Exp Clin Endocrinol Diabetes 2013; 121 - P35
DOI: 10.1055/s-0033-1336674

The effect of a TRB-3 knockdown on cholesterol absorption in THP-1 cells and macrophages

M Kafka 1, B Heinze 1, S Sbierra 1, S Hahner 1, M Fassnacht 1, B Allolio 1, D Weismann 1
  • 1Medizinische Klinik I, Universität Würzburg, Endokrinologie, Würzburg, Germany

Objective: A TRB-3 knockdown in insulin resistant mice decreased atherosclerosis. Suppression of TRB-3 in macrophages results in increased macrophage viability, increased adhesion of monocytes and increased phagocytosis. TRB-3 is induced by endoplasmatic reticulum (ER) stress.

Aim: To describe the impact of TRB-3 on cholesterol uptake in monocytes (THP-1) and monocyte derived macrophages in vitro.

Methods: THP-1 cells and monocytes were incubated with increasing concentrations of thapsigargin to promote ER-stress dependent TRB-3 expression. TRB-3 silencing by siRNA was implemented to prevent thapsigargin-induced TRB-3 induction. Expression of TRB-3 was measured by western blot (WB) and real time PCR (rt-pcr) analysis. The effect of modified TRB-3 expression on cholesterol absorption was investigated by measurement of bodipy®-labeled cholesterol influx using fluorescence spectrometry.

Results: There is a low constitutive expression of TRB-3 in monocytes and macrophages. Stimulation with thapsigargin (0.5, 1, 1.5, 2 and 5µM) resulted in a significant (p < 0.05) increase in TRB-3 expression by 4.5-, 8.5-, 10.5-, 13.5-, and 19.5- fold, respectively. Concomitantly, there was a significant increase in ER-stress markers like BAX, CHOP and ATF4 (rt-pcr). TRB-3 siRNA decreased the expression of TRB- 3 in thapsigargin-treated cells (p < 0.05). Cholesterol uptake by control macrophages was higher (3- fold) compared to control monocytes. In macrophages, stimulation with 1.5µM und 5µM Thapsigargin resulted in a significant decrease in the absorption of cholesterol (p < 0.05). However, silencing of TRB-3 did not restore cholesterol uptake in macrophages.

Conclusion: These data suggests that both in monocytes and in macrophages TRB-3 is induced by thapsigargin concomitantly to ER-stress. Furthermore, thapsigargin decreased cholesterol uptake in macrohpages. However, a knockdown of TRB-3 does not restore cholesterol uptake macrohpages in vitro.