Vitamin K refers to a group of lipophilic molecules that serve as a cofactor for γ-carboxyglutamyl carboxylase, which converts specific glutamate residues in a limited set of proteins to γ-carboxyglutamate (Shearer and Newman, 2014; Shearer and Okano, 2018). This post-translational modification is obligatory for biological functions of resultant vitamin K-dependent proteins (VKDPs), some of which play key roles in blood coagulation. Other VKDPs are implicated in processes ranging from bone and cardiovascular mineralization to energy metabolism and inflammation (Booth, 2009; Shearer and Okano, 2018). In addition, vitamin K may exert direct effects on gene expression, signal transduction, and cellular regulation.

All vitamin K forms include a common 2-methyl-1,4-naphthoquinone ring structure known as menadione (MD) (Figure 1A) and are distinguished from one another by length and saturation of the side chain attached at the 3-carbon position on the ring (Shearer and Newman, 2014). MD is a provitamin form of vitamin K as the side chain is required for vitamin K activity (Buitenhuis et al., 1990). Phylloquinone (PK, also known as vitamin K₁) (Figure 1A) contains a phytyl side chain, whereas menaquinones (MKs, collectively referred to as vitamin K₂) contain a side chain with 5-carbon isoprenyl units and are named according to the number of these units (e.g., MK-n) (Figure 1A). PK is produced by plants, whereas longer chain MKs (MK-7, MK-9, and MK-11) are predominantly of bacterial origin. Invertebrate and vertebrate animals produce MK-4 from dietary PK through a reaction involving side chain removal and re-addition with MD as an intermediate (Al Rajabi et al., 2012; Okano et al., 2008).

UbiA prenyltransferase domain-containing protein-1 (UBIAD1), a member of the UbiA superfamily of prenyltransferases (Li, 2016), transfers the 20-carbon geranylgeranyl group from geranylgeranyl pyrophosphate (GGpp) to PK-derived MD, thereby producing MK-4 (Hirota et al., 2013; Nakagawa et al., 2010; Figure 1B). The function of UBIAD1 appears to extend beyond its role in MK-4 synthesis, as indicated by association of mutations in human UBIAD1 with Schnyder corneal dystrophy (SCD) (Orr et al., 2007; Weiss et al., 2007). This rare, autosomal-dominant disease is characterized by progressive corneal opacification that results from accumulation of cholesterol. In 2015, we showed that SCD-associated UBIAD1 inhibits the sterol-accelerated, endoplasmic reticulum (ER)-associated degradation (ERAD) of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) (Schumacher et al., 2015), one of several feedback mechanisms that converge on the enzyme to assure cholesterol homeostasis (Brown and Goldstein, 1980).

Polytopic, ER-localized HMGCR produces mevalonate, an important intermediate in synthesis of cholesterol and the nonsterol isoprenoids farnesyl pyrophosphate (Fpp) and GGpp that are transferred to many cellular proteins and utilized in synthesis of other nonsterol isoprenoids including MK-4, heme, ubiquinone-10, and dolichol (Goldstein and Brown, 1990; Wang and Casey, 2016) (see Figure 1B). Sterols accelerate ERAD of HMGCR by stimulating its binding to ER membrane proteins called Insigs (Sever et al., 2003a; Sever et al., 2003b). Insig-associated ubiquitin ligases facilitate ubiquitination of HMGCR (Jiang et al., 2018; Jo et al., 2011; Song et al., 2005), marking it for extraction across ER membranes and subsequent cytosolic release for ERAD by 26S proteasomes (Morris et al., 2014). ERAD of HMGCR is enhanced by GGpp, which enhances membrane extraction of the ubiquitinated enzyme (Elsabrouty et al., 2013).

Sterols also cause HMGCR to bind UBIAD1 (Schumacher et al., 2015). This binding inhibits the ERAD of HMGCR at a post-ubiquitination step in the reaction, thereby permitting continued synthesis of mevalonate for incorporation into nonsterol isoprenoids even when intracellular sterols are abundant (Schumacher et al., 2018). GGpp triggers release of UBIAD1 from HMGCR, which allows for maximal ERAD of HMGCR and translocation of UBIAD1 from the ER to the medial-trans Golgi. SCD-associated mutations cluster around the membrane-embedded active site of UBIAD1 (Cheng and Li, 2014; Huang et al., 2014), indicating they may disrupt sensing of GGpp. Indeed, SCD-associated UBIAD1 is refractory to GGpp-induced release from HMGCR and becomes sequestered in the ER (Schumacher et al., 2016). The resultant inhibition of HMGCR ERAD leads to enhanced synthesis and intracellular accumulation of cholesterol (Schumacher et al., 2018).

To explore the in vivo function of UBIAD1, efforts were attempted to generate mice lacking Ubiad1 (Nakagawa et al., 2014). However, mouse embryos homozygous for Ubiad1 deficiency failed to survive past embryonic day 7.5. We recently observed that the ERAD of HMGCR was enhanced in transformed human fibroblasts lacking UBIAD1 (Schumacher et al., 2018). This observation led us to speculate that embryonic lethality of Ubiad1 deficiency in mice results from mevalonate depletion due to accelerated ERAD of HMGCR rather than from reduced synthesis of MK-4. We evaluate this notion here by determining whether ubiquitination/ERAD-resistant HMGCR rescues embryonic lethality of Ubiad1-deficiency.

We used CRISPR/Cas9 methods to introduce heterozygous Ubiad1 deficiency in wild type (WT) and previously described Hmgcr^Ki/Ki mice (Hwang et al., 2016), which harbor knockin mutations that prevent ubiquitination and subsequent ERAD of HMGCR (Sever et al., 2003a). These mice are designated Ubiad1^+/- and Ubiad1^+/-: :HmgcrKi^Ki/Ki. Two independent lines of mice were obtained in which the Ubiad1 gene was disrupted by a 172- (Disrupted Allele A) or 29 bp deletion (Disrupted Allele B) in exon 1 (Figure 2). If transcribed and translated, these alleles would produce protein fragments comprising amino acids 1–38 (Disrupted Allele B) or 1–39 (Disrupted Allele A) of UBIAD1 fused to a novel polypeptide of 55 or 56 amino acids (Figure 2). Table 1 shows results of breeding experiments in which mice heterozygous for Ubiad1 deletion were mated and genotypes of offspring determined by PCR analysis. Intercrosses of Ubiad1^+/- mice produced WT and Ubiad1^+/- offspring at a ratio of approximately 1:2, which is consistent with Mendelian segregation. However, Ubiad1^-/- offspring were not produced, regardless of disrupted Ubiad1 allele. In striking contrast, all three expected genotypes (Ubiad1^+/+: :Hmgcr^Ki/Ki, Ubiad1^+/-: :Hmgcr^Ki/Ki, and Ubiad1^-/-: :Hmgcr^Ki/Ki) were produced when Ubiad1^+/-: :Hmgcr^Ki/Ki mice were intercrossed (Table 1). The observed proportion of +/+ : +/- : -/- Ubiad1 alleles was 137:280:114 (Disrupted Allele A) and 23:36:20 (Disrupted Allele B), when the expected proportion is 1:2:1. These results provide genetic evidence that ubiquitination/ERAD-resistant HMGCR rescues embryonic lethality of Ubiad1 deficiency.

In Figure 3A, we measured UBIAD1 and HMGCR protein levels in livers of 8-week-old WT and Ubiad1^+/- mice. Male and female Ubiad1^+/- mice exhibited reduced levels of hepatic UBIAD1 protein as expected (Figure 3A, compare lanes 1 and 3 with lanes 2 and 4). HMGCR protein was slightly reduced in livers of the mice (lanes 1–4), which likely resulted from enhanced ERAD. Similar results were obtained with livers of mice harboring Disrupted Allele B (data not shown). Sterol regulatory element-binding protein (SREBP)−1 and −2 are transcription factors synthesized as inactive, ER-bound precursors (Brown and Goldstein, 1997). Upon lipid deprivation, transcriptionally active fragments of SREBPs are proteolytically released from membranes and migrate to the nucleus where they activate transcription of genes encoding cholesterol and fatty acid synthetic enzymes (Horton et al., 2003). The level of membrane-bound precursor and nuclear forms of SREBPs remained constant in Ubiad1^+/- mice (lanes 5–8 and 9–12). Despite reduced levels of hepatic UBIAD1 and HMGCR protein, Ubiad1^+/- mice were indistinguishable from WT littermates and had similar body weights.

The absence of UBIAD1 protein from hepatic membranes of Ubiad1^-/-: :Hmgcr^Ki/Ki was confirmed in Figure 3B (compare lanes 1–2 and 4–5 with lanes 3 and 6, respectively). The amount of HMGCR protein and nuclear SREBPs was varied in livers of the animals (lanes 1–18); however, the nature of this variation was not clear. Although ubiquitination/ERAD-resistant HMGCR rescued embryonic lethality of Ubiad1 deficiency, male and female Ubiad1^-/-: :Hmgcr^Ki/Ki mice were smaller (30% and 20%, respectively) than their Ubiad1^+/+: :Hmgcr^Ki/Ki and Ubiad1^+/-: :Hmgcr^Ki/Ki littermates at 8 weeks of age. Similar results were observed with 8-week-old Ubiad1^-/-: :Hmgcr^Ki/Ki mice harboring Disrupted Allele B (Figure 3—figure supplement 1A). Hepatic levels of cholesterol and triglycerides (Figure 3—figure supplement 1B) as well as most mRNAs encoding SREBPs, SREBP pathway components, and cholesterol/fatty acid synthetic enzymes were not globally changed in the absence of Ubiad1 (Figure 3—figure supplement 1C). Notably, the mRNA encoding Insig-2a (the major Insig-2 isoform in the liver) was reduced, whereas the minor Insig-2b transcript was slightly increased in Ubiad1-deficient mice. The variation in Insig-2 mRNA, HMGCR, and nuclear SREBPs could be related to variations in food intake or failure of Ubiad1-deficient mice to thrive (see below). For all experiments described hereafter, male and female Ubiad1^+/-: :Hmgcr^Ki/Ki mice with Disrupted Allele A were crossed to obtain Ubiad1^+/+: :Hmgcr^Ki/Ki, Ubiad1^+/-: :Hmgcr^Ki/Ki, and Ubiad1^-/-: :Hmgcr^Ki/Ki littermates for analysis.

Figure 3C compares post-weaning weight gain of Ubiad1^+/+: :Hmgcr^Ki/Ki, Ubiad1^+/-: :Hmgcr^Ki/Ki, and Ubiad1^-/-: :Hmgcr^Ki/Ki mice consuming chow diet ad libitum. The results show that Ubiad1^+/+: :Hmgcr^Ki/Ki and Ubiad1^+/-: :Hmgcr^Ki/Ki mice gained weight at similar rates up to ~8 weeks post-weaning. Ubiad1^-/-: :Hmgcr^Ki/Ki mice gained weight up to 2 weeks post-weaning (albeit at a reduced rate compared to littermates), after which weight gain plateaued. After 7.7 weeks, male and female Ubiad1^-/-: :Hmgcr^Ki/Ki mice were 30–40% smaller than Ubiad1^+/+: :Hmgcr^Ki/Ki and Ubiad1^+/-: :Hmgcr^Ki/Ki littermates.

The amount of UBIAD1 and HMGCR protein was next measured in various tissues of male Ubiad1^+/+: :Hmgcr^Ki/Ki and Ubiad1^-/-: :Hmgcr^Ki/Ki mice. As expected, UBIAD1 was not detected in membranes isolated from the liver, pancreas, brain, kidney, and spleen of Ubiad1-deficient mice (Figure 4A). Figure 4B shows that MK-4, the product of UBIAD1 enzymatic activity, accumulated to the highest level in the pancreas of Ubiad1^+/+: :Hmgcr^Ki/Ki mice. Lower levels of MK-4 were found in the brain, kidney, spleen, and liver of the animals. In contrast, MK-4 failed to accumulate to detectable levels in tissues of Ubiad1-deficient mice.

Having established the absence of UBIAD1 protein and its enzymatic product (MK-4) in Ubiad1-deficient mice, we next compared blood chemistries between Ubiad1^+/+: :Hmgcr^Ki/Ki and Ubiad1^-/-: :Hmgcr^Ki/Ki animals. Serum cholesterol levels were not significantly different between the two groups of male mice (Figure 4C). Modest, but significant increases in levels of triglycerides (55%) and nonesterified fatty acids (46%) were observed in the serum of Ubiad1^-/-: :Hmgcr^Ki/Ki mice compared to Ubiad1^+/+: :Hmgcr^Ki/Ki littermates. The Ubiad1-deficient mice exhibited larger increases in serum levels of two classic markers of liver injury, alanine aminotransferase (ALT) (174%) and aspartate aminotransferase (AST) (583%); serum alkaline phosphatase (ALP) was reduced approximately 35%. Similar results were obtained with serum from female Ubiad1^-/-: :Hmgcr^Ki/Ki mice (Figure 4—figure supplement 1A).

In Figure 4D, we conducted a second blood chemistry analysis on male Ubiad1^+/+: :Hmgcr^Ki/Ki and Ubiad1^-/-: :Hmgcr^Ki/Ki mice. Similar to results of Figure 4C, serum levels of AST and ALT were elevated 5-fold and 1.8-fold, respectively, in the absence of Ubiad1 (Figure 4D). Serum lactate dehydrogenase (LDH) was elevated approximately 2-fold in Ubiad1-deficient mice; however, a more prominent elevation (7.5-fold) of serum creatine kinase (CK) was observed. Finally, a slight reduction in the amount of serum lipase and amylase was present in Ubiad1-deficient mice; serum albumin remained unchanged. Serum from female Ubiad1^-/-: :Hmgcr^Ki/Ki mice exhibited similar characteristics (Figure 4—figure supplement 1B).

To further characterize Ubiad1^-/-: :Hmgcr^Ki/Ki mice, we conducted a complete histological analysis of all tissues from the animals. Surprisingly, abnormalities were observed in only two tissues of Ubiad1-deficient mice – skeletal muscle and bone. Hematoxylin and eosin (H and E)-staining of gastrocnemius (Figure 5A, panels 1–4) and quadriceps muscles (panels 5–8) from male Ubiad1-deficient mice revealed occasional degenerating myofibers with macrophage infiltration as well as frequent myofibers with centrally-localized nuclei. Similar results were observed in gastrocnemius and quadriceps muscles isolated from female mice (Figure 5—figure supplement 1). Overall, these histological findings are indicative of ongoing muscle injury and correlate to the elevated serum CK levels observed in Figure 4D. We used H and E together with Safranin O staining to examine growth plates in femurs from both male (Figure 5B) and female (Figure 5—figure supplement 2) Ubiad1^+/+: :Hmgcr^Ki/Ki and Ubiad1^-/-: :Hmgcr^Ki/Ki mice. The results reveal that Ubiad1 deficiency led to the disorganization of cells within proliferative and hypertrophic zones of the growth plate, persistence of cartilage within trabeculae, and a mild decrease in the number of boney trabeculae.

The genetic ablation of UBIAD1 in transformed human fibroblasts led to enhanced ERAD of HMGCR and reduced cholesterol synthesis and intracellular accumulation of cholesterol (Schumacher et al., 2018). These observations prompted us to consider the possibility that embryonic lethality associated with Ubiad1 deficiency in mice resulted from mevalonate depletion. Indeed, homozygous Hmgcr deficiency caused early embryonic lethality in mice (Ohashi et al., 2003), establishing that mevalonate-derived metabolites are crucial for embryonic development. We previously generated Hmgcr^Ki/Ki mice, which harbor knockin mutations in the Hmgcr gene that prevent sterol-induced ubiquitination and subsequent ERAD of HMGCR (Hwang et al., 2016). As a result of resistance to ERAD, HMGCR protein accumulated in tissues of Hmgcr^Ki/Ki mice that stimulated the overproduction of cholesterol and likely other sterol and nonsterol isoprenoids. Hence, we reasoned that overproduction of sterol and nonsterol isoprenoids in Hmgcr^Ki/Ki mice would rescue embryonic lethality associated with Ubiad1 deficiency. Our current studies show that homozygous Ubiad1 deletion was produced at expected Mendelian ratios in Hmgcr^Ki/Ki, but not in WT mice (Table 1). This important observation not only highlights the crucial role for UBIAD1 in regulation of HMGCR ERAD, but also confirms that abrogating the reaction allows production of a mevalonate-derived metabolite(s) that rescues embryonic lethality associated with Ubiad1 deficiency. Notably, administration of MK-4, ubiquinone-10 (Nakagawa et al., 2014), or cholesterol (data not shown) to Ubiad1^+/- mice prior to intercrossing and throughout pregnancy failed to rescue embryonic lethality of Ubiad1 deficiency. While the identity of the mevalonate-derived metabolite(s) that rescues embryonic development remains unknown, genes encoding HMGCR, enzymes required for GGpp and Fpp synthesis, and prenylation of small GTPases are essential for migration of primordial germ cells during embryonic development (Kunwar et al., 2006). Thus, depletion of GGpp and/or Fpp owing to enhanced ERAD of HMGCR and reduced prenylation of small GTPases may contribute to embryonic lethality associated with Ubiad1 deficiency.

Although ubiquitination/ERAD-resistant HMGCR rescues embryonic lethality of Ubiad1 deficiency (Table 1), Ubiad1^-/-: :Hmgcr^Ki/Ki mice 8–12 weeks of age were 20–40% smaller than their Ubiad1^+/+: :Hmgcr^Ki/Ki and Ubiad1^+/-: :Hmgcr^Ki/Ki littermates (Figure 3B and C). It will be important in future studies to determine whether Ubiad1-deficient mice consume less food, have defects in nutrient absorption, or exhibit increased energy expenditure. UBIAD1 protein and its enzymatic product MK-4 were not detected in tissues of Ubiad1-deficient mice (Figures 3B, 4A and B). However, it is important to note that despite the absence of MK-4, Ubiad1-deficient mice did not exhibit typical signs of vitamin K deficiency (i.e., excessive hemorrhaging). The diet used in this study is supplemented with MD, which does not exhibit vitamin K activity (Buitenhuis et al., 1990) and is not converted to MK-4 in absence of UBIAD1. This indicates that the diet and/or gut microbiota provide sufficient amounts of vitamin K to support γ-glutamyl carboxylation of coagulation factors in Ubiad1^-/-: :Hmgcr^Ki/Ki mice, suggesting their failure to thrive may result from disruption of carboxylation-independent activities of MK-4. Figure 4B shows that similar to previous results (Harshman et al., 2016; Okano et al., 2008), levels of MK-4 were highest in the pancreas and brain. Thus, failure of Ubiad1-deficient mice to thrive may result from reduced production of MK-4 in these or other tissues of the animals.

Recent studies show that inducible knockout of Ubiad1 in adult mice caused death within 60 days (Nakagawa et al., 2019). The most striking abnormality of these mice was a remarkable reduction in pancreas size resulting from apoptotic disappearance of acinar cells. This observation prompted the authors to conclude that UBIAD1-mediated synthesis of MK-4 is essential for survival of pancreatic acinar cells. Our current studies reveal that the pancreas of Ubiad1^-/-: :Hmgcr^Ki/Ki mice failed to produce MK-4 (Figure 4B); however, the organ was normal in size and exhibited no gross abnormalities (data not shown). These findings argue that ubiquitination/ERAD-resistant HMGCR allows for production of sterol and/or nonsterol isoprenoids distinct from MK-4 that are essential for subsistence of pancreatic acinar cells.

Blood chemistry analyses were conducted to determine whether Ubiad1 deficiency results in damage of the liver and/or other organs. Compared to Ubiad1^+/+: :Hmgcr^Ki/Ki littermates, Ubiad1^-/-: :Hmgcr^Ki/Ki mice exhibited elevated levels of ALT (1.6-fold) and AST (>5 fold) in the serum (Figure 4C and D; Figure 4—figure supplement 1). Elevated levels of serum aminotransferases are routinely applied as biomarkers for hepatocyte injury. However, livers of Ubiad1-deficient mice did not feature gross abnormalities upon histological analysis (data not shown). Further examination revealed Ubiad1 deficient mice exhibited a 2-fold increase in serum LDH and a 4–7.5-fold increase in CK that was accompanied by a 35% decrease in ALP (Figure 4 and Figure 4—figure supplement 1). These observations are consistent with muscle injury and bone dysfunction in Ubiad1^-/-: :Hmgcr^Ki/Ki mice. Indeed, degenerating skeletal muscle myofibers with macrophage infiltration and myofibers with centrally-localized nuclei as well as cell disorganization within the femoral growth plate were all observed by histological analysis of Ubiad1-deficient mice (Figure 5; Figure 5—figure supplement 1 and 2).

Statins, competitive inhibitors of HMGCR, are widely prescribed to lower plasma levels of cholesterol-rich low-density lipoprotein and reduce atherosclerotic cardiovascular disease. However, a significant fraction of patients undergoing statin therapy develop myopathy; a small portion of these patients progress to rhabdomyolysis (Thompson et al., 2003; Ward et al., 2019). Statin-induced myopathy has been attributed to depletion of mevalonate-derived metabolites resulting from inhibition of HMGCR. Skeletal muscle-specific knockout of HMGCR in mice causes severe myopathy that is rescued by mevalonate (Osaki et al., 2015). The observation that Ubiad1^-/-: :Hmgcr^Ki/Ki mice exhibit signs of muscle injury suggests statin-induced myopathy may in part, result from MK-4 depletion. Support for this possibility requires MK-4 rescue experiments in Ubiad1-deficient mice and determination of whether Hmgcr^Ki/Ki mice with skeletal muscle-specific knockout of Ubiad1 develop myopathy.

Evidence indicates that vitamin K modulates bone homeostasis and metabolism through two mechanisms. One mechanism is mediated by osteocalcin and matrix Gla protein (Fusaro et al., 2017), two VKDPs that play key roles in bone formation and mineralization. The second mechanism is mediated by the nuclear receptor known as steroid and xenobiotic receptor (SXR) in humans and pregnane X receptor (PXR) in mice. These promiscuous nuclear receptors are activated by a wide variety of xenobiotics and regulate genes involved in metabolism and clearance of the substances (Kliewer, 2003). Pxr-deficient mice present with osteopenia accompanied by reduced bone formation and increased bone resorption (Azuma et al., 2010). Considering that MK-4 has been reported to bind to and activate PXR (Tabb et al., 2003), it will be important to determine whether Ubiad1-deficiency phenocopies Pxr-deficiency with regard to bone homeostasis.

The characterization of genetically-manipulated mice underscores the physiological significance of UBIAD1 as an inhibitor of HMGCR ERAD. We recently generated mice (designated Ubiad1^Ki/Ki) harboring a knockin mutation that changes asparagine-100 (N100) to serine (N100S) (Jo et al., 2019). The N100S mutation in mouse UBIAD1 corresponds to SCD-associated N102S mutation in human UBIAD1 that abolishes sensing of membrane-embedded GGpp. UBIAD1 (N100S) was sequestered in ER membranes to inhibit ERAD of HMGCR, causing the protein’s accumulation and overproduction of sterol and nonsterol isoprenoids in the liver and other tissues of Ubiad1^Ki/Ki mice. Significant corneal opacification was observed in Ubiad1^Ki/Ki mice greater than 50 weeks of age (Hmgcr^Ki/Ki mice used in the current study were not aged and thus, not examined for corneal opacification). Considered together with current studies, these findings unequivocally position UBIAD1 as a major regulator of HMGCR and mevalonate metabolism in vivo, provide new links between synthesis of sterols and MK-4, and establish Ubiad1^-/-: :Hmgcr^Ki/Ki mice as a model of MK-4 deficiency. Further analysis of these mice may reveal new physiological roles for MK-4 and additional pathways modulated by the vitamin K subtype.