Apical trehalase expression associated with cell patterning after inducer treatment of LLC‐PK1 monolayers

Abstract

Trehalase, a differentiation-specific marker of renal proximal tubule brush border membrane, is expressed in confluent long-term cultures of the renal epithelial cell line LLC-PK₁. The level of trehalase is greatly increased after treatment of cultures with differentiation inducers such as hexamethylene bisacetamide (HMBA), accompanied by increases in other apical membraneassociated differentiated functions (Yoneyama and Lever: J. Cell. Physiol. 121: 64–73, 1984). In the present study, we utilize a polyclonal antibody specific for renal trehalase to demonstrate that trehalase expression induced in LLC-PK₁ cultures after HMBA treatment is localized in cells forming a three-dimensional network of strands across the confluent monolayer. The antitrehalase antibody recognized an apical membrane antigen of apparent molecular weight 100–110 kD both in LLC-PK₁ cultures and in the corresponding pig renal brush border membranes. Strand formation and total trehalase activity increased in parallel as a function of inducer concentration and duration of exposure. Strand formation and trehalase expression were also greatly enhanced in monolayers grown on a Nuclepore filter support even in the absence of inducer. Strand formation was not a prerequisite for induced trehalase expression in culture, since strands did not develop in cultures treated with N, N'-dimethylformamide (DMF) an equally potent inducer of trehalase expression. In this case, cells which expressed increased levels of trehalase were dispersed at random over the monolayer. Induction of strand formation and trehalase expression by HMBA required a minimum exposure period of 48 hr and persisted up to a week after removal of inducer. By contrast, the response to DMF required continuous presence of inducer. Levels of trehalase declined even in the continuous presence of inducer in local regions of low cell density created by wound-repair of the monolayer. In addition to the membrane-bound form, trehalase activity was also recoverable from the culture medium, but release of trehalase was not affected by inducers. These observations are consistent with the view that a cell type committed to express a program of differentiation after HMBA treatment or growth on a permeable support is organized in specific cell patterns visible as strands over the confluent cell monolayer.