24S-hydroxycholesterol effects on lipid metabolism genes are modeled in traumatic brain injury

Abstract

Membrane damage during traumatic brain injury (TBI) alters the brain homeostasis of cholesterol and other lipids. Cholesterol 24S-hydroxylase (Cyp46) is a cholesterol metabolic enzyme that is increased after TBI. Here, we systematically examined the effects of the enzymatic product of Cyp46, 24S-hydroxycholesterol, on the cholesterol regulatory genes, SREBP-1 and 2, their posttranslational regulation, and their effects on gene transcription. 24S-hydroxycholesterol increased levels of SREBP-1 mRNA and full-length protein but did not change levels of cleaved SREBP-1, consistent with the role of 24-hydroxycholesterol as an LXR agonist. In contrast, 24S-hydroxycholesterol decreased levels of LXR-independent SREBP-2 mRNA, full-length protein, and SREBP-2 active cleavage product. We examined the downstream effects of changes to these lipid regulatory factors by studying cholesterol and fatty acid synthesis genes. In neuroblastoma cells, 24S-hydroxycholesterol decreased mRNA levels of the cholesterol synthesis genes HMG CoA reductase, squalene synthase, and FPP synthase but did not alter levels of the mRNA of fatty acid synthesis genes acetyl CoA carboxylase or fatty acid synthase. After TBI, as after 24S-hydroxycholesterol treatment in vitro, SREBP-1 mRNA levels were increased while SREBP-2 mRNA levels were decreased. Also similar to the in vitro results with 24S-hydroxycholesterol, HMG CoA reductase and squalene synthase mRNA levels were significantly decreased. Fatty acid synthase mRNA levels were not altered but acetyl CoA carboxylase mRNA levels were significantly decreased. Thus, changes to transcription of cholesterol synthesis genes after TBI were consistent with increases in Cyp46 activity, but changes to fatty acid synthesis genes must be regulated by other mechanisms.

Figure 1. 24S-hydroxycholesterol increases SREBP-1 protein levels while uppressing SREBP-2 levels

293 cells were treated for 48 hr with 5µM 24S-hydroxycholesterol (24) or control vehicle (C). A) Levels of precursor SREBP-1 (pSREBP-1) and mature SREBP-1 (mSREBP) as well as precursor SREBP-2 (pSREBP-2) and mature SREBP-2 (mSREBP-2) were measured in cell lysate by immunoblot. B) Quantification of pSREBP-1 levels showed an increase of 38% after 24S-hydroxcholesterol treatment (Student’s t-test, *p<0.05, n=8) while there were no significant changes in mSREBP-1 levels. C) Quantification of pSREBP-2 levels showed a decrease of 51% after 24S-hydroxycholesterol treatment (Student’s t-test, *p<0.0001, n=7) while mSREBP-2 levels showed a decrease of 91% (Student’s t-test, *p<0.0001, n=7). Error bars (standard error of the mean).

Figure 2. 24S-hydroxycholesterol and LXR agonist TO-901317 both increase SREBP-1 mRNA levels while 24S-hydroxycholesterol decreases SREBP-2 mRNA levels

293 cells were treated for 48 hr with 5 µM 24S-hydroxycholesterol (24), 1 µM TO-901317 (TO) or control vehicle (C). mRNA from these cells was converted to cDNA and amplified using primers against either SREBP-1 or SREBP-2. A) Relative levels of SREBP-1 mRNA showed a clear increase following 24S-hydroxycholesterol and a greater increase following TO-901317 treatment. Relative levels of SREBP-2 mRNA showed small decreases with 24S-hydroxycholesterol treatment and small increases with TO-901317 treatment. B) Real-time PCR quantification of SREBP-1 mRNA levels showed an increase of 280% with 24S-hydroxycholesterol treatment (Newman-Keuls, *p<0.001, n=6) and an increase of 370% with TO-901317 treatment (Newman-Keuls, *p<0.001, n=6). Real-time PCR quantification of SREBP-2 mRNA levels showed a decrease of 18% with 24S-hydroxycholesterol treatment (Newman-Keuls, *p<0.05, n=6) but no significant change with TO-901317 treatment. Control relative quantity=1. Error bars (standard error of the mean).

Figure 3. 24S-hydroxycholesterol but not cholesterol or TO-901317 suppresses SREBP-2 protein levels

293 cells were treated for 48 hr with 5 µM 24S-hydroxycholesterol (24), 5 µM 27-hydroxycholesterol (27), 5 µM cholesterol (Ch), 1 µM TO-901317 (T) or control vehicle (C). A) Levels of precursor SREBP-2 (pSREBP-2) and mature SREBP-2 (mSREBP-2) were measured in cell lysate by immunoblot. B) Quantification of pSREBP-2 levels showed a decrease of 50% with 24S-hydroxycholesterol treatment (Newman-Keuls, *p<0.001, n=6) while 27-hydroxycholesterol failed to significantly decrease pSREBP-2 levels. Neither cholesterol nor TO-901317 had a significant effect on pSREBP-2 levels. C) mSREBP-2 levels showed a decrease of 84% with 24S-hydroxycholesterol treatment (Newman-Keuls, *p<0.001, n=6) and a decrease of 80% with 27-hydroxycholesterol treatment (Newman-Keuls, *p<0.001, n=6). TO-901317 also decreased mSREBP-2 levels by 36% (Newman-Keuls, *p<0.05, n=6). Decreases in mSREBP-2 levels by 24S-hydroxycholesterol and 27-hydroxycholesterol were significantly lower than those seen with TO-901317 (Newman-Keuls, ❖p<0.01, n=6). Cholesterol did not have a significant effect on mSREBP-2 levels. Error bars (standard error of the mean).

Figure 4. 24S-hydroxycholesterol increases SREBP-1 protein levels while suppressing SREBP-2 levels in SY5Y cells

SY5Y cells were treated for 48 hr with 5 µM 24S-hydroxycholesterol (24S) or control vehicle (C). Results seen in this representative blot were similar to those seen in 293 cells. pSREBP-1 levels were clearly increased while mSREBP-1 levels remained relatively stable with 24S-hydroxycholesterol treatment. Both pSREBP-2 and mSREBP-2 were suppressed by 24S-hydroxycholesterol treatment.

Figure 5. Effects of 24S-hydroxycholesterol on cholesterol and fatty acid synthesis genes in human neuroblastoma SY5Y cells

SY5Y cells were treated for 48 hr with 5 µM 24S-hydroxycholesterol or vehicle control and mRNA levels were measured by real-time PCR. A) 24S-hydroxycholesterol increased SREBP-1 mRNA levels by 210% (student’s t-test, *p<0.001, n=6) and decreased SREBP-2 mRNA levels by 31% (student’s t-test, *p<0.001, n=6) in comparison to controls. B) 24S-hydroxycholesterol decreased mRNA levels of the rate limiting cholesterol synthesis enzyme HMG CoA reductase by 18% (student’s t-test, *p<0.005, n=6), decreased squalene synthase by 31% (student’s t-test, *p<0.005, n=6) and FPP synthase by 17% (student’s t-test, *p<0.01, n=6). C) 24S-hydroxycholesterol did not significantly change mRNA levels fatty acid synthesis enzymes acetyl CoA carboxylase or fatty acid synthase in comparison to controls. Control relative quantity=1. Error bars (standard error of the mean).

Figure 6. Regulation of SREBP-1 and SREBP-2 mRNA levels 7 days after TBI

SREBP-1 and SREBP-2 mRNA levels were measured by real-time PCR in cortex ipsilateral to the site of injury 7 days after TBI (tbi icx), contralateral cortex (tbi ccx) and sham cortex ipsilateral to craniotomy (sham icx). A) SREBP-1 mRNA levels at the site of injury were significantly increased 86% in comparison to sham controls (Newman-Keuls, *p<0.01, n=5). B) SREBP-2 mRNA levels at the site of injury were significantly decreased 43% in comparison to sham controls (Newman-Keuls, *p<0.05, n=5).

Figure 7. Regulation of HMG CoA reductase and acetyl CoA carboxylase mRNA levels 7 days after TBI

mRNA levels of acetyl CoA carboxylase, fatty acid synthase, HMG CoA reductase and squalene synthase were measured by real-time PCR in cortex ipsilateral to the site of injury 7 days after TBI (tbi icx), and sham cortex ipsilateral to craniotomy (sham icx). A) HMG CoA reductase mRNA levels were significantly decreased 38% (Student’s t-test, *p<0.005, n=5) and squalene synthase mRNA level were significantly decreased 33% (student’s t-test, *p<0.05) at the site of injury in comparison to sham controls. B) acetyl CoA reductase mRNA levels at the site of injury were significantly decreased 26% (Student’s t-test, *p<0.05, n=5) while there was no change in fatty acid synthesis mRNA levels at the site of injury in comparison to sham controls. Error bars (standard error of the mean).