Characterization of the Functional Role of the Intracellular Cholesterol Transporter StARD4 in Knockout Mice, and Investigation of Epigenetic Modulation of ApoA-I Transcription

Cholesterol is crucial for mammalian survival by playing important roles, such as regulating membrane fluidity and as a precursor for the synthesis of steroid and sex hormones, bile acids, and Vitamin D. In addition, cellular and organismal regulation of cholesterol is important for health. For example, increased levels of plasma LDL cholesterol are a risk factor for coronary heart disease and stroke. Intracellular cholesterol levels are regulated by a variety of mechanisms, but numerous studies indicate a very important role for transcriptional regulation by Sterol Regulatory Binding Proteins (SREBPs), Liver X Receptors (LXRs) and Unfolded Protein Response (UPR) or ER stress families of transcription factors. StARD4 is regulated by SREBPs and we have chosen to make a mouse knockout model to characterize its role in-vivo. StARD4, expressed primarily in liver and macrophages, is a known intracellular cholesterol transporter previously shown to be down-regulated ~2 fold in liver, by high cholesterol feeding. It is thought to be involved in the dynamics of cholesterol movement between ER, plasma membrane, endosomes and lipid droplets. Based on these observations, I hypothesized that a knockout of StARD4 in a mouse model would show altered intracellular cholesterol sorting, and figuring out the basis of such a defect would provide insight into the general mechanisms of intracellular sterol transport. To my surprise, StARD4 knockouts were viable and for the most part phenotypically normal. They showed no alteration in plasma or liver cholesterol or triglycerides. In addition, no abnormalities were found in glucose metabolism, macrophage cholesterol efflux, or atherosclerosis susceptibility. Based on these observations, I hypothesize that in-vivo, the absence of StARD4 is compensated for by other genes and/or pathways. In the future, it will be necessary to identify these compensatory mechanism(s) to truly understand the physiological role of StARD4.I also studied another aspect of cholesterol metabolism related to its transport in plasma in high density lipoproteins (HDLs). HDL is involved in the reverse cholesterol transport mechanism, whereby excess cholesterol is removed from peripheral tissues and transported to the liver for excretion. The major protein of HDL is apoA-I and in mouse models it has been shown that animals transgenic for apoA-I have increased HDL levels. This suggests increased apoA-I transcription as a mechanism for increasing HDL, which might be preventive or therapeutic for coronary heart disease. With this as a goal, as part of my thesis I studied the epigenetic regulation of apoA-I transcription. I found that increased apoA-I transcription in liver cell culture cell lines was associated with highly unmethylated CpGs in the apoA-I promoter, and the reverse, in cultures with poor apoA-I expression. I also found histone marks associated with apoA-I expression. This project was discontinued in favor of the StARD4 knockout mouse project. However, it might be continued in the future to reveal drug targets that alter epigenetic regulation of apoA-I in a manner that raises HDL levels

Targeted disruption of steroidogenic acute regulatory protein D4 leads to modest weight reduction and minor alterations in lipid metabolism[S]

Steroidogenic acute regulatory protein (StAR)D4 is a member of the StAR related lipid transfer family. Homology comes from the ∼210 amino acid lipid binding domain implicated in intracellular transport, cell signaling, and lipid metabolism. StARD4 was identified as a gene downregulated 2-fold by dietary cholesterol (Soccio, R. E., R. M. Adams, K. N. Maxwell, and J. L. Breslow. 2005. Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress. J. Biol. Chem. 280: 19410–19418). A mouse knockout was created to investigate StARD4’s functionality and role in lipid metabolism. Homozygous knockout mice exhibited normal Mendelian mating genetics, but weighed less than wild-type littermates, an effect not accounted for by energy metabolism or food intake. Body composition as analyzed by DEXA scan showed no significant difference. No significant alterations in plasma or liver lipid content were observed on a chow diet, but female knockout mice showed a decrease in gallbladder bile cholesterol and phospholipid concentration. When challenged with a 0.2% lova­statin diet, StARD4 homozygous mice exhibited no changes. However, when challenged with a 0.5% cholesterol diet, female StARD4 homozygous mice showed a moderate decrease in total cholesterol, LDL, and cholesterol ester concentrations. Microarray analysis of liver RNA found few changes. However, NPC1’s expression, a gene not on the microarray, was decreased ∼2.5-fold in knockouts. These observations suggest that StARD4’s role can largely be compensated for by other intracellular cholesterol transporters