENaC activity is strictly dependent upon its alpha-subunit expression, little is known about the molecular mechanisms by which cells modulate alpha-ENaC gene expression. Previously, we have shown that salivary alpha-ENaC expression is transcriptionally repressed by the activation of Raf/extracellular signal-regulated protein kinase pathway. Here, this work further investigates the molecular mechanism(s) by which alpha-ENaC expression is regulated in salivary epithelial Pa-4 cells. A region located between -1.5 and -1.0 kilobase pairs of the alpha-ENaC 5'-flanking region is demonstrated to be indispensable for the maximal and Ras-repressible reporter expression. Deletional analyses using heterologous promoter constructs reveal that a DNA sequence between -1355 and -1269 base pairs functions as an enhancer conferring the high level of expression on reporter constructs, and this induction effect is inhibited by Ras pathway activation. Mutational analyses indicate that full induction and Ras-mediated repression require a glucocorticoid response element (GRE) located between -1323 and -1309 base pairs. The identified alpha-ENaC GRE encompassing sequence (-1334/-1306) is sufficient to confer glucocorticoid receptor/dexamethasone-dependent and Ras-repressible expression on both heterologous and homologous promoters. This report demon- strates for the first time that the cross-talk between glucocorticoid receptor and Ras/extracellular signal-regulated protein kinase signaling pathways results in an antagonistic effect at the transcriptional level to modulate alpha-ENaC expression through the identified GRE. In summary, this study presents a mechanism by which alpha-ENaC expression is regulated in salivary epithelial cells.
Highlights
Sodium balance is important for the maintenance of body electrolyte, extracellular volume, and blood pressure
In order to more precisely evaluate the ability of Ras and its different effector pathways to modulate ␣-ENaC expression in mammalian cells, salivary epithelial Pa-4 cells were cotransfected with an ␣-ENaC/chloramphenicol acetyltransferase (CAT) reporter construct and an expression plasmid encoding a constitutively activated form of Ras (Ras V12) or a “single-effector” mutant, i.e. Ras S35, Ras G37, and Ras C40
It is possible that the known differences in the activity and expression level of these mutant proteins [28] may have contributed to these results, it is more likely that both Ral-GDS and Raf/extracellular signalregulated protein kinase (ERK) signaling pathways are involved in down-regulating ␣-ENaC expression
Summary
Sodium balance is important for the maintenance of body electrolyte, extracellular volume, and blood pressure. When expressed individually in a Xenopus oocyte system, only the ␣-subunit can produce an amiloride-sensitive current Both - and ␥-subunits are not functional on their own, but augment the channel activity of ␣-ENaC [3, 8]. Structure, and activity of ENaC have been well studied, the molecular basis for the regulatory mechanisms underlying the gene expression of ENaC subunits remains unclear. Results from studies by us and others have suggested that the transcriptional control of ENaC gene expression occurs in a subunit- and tissue-specific manner Studies that more precisely define the interaction between the cellular transcriptional machinery and signaling pathways are needed to provide a framework for understanding the mechanisms underlying the regulation of ENaC expression and the resultant electrolyte homeostasis. The GR is expressed in virtually all mammalian cell types, it is possible that the expression of a distinct set of GR-responsive genes is cell context-specific and may be modulated by other signaling pathway(s)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
