bThe mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.T he mevalonate pathway leading to cholesterol synthesis is controlled transcriptionally and, for more rapid shutdown, posttranslationally (1). The third step in the pathway, catalyzed by 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), is generally regarded as the rate-limiting step in cholesterol synthesis and has been intensively studied (2, 3). However, squalene monooxygenase (SM) is a neglected rate-limiting enzyme in cholesterol synthesis downstream of HMGCR. A flavin monooxygenase located in the endoplasmic reticulum (ER), SM catalyzes the conversion of squalene into monooxidosqualene (MOS), the step in the mevalonate pathway preceding cyclization to form the steroid backbone. It can also act on its product to yield dioxidosqualene (DOS), the precursor for the potent oxysterol regulator 24(S),25-epoxycholesterol, which fine-tunes acute cholesterol synthesis (4). SM resides after the isoprenoid branch of the mevalonate pathway, committing products to sterol synthesis. This may allow differential control of cholesterol synthesis from that of essential nonsterol products (5).We recently reported that SM's activity is controlled at the posttranslational level via accelerated cholesterol-dependent ubiquitination and proteasomal degradation (5). This regulation requires the first 100 amino acids of the protein-a region that is highly conserved in vertebrates but lacking in lower organisms, such as yeast (Saccharomyces cerevisiae). Moreover, this sequence is sufficient to confer cholesterol-dependent turnover when fused to green fluorescent protein (GFP) (5). We have established that the process of degradation is distinct from the sterol-regulated ubiquitination and proteasomal degradation of HMGCR, as it does not require...

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article, we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively. Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system.

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Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake. Cholesterol is an essential constituent of cellular membranes and signaling pathways and is a precursor of sterol-derived molecules (1). Yet elevated levels of cholesterol are toxic to cells, and dysregulated cholesterol metabolism is associated, most evidently, with development of cardiovascular disease. As such, multiple transcriptional networks and posttranscriptional processes regulate the synthesis, uptake, and efflux of cholesterol. Transcriptionally, these processes are largely governed by the opposing actions of the transcription factors sterol regulatory element binding proteins (SREBPs) and the liver X receptors (LXRs) (2-6). Upon sensing low cholesterol levels in the endoplasmic reticulum (ER), SREBPs are processed into their mature, transcriptionally active form. This results in induction of the full set of genes required for de novo biosynthesis of cholesterol via the mevalonate pathway and of the low-density lipoprotein receptor (LDLR) that is required for uptake of LDL-derived cholesterol (7, 8). In contrast, LXRs, members of the nuclear receptor family, are activated when cellular cholesterol levels are elevated. Once activated by their cognate oxysterol ligands, LXRs induce cholesterol efflux pathways (e.g., via the transporters ABCA1 and ABCG1) and limit LDL uptake by inducing expression of the E3 ubiquitin ligase (E3)-inducible degrader of the LDLR (IDOL) (6, 9, 10). The coordinated action of these two transcription factor families ensures that cellular cholesterol is adequately maintained at an appropriate level.Next to transcriptional regulation, posttranscriptional mechanisms are ...

Cellular cholesterol metabolism is subject to tight regulation to maintain adequate levels of this central lipid molecule. Herein, the sterol-responsive Liver X Receptors (LXRs) play an important role owing to their ability to reduce cellular cholesterol load. In this context, identifying the full set of LXR-regulated genes will contribute to our understanding of their role in cholesterol metabolism. Using global transcriptional analysis we report here the identification of RNF145 as an LXR-regulated target gene. We demonstrate that RNF145 is regulated by LXRs in both human and mouse primary cells and cell lines, and in vivo in mice. Regulation of RNF145 by LXR depends on a functional LXR-element in its proximal promotor. Consistent with LXR-dependent regulation of Rnf145 we show that regulation is lost in macrophages and fibroblasts from Lxrαβ(-/-) mice, and also in vivo in livers of Lxrα(-/-) mice treated with the LXR synthetic ligand T0901317. RNF145 is closely related to RNF139/TRC8, an E3 ligase implicated in control of SREBP processing. However, silencing of RNF145 in HepG2 or HeLa cells does not impair SREBP1/2 processing and sterol-responsive gene expression in these cells. Similar to TRC8, we demonstrate that RNF145 is localized to the ER and that it possesses intrinsic E3 ubiquitin ligase activity. In summary, we report the identification of RNF145 as an ER-resident E3 ubiquitin ligase that is transcriptionally controlled by LXR.

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The vitamin A metabolite retinoic acid (RA) has been reported to suppress Th1 responses and enhance Th2 responses. Here, we investigated whether differences in vitamin A metabolism could underlie the differences between C57BL/6 and BALB/c mice, which are reportedly seen as Th1 and Th2 responders, respectively. BALB/c mice were shown to have higher intestinal epithelial expression of RALDH1 (where RALDH is retinaldehyde dehydrogenase), and, consequently, higher RALDH activity in MLN-DCs, leading to an increased ability to induce IgA class switching in B cells. Furthermore, within BALB/c mice, induction of IgA secretion as well as increased accumulation of regulatory T cells (Treg) in the intestinal lamina propria was observed. Additionally, as BALB/c mice are more resistant to dextran sulphate sodium (DSS) induced colitis, mice that lacked vitamin A in their diet had a more severe form of DSS-induced colitis compared to control mice. Therefore, the level of RA production and consequently the degree of RA-mediated signaling is crucial for the efficiency of the mucosal immune system. Keywords: BALB/c r C57BL/6 r FoxP3 r IgA r Intestine r Retinaldehyde dehydrogenase r Vitamin A Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionVitamin A has long been known for its role in immunity, especially since vitamin A deficiency ablates proper mucosal immune responses, leading to diarrhea, infections, and early childhood mortality [1][2][3]. Vitamin A is a fat-soluble vitamin and is absorbed from the gastrointestinal tract. It can be absorbed in the form of Correspondence: Prof. Reina E. Mebius e-mail: r.mebius@vumc.nl preformed retinyl esters, as found in animal source foods such as liver, egg, fish, and whole fat dairy products [3]. Dietary vitamin A can also be obtained from provitamin A carotenoids as found in vegetables and fruits. Vitamin A is metabolized into the active metabolite retinoic acid (RA) in two oxidative steps. Vitamin A is first reversibly oxidized by alcohol dehydrogenases (ADH) to form retinaldehyde. Next, retinaldehyde is irreversibly metabolized to * These authors contributed equally to this work.C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 90Gera Goverse et al. Eur. J. Immunol. 2015. 45: 89-100 RA by one of the three members of the aldehyde dehydrogenase gene family, RALDH1, RALDH2, and RALDH3 (where RALDH is retinaldehyde dehydrogenase) [4][5][6]. The active metabolite RA binds to retinoic acid receptors (Rars) as well as retinoic X receptors, which in turn act as transcription factors that bind RA responsive elements within the promoter regions of target genes [7][8][9]. Expression of the genes encoding RALDH (aldh1a1-3) is associated with the mucosal immune system and the RALDH1 enzyme is reported to be functionally active within intestinal epithelial cells [10][11][12]. Also, DCs in Peyer's patches, MLNs, and intestinal lamina propria express RALDH enzymes, while splenic or peripheral LN DCs disp...

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