br ABCA is a sterol responsive gene The
ABCA2 is a sterol-responsive gene The first evidence to demonstrate that ABCA2 expression responded to sterol loading was provided in an experiment involving monocyte to macrophage differentiation in vitro. Upon sterol loading with enzymatically degraded LDL (eLDL), ABCA2 expression increased only in differentiated macrophages and not in monocytes ; however, the mechanism for the increase in expression or its relevance in vivo was not investigated. To investigate whether ABCA2 was coordinately regulated with other sterol-responsive genes, we measured the mRNA levels of several genes in HepG2 liver hepatocarcinoma cells cultured in lipoprotein-deficient medium (LPDS) and in medium supplemented with exogenous sterols. The mRNA levels of both the Low-density lipoprotein receptor (LDLR) and Hydroxymethylglutaryl coenzyme A synthase (HMGCS) genes are sensitive to changes in the level of cellular cholesterol. Transcription of these genes is increased when cells are cultured in lipoprotein-deficient medium and repressed when exogenous sterols are added to the culture medium. Up-regulation of the LDLR under lipoprotein-deficient conditions facilitates the increased uptake of extracellular LDL (salvage pathway) to restore cellular cholesterol levels. HMGCS is one of a battery of enzymes whose expression is up regulated under sterol-deficient conditions to synthesize cholesterol de novo from Fluorescein TSA precursors to restore cellular cholesterol levels (synthetic pathway). Under lipoprotein-deficient culture conditions we measured increased mRNA levels for ABCA2, LDLR and HMGCS that were repressed upon supplementation of culture medium with exogenous cholesterol . The results of these studies provided further evidence that ABCA2 was a sterol-responsive gene involved in cholesterol homeostasis. To determine whether the constitutive overexpression of ABCA2 in cells alters the expression of other sterol-responsive genes, we generated a CHO cell line that constitutively overexpressed human ABCA2 under control of the cytomegalovirus (CMV) promoter control element. The mRNA of the transcription factor, Sterol response element binding protein-2 (SREBP2), is elevated in cells under sterol-depleted conditions and repressed upon sterol loading . SREBP2 regulates the transcription of both the LDLR as well as other enzymes in the cholesterol synthetic pathway, including HMGCS. We hypothesized that constitutive overexpression of ABCA2 in CHO cells in culture medium containing serum (a source of LDL-derived free cholesterol) would mimic sterol-deprivation. Under these conditions, ABCA2-mediated inhibition of cholesterol trafficking from the LE/LY to cholesterol sensors in the endoplasmic reticulum would activate the cholesterol synthetic pathway and sterol-responsive gene expression would be up regulated. In fact, this was what we observed, i.e., both the LDLR and SREBP-2 mRNAs were elevated in CHO cells overexpressing ABCA2 compared to control CHO cells . The results were recapitulated in a study of the human LDLR promoter coupled to the luciferase gene in CHO cells under the same cell culture conditions. These findings indicated that constitutive overexpression of ABCA2 in cells was sufficient to increase the expression of genes involved in cholesterol homeostasis.
Progesterone and U18666a increase expression of ABCA2 mRNA To gain insight into a functional role for ABCA2, we treated cells with agents that are known to modulate cholesterol homeostasis in the LE/LY. Numerous investigators have reported that treatment of cells in culture with either the hormone progesterone or a pharmacological reagent, the cationic amphiphile U18666a, resulted in the sequestration of free-cholesterol in the LE/LY , , , . More recent work has elucidated the mechanism of U18666a function in inhibiting cholesterol exit from the LE/LY. When an U18666a-derivative, synthesized to contain an ultraviolet light-sensitive benzophenone moiety was added to CHO cells, exposure to UV light induced the crosslinking of the derivative to the sterol-sensing domain (SSD) of the Niemann Pick Type C1 protein (NPC1), abrogating its activity . We hypothesized that these LE/LY cholesterol-sequestering agents would mimic the cellular physiological effects of ABCA2 in cells and may in fact be modulators of ABCA2 expression. Treatment of CHO cells with either progesterone or U18666a resulted in an elevation of ABCA2 mRNA expression over 2-fold and 3.5-fold, respectively . These results indicated that ABCA2 expression is responsive to hormonal and pharmacological agents that sequester cholesterol in the LE/LY.