Synthesis, function, and regulation of sterol and nonsterol isoprenoids.

Cholesterol, the bulk end-product of the mevalonate pathway, is a key component of cellular membranes and lipoproteins that transport lipids throughout the body. It is also a precursor of steroid hormones, vitamin D, and bile acids. In addition to cholesterol, the mevalonate pathway yields a variety of nonsterol isoprenoids that are essential to cell survival. Flux through the mevalonate pathway is tightly controlled to ensure cells continuously synthesize nonsterol isoprenoids but avoid overproducing cholesterol and other sterols. Endoplasmic reticulum (ER)-localized 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGCR), the rate limiting enzyme in the mevalonate pathway, is the focus of a complex feedback regulatory system governed by sterol and nonsterol isoprenoids. This review highlights transcriptional and post-translational regulation of HMGCR. Transcriptional regulation of HMGCR is mediated by the Scap-SREBP pathway. Post-translational control is initiated by the intracellular accumulation of sterols, which causes HMGCR to become ubiquitinated and subjected to proteasome-mediated ER-associated degradation (ERAD). Sterols also cause a subfraction of HMGCR molecules to bind the vitamin K2 synthetic enzyme, UbiA prenyltransferase domain-containing protein-1 (UBIAD1). This binding inhibits ERAD of HMGCR, which allows cells to continuously synthesize nonsterol isoprenoids such as geranylgeranyl pyrophosphate (GGPP), even when sterols are abundant. Recent studies reveal that UBIAD1 is a GGPP sensor, dissociating from HMGCR when GGPP thresholds are met to allow maximal ERAD. Animal studies using genetically manipulated mice disclose the physiological significance of the HMGCR regulatory system and we describe how dysregulation of these pathways contributes to disease.

Post-Translational Regulation of HMG CoA Reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is an endoplasmic reticulum (ER)-localized integral membrane protein that catalyzes the rate-limiting step in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). HMGCR is subjected to strict feedback control through multiple mechanisms to ensure cells constantly produce essential nonsterol isoprenoids, but do not overaccumulate cholesterol. Here, we focus on the mechanism of feedback control of HMGCR that involves its sterol-induced ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We will also discuss the how GGpp-regulated intracellular trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) inhibits HMGCR ERAD to balance the synthesis of sterol and nonsterol isoprenoids. Finally, we will summarize various mouse models, the characterization of which establish that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMGCR and cholesterol metabolism in vivo.

Type 1 polyisoprenoid diphosphate phosphatase modulates geranylgeranyl-mediated control of HMG CoA reductase and UBIAD1.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. The prenyltransferase has emerged as a key regulator of sterol-accelerated, endoplasmic reticulum (ER)-associated degradation (ERAD) of HMG CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids including GGpp. Sterols induce binding of UBIAD1 to reductase, inhibiting its ERAD. Geranylgeraniol (GGOH), the alcohol derivative of GGpp, disrupts this binding and thereby stimulates ERAD of reductase and translocation of UBIAD1 to Golgi. We now show that overexpression of Type 1 polyisoprenoid diphosphate phosphatase (PDP1), which dephosphorylates GGpp and other isoprenyl pyrophosphates to corresponding isoprenols, abolishes protein geranylgeranylation as well as GGOH-induced ERAD of reductase and Golgi transport of UBIAD1. Conversely, these reactions are enhanced in the absence of PDP1. Our findings indicate PDP1-mediated hydrolysis of GGpp significantly contributes to a feedback mechanism that maintains optimal intracellular levels of the nonsterol isoprenoid.

Enhanced ER-associated degradation of HMG CoA reductase causes embryonic lethality associated with Ubiad1 deficiency.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) synthesizes the vitamin K subtype menaquinone-4 (MK-4). Previous studies in cultured cells (Schumacher et al., 2015) revealed that UBIAD1 also inhibits endoplasmic reticulum (ER)-associated degradation (ERAD) of ubiquitinated HMG CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway that produces cholesterol and essential nonsterol isoprenoids. Gene knockout studies were previously attempted to explore the function of UBIAD1 in mice; however, homozygous germ-line elimination of the Ubiad1 gene caused embryonic lethality. We now report that homozygous deletion of Ubiad1 is produced in knockin mice expressing ubiquitination/ERAD-resistant HMGCR. Thus, embryonic lethality of Ubiad1 deficiency results from depletion of mevalonate-derived products owing to enhanced ERAD of HMGCR rather than from reduced synthesis of MK-4. These findings provide genetic evidence for the significance of UBIAD1 in regulation of cholesterol synthesis and offer the opportunity in future studies for the discovery of new physiological roles of MK-4.

Schnyder corneal dystrophy-associated UBIAD1 inhibits ER-associated degradation of HMG CoA reductase in mice.

Autosomal-dominant Schnyder corneal dystrophy (SCD) is characterized by corneal opacification owing to overaccumulation of cholesterol. SCD is caused by mutations in UBIAD1, which utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K2. Using cultured cells, we previously showed that sterols trigger binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase (HMGCR), thereby inhibiting its endoplasmic reticulum (ER)-associated degradation (ERAD) (Schumacher et al. 2015). GGpp triggers release of UBIAD1 from HMGCR, allowing maximal ERAD and ER-to-Golgi transport of UBIAD1. SCD-associated UBIAD1 resists GGpp-induced release and is sequestered in ER to inhibit ERAD. We now report knockin mice expressing SCD-associated UBIAD1 accumulate HMGCR in several tissues resulting from ER sequestration of mutant UBIAD1 and inhibition of HMGCR ERAD. Corneas from aged knockin mice exhibit signs of opacification and sterol overaccumulation. These results establish the physiological significance of UBIAD1 in cholesterol homeostasis and indicate inhibition of HMGCR ERAD contributes to SCD pathogenesis.

Hypoxia-inducible factor 1α activates insulin-induced gene 2 (Insig-2) transcription for degradation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase in the liver.

Cholesterol synthesis is a highly oxygen-consuming process. As such, oxygen deprivation (hypoxia) limits cholesterol synthesis through incompletely understood mechanisms mediated by the oxygen-sensitive transcription factor hypoxia-inducible factor 1α (HIF-1α). We show here that HIF-1α links pathways for oxygen sensing and feedback control of cholesterol synthesis in human fibroblasts by directly activating transcription of the INSIG-2 gene. Insig-2 is one of two endoplasmic reticulum membrane proteins that inhibit cholesterol synthesis by mediating sterol-induced ubiquitination and subsequent endoplasmic reticulum-associated degradation of the rate-limiting enzyme in the pathway, HMG-CoA reductase (HMGCR). Consistent with the results in cultured cells, hepatic levels of Insig-2 mRNA were enhanced in mouse models of hypoxia. Moreover, pharmacologic stabilization of HIF-1α in the liver stimulated HMGCR degradation via a reaction that requires the protein's prior ubiquitination and the presence of the Insig-2 protein. In summary, our results show that HIF-1α activates INSIG-2 transcription, leading to accumulation of Insig-2 protein, which binds to HMGCR and triggers its accelerated ubiquitination and degradation. These results indicate that HIF-mediated induction of Insig-2 and degradation of HMGCR are physiologically relevant events that guard against wasteful oxygen consumption and inappropriate cell growth during hypoxia.

Regulation of INSIG2 by microRNA-96.

Mature forms of the microRNAs miR-96, -182, and -183 originate from a single genomic locus and have been shown to be elevated approximately 50-fold in the livers of sterol regulatory element-binding protein-1a and -2 (SREBP-1a and -2) transgenic mice. Our study attempted to identify the possible targets of these microRNAs using miRNA target prediction software. This revealed putative sites in insulin-induced genes (INSIGs). The 3' untranslated region (UTR) of insulin-induced gene 1 (INSIG1) contained sites corresponding to miR-182, and -183, while the 3' UTR of INSIG2 featured an miR-96 site. Among these putative sites, only miR-96 demonstrated an inhibitory effect that was specific to the 3' UTR of INSIG2. As INSIG proteins are the main components of SREBP cleavage complexes that act to release active SREBPs, we assessed the effects of miR-96 on INSIG and SREBP levels and activities. We found that miR-96 reduced the levels of INSIG2 in INSIG1 knockout human fibroblasts, resulting in an increase in SREBP-1 and -2 nuclear forms and a subsequent increase in the abundance of the mRNA of their target genes. These results suggest that miR-96, an miRNA induced by SREBP-2 activation, regulates downstream targets of SREBPs and may increase the abundance of active SREBP.

Geranylgeranyl-regulated transport of the prenyltransferase UBIAD1 between membranes of the ER and Golgi.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. Previously, we found that sterols trigger binding of UBIAD1 to endoplasmic reticulum (ER)-localized HMG-CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids, including GGpp. This binding inhibits sterol-accelerated degradation of reductase, which contributes to feedback regulation of the enzyme. The addition to cells of geranylgeraniol (GGOH), which can become converted to GGpp, triggers release of UBIAD1 from reductase, allowing for its maximal degradation and permitting ER-to-Golgi transport of UBIAD1. Here, we further characterize geranylgeranyl-regulated transport of UBIAD1. Results of this characterization support a model in which UBIAD1 continuously cycles between the ER and medial-trans Golgi of isoprenoid-replete cells. Upon sensing a decline of GGpp in ER membranes, UBIAD1 becomes trapped in the organelle where it inhibits reductase degradation. Mutant forms of UBIAD1 associated with Schnyder corneal dystrophy (SCD), a human eye disease characterized by corneal accumulation of cholesterol, are sequestered in the ER and block reductase degradation. Collectively, these findings disclose a novel sensing mechanism that allows for stringent metabolic control of intracellular trafficking of UBIAD1, which directly modulates reductase degradation and becomes disrupted in SCD.

The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells.

The polytopic endoplasmic reticulum (ER)-localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.

Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells.

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein's cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.

Ancient ubiquitous protein-1 mediates sterol-induced ubiquitination of 3-hydroxy-3-methylglutaryl CoA reductase in lipid droplet-associated endoplasmic reticulum membranes.

Sterol-induced binding to Insigs in endoplasmic reticulum (ER) membranes triggers ubiquitination of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase. This ubiquitination, which is mediated by Insig-associated ubiquitin ligases gp78 and Trc8, is obligatory for extraction of reductase from lipid droplet-associated ER membranes into the cytosol for proteasome-mediated, ER-associated degradation (ERAD). In this study, we identify lipid droplet-associated, ancient, ubiquitous protein-1 (Aup1) as one of several proteins that copurify with gp78. RNA interference (RNAi) studies show that Aup1 recruits the ubiquitin-conjugating enzyme Ubc7 to lipid droplets and facilitates its binding to both gp78 and Trc8. The functional significance of these interactions is revealed by the observation that RNAi-mediated knockdown of Aup1 blunts sterol-accelerated ubiquitination of reductase, which appears to occur in lipid droplet-associated membranes and subsequent ERAD of the enzyme. In addition, Aup1 knockdown inhibits ERAD of Insig-1, another substrate for gp78, as well as that of membrane-bound precursor forms of sterol-regulatory, element-binding protein-1 and -2, transcription factors that modulate expression of genes encoding enzymes required for cholesterol synthesis. Considered together, these findings not only implicate a role for Aup1 in maintenance of intracellular cholesterol homeostasis, but they also highlight the close connections among ERAD, lipid droplets, and lipid droplet-associated proteins.

Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8.

Accumulation of sterols in membranes of the endoplasmic reticulum (ER) leads to the accelerated ubiquitination and proteasomal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids. This degradation results from sterol-induced binding of reductase to the Insig-1 or Insig-2 proteins of ER membranes. We previously reported that in immortalized human fibroblasts (SV-589 cells) Insig-1, but not Insig-2, recruits gp78, a membrane-bound RING-finger ubiquitin ligase. We now report that both Insig-1 and Insig-2 bind another membrane-bound RING-finger ubiquitin ligase called Trc8. Knockdown of either gp78 or Trc8 in SV-589 cells through RNA interference (RNAi) inhibited sterol-induced ubiquitination of reductase and inhibited sterol-induced degradation by 50-60%. The combined knockdown of gp78 and Trc8 produced a more complete inhibition of degradation (> 90%). Knockdown of gp78 led to a three to fourfold increase in levels of Trc8 and Insig-1 proteins, which opposed the inhibitory action of gp78. In contrast, knockdown of Trc8 had no effect on gp78 or Insig-1. The current results suggest that sterol-induced ubiquitination and proteasomal degradation of reductase is dictated by the complex interplay of at least four proteins: Insig-1, Insig-2, gp78, and Trc8. Variations in the concentrations of any one of these proteins may account for differences in cell- and/or tissue-specific regulation of reductase degradation.

Membrane-associated ubiquitin ligase complex containing gp78 mediates sterol-accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway in the yeast Saccharomyces cerevisiae is mediated by two membrane-bound ubiquitin ligases, Doa10 and Hrd1. These enzymes are found in distinct multiprotein complexes that allow them to recognize and target a variety of substrates for proteasomal degradation. Although multiprotein complexes containing mammalian ERAD ubiquitin ligases likely exist, they have yet to be identified and characterized in detail. Here, we identify two ER membrane proteins, SPFH2 and TMUB1, as associated proteins of mammalian gp78, a membrane-bound ubiquitin ligase that bears significant sequence homology with mammalian Hrd1 and mediates sterol-accelerated ERAD of the cholesterol biosynthetic enzyme HMG-CoA reductase. Co-immunoprecipitation studies indicate that TMUB1 bridges SPFH2 to gp78 in ER membranes. The functional significance of these interactions is revealed by the observation that RNA interference (RNAi)-mediated knockdown of SPFH2 and TMUB1 blunts both the sterol-induced ubiquitination and degradation of endogenous reductase in HEK-293 cells. These studies mark the initial steps in the characterization of the mammalian gp78 ubiquitin ligase complex, the further elucidation of which may yield important insights into mechanisms underlying gp78-mediated ERAD.

Control of cholesterol synthesis through regulated ER-associated degradation of HMG CoA reductase.

Multiple mechanisms for feedback control of cholesterol synthesis converge on the rate-limiting enzyme in the pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase. This complex feedback regulatory system is mediated by sterol and nonsterol metabolites of mevalonate, the immediate product of reductase activity. One mechanism for feedback control of reductase involves rapid degradation of the enzyme from membranes of the endoplasmic reticulum (ER). This degradation results from the accumulation of sterols in ER membranes, which triggers binding of reductase to ER membrane proteins called Insig-1 and Insig-2. Insig binding leads to the recruitment of a membrane-associated ubiquitin ligase called gp78 that initiates ubiquitination of reductase. Ubiquitinated reductase then becomes extracted from ER membranes and is delivered to cytosolic 26S proteasomes through an unknown mechanism that is mediated by the gp78-associated ATPase Valosin-containing protein/p97 and appears to be augmented by nonsterol isoprenoids. Here, we will highlight several advances that have led to the current view of mechanisms for sterol-accelerated, ER-associated degradation of reductase. In addition, we will discuss potential mechanisms for other aspects of the pathway such as selection of reductase for gp78-mediated ubiquitination, extraction of the ubiquitinated enzyme from ER membranes, and the contribution of Insig-mediated degradation to overall regulation of reductase in whole animals.

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from endoplasmic reticulum membranes into the cytosol through a subcellular compartment resembling lipid droplets.

Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets.

Role of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 in protein kinase A- and protein kinase C-mediated regulation of the steroidogenic acute regulatory protein expression in mouse Leydig tumor cells: mechanism of action.

Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) is an orphan nuclear receptor that has been demonstrated to be instrumental to the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. However, its mechanism of action remains obscure. The present investigation was aimed at exploring the molecular involvement of DAX-1 in protein kinase A (PKA)- and protein kinase C (PKC)-mediated regulation of StAR expression and its concomitant impact on steroid synthesis using MA-10 mouse Leydig tumor cells. We demonstrate that activation of the PKA and PKC pathways, by a cAMP analog dibutyryl (Bu)2cAMP [(Bu)2cAMP] and phorbol 12-myristate 13-acetate (PMA), respectively, markedly decreased DAX-1 expression, an event that was inversely correlated with StAR protein, StAR mRNA, and progesterone levels. Notably, the suppression of DAX-1 requires de novo transcription and translation, suggesting that the effect of DAX-1 in regulating StAR expression is dynamic. Chromatin immunoprecipitation studies revealed the association of DAX-1 with the proximal but not the distal region of the StAR promoter, and both (Bu)2cAMP and PMA decreased in vivo DAX-1-DNA interactions. EMSA and reporter gene analyses demonstrated the functional integrity of this interaction by showing that DAX-1 binds to a DNA hairpin at position -44/-20 bp of the mouse StAR promoter and that the binding of DAX-1 to this region decreases progesterone synthesis by impairing transcription of the StAR gene. In support of this, targeted silencing of endogenous DAX-1 elevated basal, (Bu)2cAMP-, and PMA-stimulated StAR expression and progesterone synthesis. Transrepression of the StAR gene by DAX-1 was tightly associated with expression of the nuclear receptors Nur77 and steroidogenic factor-1, demonstrating these factors negatively modulate the steroidogenic response. These findings provide insight into the molecular events by which DAX-1 influences the PKA and PKC signaling pathways involved in the regulation of the StAR protein and steroidogenesis in mouse Leydig tumor cells.

Complex role of the mitochondrial targeting signal in the function of steroidogenic acute regulatory protein revealed by bacterial artificial chromosome transgenesis in vivo.

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR-/-), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR-/- mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR-/- mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

Regulation of Leydig cell steroidogenesis by extracellular signal-regulated kinase 1/2: role of protein kinase A and protein kinase C signaling.

The steroidogenic acute regulatory (StAR) protein plays a central role in the regulation of steroid biosynthesis. While steroidogenesis is influenced by many processes, their modes of actions, in a few cases, remain obscure. In this study, we explored the mechanism of action of one such signaling pathway, the extracellular signal-regulated kinase 1/2 (ERK1/2), in regulating StAR expression and steroidogenesis in conjunction with the protein kinase A (PKA) and protein kinase C (PKC) pathways. Using MA-10 mouse Leydig tumor cells, we demonstrate that the activation of PKC and PKA signaling, by phorbol-12-myristate-13-acetate (PMA) and dibutyryl cAMP (dbcAMP)/human chorionic gonadotropin (hCG) respectively, was able to phosphorylate ERK1/2, an event markedly decreased by an upstream kinase inhibitor, U0126. Treatment with PMA enhanced StAR protein expression (associated with a slight increase in progesterone synthesis) but not its phosphorylation (P-StAR), which, in contrast, coordinately increased in response to dbcAMP/hCG. Inhibition of ERK1/2 activity by U0126 decreased PMA-treated StAR expression but increased dbcAMP/hCG-mediated StAR and P-StAR; however, progesterone levels were attenuated. U0126 was found to affect StAR expression and steroidogenesis both at the transcriptional and translational levels. Further studies demonstrated that the effect of U0126 on PMA- and dbcAMP/hCG-mediated StAR expression and steroid synthesis was tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). In fact, both DAX-1 and SR-B1 appear to play important roles in hormone-regulated steroidogenesis. These findings clearly demonstrate that the ERK1/2 signaling cascade involved in regulating StAR expression and steroid synthesis is mediated by multiple factors and pathways and is stimulus specific in mouse Leydig cells.