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Jorge Alberto Lobato, Arlet Loza-Huerta, José Bonilla-Delgado, Ricardo Gonzalez, Liora Shoshani; The apical polarity of Na+,K+-ATPase in ARPE-19 cells is regulated by the expression of the β2 subunit. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6309.
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The Na+, K+ ATPase or sodium pump plays a very important role in the maintenance of epithelial phenotype. In most epithelia, the pump is located in the lateral domain. We have shown that the β1-subunit plays an important role in the polarization mechanism of the pump. In the retinal pigment epithelium (RPE) the sodium pump is located at the apical domain, nonetheless, the mechanism is still unknown. Our working hypothesis is that the pump apical polarity in RPE cells is regulated by its β2 subunit. We got evidences that related the apical localization of the pump in RPE with the expression of the β2 subunit, now we are focused on the mechanism by which β2 subunit regulates the pump polarity.
ARPE-19 cells are cultured on laminin covered clear inserts with a mixture of DMEM/F-12 medium and 1% BSA. The expression of β1 and β2 isoforms was determined by WB. The membrane localization of each isoform was determined by IF staining with specific antibodies for each isoform. mRNAs were quantified by RT-qPCR using human specific primers designed for each isoform. siRNAs for silencing the human β1 and β2 isoforms were purchased from Quiagen.
ARPE-19 cells are culture for 8 weeks to acquire the RPE phenotype and express the sodium pump in the apical domain. We show that adding ITS (mix of Insulin, Transferrin and Selenic acid) to the culture medium reduces that time to 4 weeks. Western Blot (WB) and Immunofluorescence (IF) analyses show that under these conditions (+ITS), the β2 isoform is detected in the apical domain earlier, since the 2nd week. Interestingly, we found that this isoform is co-expressed with the α2 subunit at the apical domain. Nevertheless, β1 subunit is detected from the 2nd week at the basolateral domain and is co-localized with α1 subunit. In order to find out if the β2 expression is regulated on a transcriptional or translational level, we analyzed as function of time, the mRNAs level of the β subunit isoforms by RTqPCR and the protein expression level by WB. We observed that in the presence of ITS, β2 mRNAs are incremented during the 6 weeks, while the protein level is constant.
Our results suggest that the expression of the β2 isoform in ARPE-19 is regulated on a translational level. Nevertheless, we do not exclude the possibility that transcription factors such as SP1 are also involved.
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