Increase in cholesterol sulfotransferase activity during in vitro squamous differentiation of rabbit tracheal epithelial cells and its inhibition by retinoic acid.

J I Rearick,P W Albro,A M Jetten

doi:10.1016/s0021-9258(18)45168-x

Abstract

It has previously been demonstrated that rabbit tracheal epithelial cells in primary culture undergo terminal differentiation at confluence to yield cornified cells much in analogy to epidermal keratinocytes and that one biochemical marker of this process seems to be the accumulation of cholesterol sulfate by the cells. The current work addresses the possible causes of this accumulation. Our studies show that the stimulation of cholesterol sulfate is paralleled by an increased activity of the biosynthetic enzyme cholesterol sulfotransferase. Squamous differentiated cells exhibited 20- to 30- fold higher levels of this enzyme activity than that in undifferentiated cells. As with other markers of squamous cell differentiation, the increase in cholesterol sulfotransferase can be prevented by the inclusion of retinoids in the cell culture medium. Inhibition of sulfotransferase levels can be observed at concentration of retinoic acid as low as 10(-11) M. The enzyme activity is optimal at pH 7 in buffers containing 0.2 M NaCl and 0.01% Triton X-100. Apparent Michaelis constants for the substrates 3'-phosphoadenosine-5'-phosphosulfate and cholesterol are 1 microM and 0.6 mM, respectively. Our results indicate that the increase in cholesterol sulfotransferase is the proximate cause for the accumulation of cholesterol sulfate in rabbit tracheal epithelial cells during squamous cell differentiation.

Highlights

NaCl and 0.01‘70Triton X-100.ApparentMichaelis of cholesterol sulfate synthesis with other well-established constants for the substrates 3’-phosphoadenosine-5’- markers of squamous cell differentiation leads us to propose phosphosulfate and cholesterol are 1 FM and 0.6 mM, that itbe considered a new marker of this process
In the current work we address theotherthree possibilities mentioned above and conclude that increases in cholesterol sulfotransferase activity which occurduring i n vitro squamous cell differentiation of RbTE cells appear to be the main cause for theincreased levels of cholesterol sulfate
Crude sonicates of confluent RbTE cells are able to transfer amounts of enzyme from the supernatant described above in a final 35S04from PAPS to lipophilic acceptors as detected by the volume of 100 pl

Summary

THEJOURNAOFLBroLOClCAL CHEMISTRY

13069-13074 1987 Printed in d.S.A. Increase in Cholesterol Sulfotransferase Activity during in Vitro Squamous Differentiation of Rabbit TrachealEpithelial Cells and Its Inhibition by Retinoic Acid*. We have observed that increases in [35S]sulfateincorporation into cholesterol sulfate correlate well with squamous cell differentiation in vitro, in particular with the formation of crosssion of retinoids in thecell culture medium. To study the regulation of differentiation of tracheobronchial epithelial cells, an in vitro model was developed using rabbit previously been ruled out since no overall increase in other sulfation reactions in the RbTE cells was observed [13]. In the current work we address theotherthree possibilities mentioned above and conclude that increases in cholesterol sulfotransferase activity which occurduring i n vitro squamous cell differentiation of RbTE cells appear to be the main cause for theincreased levels of cholesterol sulfate. Standard assay mixtures contained 50 mM HEPES, pH 7.3,0.01%Triton X-100,200 mM NaCI, 2.6 mM cholesterol, 1.8p M PAP=S (10' dpm), and varying

RESULTS

Endogenous acceDtof

DISCUSSION