Characterization of UT2 Cells

Apomine, a novel 1,1-bisphosphonate ester, has been shown to lower plasma cholesterol concentration in several species. Here we show that Apomine reduced the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme in the mevalonate pathway, both in rat liver and in cultured cells. Apomine resembles sterols such as 25-hydroxycholesterol in its ability to potently accelerate the rate of HMGR degradation by the ubiquitin-proteasome pathway, a process that depends on the transmembrane domain of the enzyme. The similarity between Apomine and sterols in promoting rapid HMGR degradation extends to its acute requirements for ongoing protein synthesis and mevalonate-derived non-sterol product(s) as a co-regulator. Yet, at suboptimal concentrations, sterols potentiated the effect of Apomine in stimulating HMGR degradation, indicating that these agents act via distinct modes. Furthermore, unlike sterols, Apomine inhibited the activity of acyl-CoA:cholesterol acyltransferase in intact cells but not in cell-free extracts. Apomine stimulated the cleavage of the precursor of sterolregulatory element-binding protein-2 and increased the activity of low density lipoprotein receptor pathway. This Apomine-enhanced activation of sterol-regulatory element-binding protein-2 was prevented by sterols or mevalonate. Taken together, our results provide a molecular mechanism for the hypocholesterolemic activity of Apomine.In mammalian cells, cholesterol homeostasis is maintained by balancing cholesterol uptake and production. Cholesterol uptake is regulated through modulating the levels of the cell surface low density lipoprotein (LDL) 1 receptor (LDLR), and cholesterol synthesis is regulated primarily by changes in levels and activity of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) (1, 2). HMGR is an endoplasmic reticulum (ER) enzyme that catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA into mevalonate (MVA), the first committed step in the MVA pathway that leads to the synthesis of cholesterol as well as of essential non-sterol isoprenoid compounds (1, 2). When cells are starved for cholesterol and/or MVA, levels of HMGR and LDLR are elevated, thus increasing the rate of endogenous sterol synthesis and of LDL uptake (3, 4). Conversely, in cholesterol-replete cells, the levels of HMGR and LDLR decline, thereby lowering sterol production and LDL internalization.HMGR and LDLR are regulated at the transcriptional level by sterol-regulatory elements (SREs) in the promoter of their genes (1, 5). Specific transcription factors, designated SREbinding proteins (SREBPs), bind to these elements and activate transcription (5, 6). The SREBP family comprises three members that control different sets of genes. SREBP-2 regulates the transcription of genes mainly involved in sterol synthesis, whereas SREBP-1a and -1c regulate principally fatty acid biosynthetic genes (7). The nuclear, transcriptionally active SREBPs are derived from the NH 2 -terminal domain of large ER membrane-bound prec...

Section: Resultsmentioning

confidence: 99%

Apomine, a Novel Hypocholesterolemic Agent, Accelerates Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase and Stimulates Low Density Lipoprotein Receptor Activity

Roitelman

Masson²,

Avner

et al. 2004

“…One such cell line, designated as UT-2 cells, is a mutant clone of Chinese hamster ovary cells that expresses only 2-5% of the HMG-CoA reductase activity of parental Chinese hamster ovary cells and requires exogenous mevalonate for growth (52). Recently, Engfelt et al (16) identified the responsible mutations in the structural gene for reductase. By growing UT-2 cells in the absence of mevalonate, they have established a new cell line, designated as UT-2* cells, and demonstrated that UT-2* cells had up-regulated HMGCoA reductase activity, which was localized exclusively to peroxisomes and the peroxisomal enzyme in these cells was sufficient for survival without mevalonate (15,17).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, Engfelt et al (16) identified the responsible mutations in the structural gene for reductase. By growing UT-2 cells in the absence of mevalonate, they have established a new cell line, designated as UT-2* cells, and demonstrated that UT-2* cells had up-regulated HMGCoA reductase activity, which was localized exclusively to peroxisomes and the peroxisomal enzyme in these cells was sufficient for survival without mevalonate (15,17). Based on the detailed characterization of UT-2* cells, they suggested the existence of a second, peroxisome-specific gene for the reductase.…”

Section: Discussionmentioning

confidence: 99%

“…Given the major role of HMG-CoA reductase in the mevalonate pathway, it is tempting to hypothesize that mammalian cells also have a second gene for the enzyme. In fact, although the classic form of the enzyme is a transmembrane protein anchored to the endoplasmic reticulum (ER), recent studies using a mutant cell line that lacks the ER isoform of the enzyme indicate the existence of a second isoform of the reductase exclusively localized in peroxisomes and that the peroxisomal activity might be due to a second gene (15)(16)(17).…”

Section: -3)mentioning

confidence: 99%

See 1 more Smart Citation

Early Embryonic Lethality Caused by Targeted Disruption of the 3-Hydroxy-3-methylglutaryl-CoA Reductase Gene

Ohashi

Osuga

Tozawa

et al. 2003

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr؉/؊) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr؉/؊ embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr؉/؊ mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr؉/؊ and Hmgcr؉/؉ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.The mevalonate pathway produces isoprenoids that are essential for diverse cellular functions, ranging from cholesterol synthesis to growth control. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 1 reductase (EC 1.1.1.34), which catalyzes the conversion of HMG-CoA to mevalonate, is the rate-limiting enzyme in the mevalonate pathway (1). Because of its major role in cholesterol biosynthesis, the regulation of HMG-CoA reductase has been intensely studied. To ensure a steady mevalonate supply, the non-sterol and sterol end-products of mevalonate metabolism exert feedback regulation on the activity of this enzyme through multivalent mechanisms, including inhibition of transcription of the HMG-CoA reductase mRNA, blocking of translation, and acceleration of protein degradation, thus regulating the amount of reductase protein over a several hundred-fold range (reviewed in Refs.

“…To address which steps in ERAD of HMGR are regulated by which MVA-derived metabolites, we examined the degradation of HMGal that is stably expressed in UT-2 cells. UT-2 cells are deficient in HMGR protein (36) due to point mutations that abrogate the correct splicing of reductase mRNA (41), and depend on exogenous MVA for viability. Thus, intracellular MVA levels can be efficiently manipulated by changing MVA concentrations in the growth medium.…”

Section: Mevalonate Ismentioning

confidence: 99%

Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase

Leichner

Avner

Harats

et al. 2011

In mammalian cells, the enzyme 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR), which catalyzes the rate-limiting step in the mevalonate pathway, is ubiquitylated and degraded by the 26 S proteasome when mevalonate-derived metabolites accumulate, representing a case of metabolically regulated endoplasmic reticulum-associated degradation (ERAD). Here, we studied which mevalonate-derived metabolites signal for HMGR degradation and the ERAD step(s) in which these metabolites are required. In HMGR-deficient UT-2 cells that stably express HMGal, a chimeric protein between ␤-galactosidase and the membrane region of HMGR, which is necessary and sufficient for the regulated ERAD, we tested inhibitors specific to different steps in the mevalonate pathway. We found that metabolites downstream of farnesyl pyrophosphate but upstream to lanosterol were highly effective in initiating ubiquitylation, dislocation, and degradation of HMGal. Similar results were observed for endogenous HMGR in cells that express this protein. Ubiquitylation, dislocation, and proteasomal degradation of HMGal were severely hampered when production of geranylgeranyl pyrophosphate was inhibited. Importantly, inhibition of protein geranylgeranylation markedly attenuated ubiquitylation and dislocation, implicating for the first time a geranylgeranylated protein(s) in the metabolically regulated ERAD of HMGR.Mammalian cells satisfy most of their cholesterol requirements through receptor-mediated endocytosis of cholesterolrich plasma lipoproteins. However, when exogenous cholesterol supply is short, or when there is an increased demand for particular nonsterol isoprenoid(s), cells up-regulate the activity of the mevalonate (MVA) 3 pathway ( Fig. 1), in which sterols and essential MVA-derived nonsterol isoprenoids are produced (1, 2). Intracellular MVA levels are finely tuned and tightly regulated by a feedback mechanism that governs the rate of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reduction into MVA, the major rate-limiting step for the entire pathway (3, 4). This reaction is catalyzed by HMG-CoA reductase (HMGR), an integral glycoprotein resident to the endoplasmic reticulum (ER). Each of the 97-kDa subunits of HMGR is embedded in the ER membrane via a ϳ350 residue N-terminal noncatalytic region that spans the membrane 8 times (5-7), whereas the rest of the polypeptide faces the cytosol where it tetramerizes to form the active sites of the enzyme (5, 8, 9). To allow sufficient production of isoprenoids yet avoid overaccumulation of potentially toxic products, the activity of HMGR is stringently controlled and varies according to the cellular needs for sterol and the nonsterol products. HMGR activity increases when cells are starved for cholesterol or MVA. Conversely, abundant cholesterol and/or MVA acutely suppress the enzyme (4, 10). At the post-translational level, regulation of HMGR mainly involves modulation of its stability. Thus, in cells deprived of sterols and/or MVA, HMGR is a rather stable protein that turns over with a half-l...