Isolation of the Human Aquaporin-1 Promoter and Functional Characterization in Human Erythroleukemia Cell Lines

Abstract

Water channel aquaporin-1 (AQP1) is expressed in erythrocytes and various epithelia and endothelia. To study AQP1 gene regulation, human cell lines were screened for inducible AQP1 expression. Human erythroleukemia HEL cells showed AQP1 transcript expression on RNase protection assay. After butyrate-induced erythroid differentiation, AQP1 transcript expression increased strongly, producing water-permeable cells with plasma membrane localization of immunoreactive AQP1. In addition, a clonal subline of K562 cells [K562(S)] showed strong butyrate-induced expression of functional AQP1. A 1.8-kb DNA fragment of the 5′ flanking region of the human AQP1 gene was isolated, sequenced, and analyzed functionally by the CAT reporter assay. The AQP1 promoter contained TATA and CCAAT boxes; Sp1, AP1, AP2, and E-box elements; and erythrocyte-specific CACCC and Kruppel-like (CCCCACCCA) elements. AQP1 promoter activity was more than 24-fold higher in HEL and K562(S) cells than in nonerythroid (HeLa) cells, indicating the presence of erythroid-specific factors. In K562(S) cells, CAT activities for promoter fragments to bp +23 [relative to β-gal and normalized to 100% for the plasmid CP-282 (bp −282 to +23)] were 22 (−1779), 73 (−1402), 61 (−1129), 31 (−789), 87 (−487), 100 (−282), 73 (−229), 52 (−152), and 60% (−79). After butyrate-induced differentiation, CAT activities were stimulated ∼10-fold for constructs −229/+23 and longer, compared to ∼5-fold for −152/+23 and −79/+23; glucocorticoids did not affect CAT activities. These results suggest a basis for erythroid-specific AQP1 expression and the presence of a butyrate-response sequence involved in inducible AQP1 regulation in erythroleukemia cells.