Background and aims: Dehydroepiandrosterone fatty acid esters (DHEA-FAEs) belong to a unique family of naturally occurring hydrophobic steroid hormone derivatives with long-lasting hormonal activity. DHEA-FAEs function through hydrolysis of DHEA-FAEs into free DHEA and further metabolized to biologically active steroids. The lysosomal acid lipase (LAL) mediates the intracellular hydrolysis of cholesterol esters. This study aims to study the possible role of LAL on the hydrolysis of DHEA-FAE in cultured HeLa cells.
Methods: We used LAL short-interfering RNA (siRNA) oligonucleotides to knock down the expression of LAL in HeLa cells. To control for unspecific RNAi effects, control cells were transfected with nonsilencing siRNA oligos without known similarities to human sequences. We incorporated labelled DHEA-FAEs into LDL by incubating 3H-DHEA with VLDL-free plasma. These siRNA-treated cells were labelled with 3H-DHEA -FAE-LDL for 6 h and chased for 14 and 20 h in the presence of Acyl-CoA:cholesterol acyltransferase inhibitor, PKF 58-035. Cellular and medium fractions were collected and immediately extracted. We used a variety of chromatographic techniques to identify metabolites in the cellular and medium fractions. 3H-cholesterol-oleate-LDL was used as control in parallel experiments to test the validity of the assay.
Results: Anti-LAL western blot showed that most of the LAL protein was depleted by LAL siRNA. After 14 hours chase, more 3H-DHEA-FAE remained unhydrolyzed in the LAL siRNA experiment (71.4%) than that in control experiment (51.8%). When the chase time was extended to 20 h, the difference between two groups disappeared (14.9% and 11.9%, respectively).
Conclusion: Our preliminary studies suggest that LAL is involved in the hydrolysis of 3H-DHEA-FAEs in cultured HeLa cells.
03 - 07 May 2008
European Society of Endocrinology