Mevalonate kinase (MK) deficiency is an autosomal-recessive inborn error of metabolism. Patients with MK deficiency present with characteristic autoinflammatory symptoms, including recurring episodes of high fever associated with headache, arthritis, nausea, abdominal pain, diarrhea, and skin rash (1, 2). Originally, 2 distinct clinical entities associated with this defect were defined: classic mevalonic aciduria (3) and hyperimmunoglobulin D with periodic fever syndrome (HIDS) (1). However, it is now clear that those 2 entities represent the severe and mild ends, respectively, of the clinical and biochemical spectrum of MK deficiency. Indeed, patients with the HIDS presentation typically have recurrent episodes of fever with associated symptoms (1), whereas patients with the mevalonic aciduria presentation show, in addition to these episodes, developmental delay, dysmorphic features, ataxia, cerebellar atrophy, and psychomotor retardation; patients with mevalonic aciduria may die during early childhood (2).
This difference in clinical presentation can be explained by the fact that at the biochemical level, cells from patients with the HIDS presentation still show residual MK enzyme activity that is 1–8% of the activity in cells from healthy controls (4–7). In contrast, enzyme activity in cells from patients with the mevalonic aciduria presentation is below the level of detection (2). This difference in residual enzyme activity is also reflected by high levels of mevalonic acid in plasma and urine from patients with the mevalonic aciduria presentation (2) and low to moderate levels of mevalonic acid in plasma and urine from patients with the HIDS presentation (6).
MK catalyzes the phosphorylation of mevalonate to produce 5-phosphomevalonate and is the next enzyme in the isoprenoid biosynthesis pathway after 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Figure 1), which, under normal conditions, is the rate-limiting enzyme in this pathway (8). The isoprenoid biosynthesis pathway provides cells with a variety of essential bioactive molecules, including sterols and nonsterol compounds, that have pivotal functions in multiple cellular processes, ranging from cell growth and differentiation to protein glycosylation and numerous signal transduction pathways.
Figure 1. The isoprenoid biosynthesis pathway. HMG-CoA = 3-hydroxy-3-methylglutaryl coenzyme A; SREBPs = sterol regulatory element binding proteins; ZAA = zaragozic acid A.
Download figure to PowerPoint
To prevent a shortage of end products or overaccumulation of intermediates, the flux through the isoprenoid biosynthesis pathway is tightly regulated by the levels of its end products (8). HMG-CoA reductase plays a central role in this regulation and is subject to different regulatory mechanisms. For example, the efficiency of HMG-CoA reductase messenger RNA (mRNA) translation is dictated by the requirement of the cell for nonsterol isoprenoids, whereas the degradation rate of HMG-CoA reductase protein is dependent on the requirement for both sterol and nonsterol isoprenoids (8). At the transcription level, the HMG-CoA reductase gene and all other genes encoding enzymes of the isoprenoid biosynthesis pathway are subject to transcriptional feedback regulation coordinated by the so-called sterol regulatory element binding proteins (SREBPs), in particular SREBP type 2 (9–11). SREBPs are conditional positive transcription factors that enhance gene transcription in the absence of sterols but are not required for basal transcription when sterols are present (9, 10).
Previously, we postulated that the episodes of fever in MK-deficient patients are attributable to a temporary shortage of 1 or more of the nonsterol isoprenoids that are required for down-regulation of the inflammatory response precipitated by fairly harmless events such as physical stress or vaccination (12–14). This lack of down-regulation can be explained by the fact that in patients with MK deficiency, MK has become the rate-limiting enzyme in the isoprenoid biosynthesis pathway instead of HMG-CoA reductase. Under normal conditions, the flux through this pathway in MK-deficient patients will be sufficiently high for most cellular processes requiring isoprenoids (12, 13). However, any small increase in body temperature will result in a rapid decrease in residual MK activity, due to the deleterious, temperature-sensitive effect on MK protein maturation and stability of most mutations found in these patients, which will lead to a rather instant disturbance of the flux through the pathway and, as a consequence, a shortage of end products (12).
Based on this postulate, we investigated the effect of 2 specific inhibitors of isoprenoid biosynthetic enzymes on the residual activity of MK in cells from MK-deficient patients. For these studies, we used cultured primary skin fibroblasts from such patients, in which the isoprenoid biosynthesis pathway can be readily manipulated by varying the culturing conditions, as reported previously (12, 13). Our results show that both simvastatin, an inhibitor of HMG-CoA reductase, and zaragozic acid A (ZAA), an inhibitor of squalene synthase, give rise to increased MK activities, which is attributable to enhanced transcription of the gene MVK. This increase in MK activity renders the flux through the pathway less dependent on the conversion of mevalonate to phosphomevalonate. Our in vitro results suggest that treatment of patients with inhibitors of isoprenoid biosynthesis may provide a therapeutic option that could lead to less frequent and/or shorter episodes of fever.
- Top of page
- PATIENTS AND METHODS
In this study, we sought to determine whether it is possible to up-regulate residual MK activity in cells from MK-deficient patients by manipulating the isoprenoid biosynthesis pathway. The rationale for this study is based on our earlier observations, which indicated that in MK-deficient patients, MK has become the rate-limiting enzyme in isoprenoid biosynthesis that determines the flux through the pathway (12, 18). Thus, in principle, up-regulation of residual MK activity in patients would shift the rate-limiting step back to HMG-CoA reductase and make the pathway flux less dependent on MK activity. As a consequence, patients would be less susceptible to external precipitating events that negatively influence MK activity, which affects the flux through the pathway and the production of certain end products.
We studied the effect of 2 specific inhibitors of the isoprenoid biosynthesis pathway, simvastatin and ZAA. Simvastatin is a widely used competitive inhibitor of HMG-CoA reductase, the enzyme preceding MK in the pathway. ZAA is a competitive inhibitor of squalene synthase (19), the first enzyme dedicated exclusively to the production of sterol isoprenoids. Treatment of fibroblasts from MK-deficient patients with either of the inhibitors led to a marked increase in residual MK enzyme activity, mainly due to increased MVK gene transcription. This effect was already evident in cells cultured in standard (lipoprotein-rich) medium but was further enhanced when the cells were cultured in lipoprotein-depleted medium.
These findings are consistent with the known feedback regulation of isoprenoid biosynthesis genes by SREBPs, which become activated under these conditions, because the production of sterol end products will further decrease due to the inhibitors. Remarkably, however, after incubation with simvastatin and ZAA in lipoprotein-depleted medium, the relative increase in MK activity in cells from patients with HIDS and those from the patient with mevalonic aciduria was much higher than that in control cells cultured under the same conditions. This cannot be explained by increased MVK transcription alone, as was evident from the increase in MK activity over MVK mRNA produced in these cells. This suggests that inhibition of isoprenoid biosynthesis somehow promotes the formation of stable mutant MK protein.
Because our previous studies indicated that the shortage in MK-deficient patients involves primarily, if not exclusively, nonsterol isoprenoids including geranylgeranyl groups (13, 14), we also studied the effect of the 2 inhibitors on the flux toward nonsterol isoprenoid biosynthesis. This was done by determining the presence of the small GTPases RhoA and Rac1 in cell membranes. Both proteins are synthesized as soluble proteins and require a covalently bound geranylgeranyl moiety to become localized in the cellular membrane, where they exert their effect. Our results showed that although treatment with simvastatin led to up-regulation of MK, it apparently had a negative effect on the overall flux toward geranylgeranyl synthesis at this particular concentration, because both RhoA and Rac1 became less abundant in the cellular membrane.
Thus, whereas treatment with simvastatin seemed promising with regard to increasing MK activity, the consequence of inhibiting the preceding enzyme HMG-CoA reductase appears to be negative for the flux. This finding indicates that one needs to be cautious when treating MK-deficient patients with simvastatin, because the balance between inhibiting HMG-CoA reductase and inducing MK activity may be critical, especially in MK-deficient patients in whom the pathway flux is very sensitive to external influences. This was also suggested by the negative outcome of treatment of 2 patients with mevalonic aciduria using lovastatin, a drug that is similar to simvastatin (2). This treatment provoked severe clinical crises in those patients. However, it was recently reported that treatment of 6 HIDS patients with simvastatin (20) did not provoke clinical crises in those patients, in contrast to the patients with mevalonic aciduria, which is a more severe disease. Although no statistical difference was observed with respect to the severity, frequency, and occurrence of febrile attacks, a clear decrease in the total number of febrile days was observed. These findings in patients with mevalonic aciduria and in patients with HIDS are consistent with our previous observations that, in vitro, cells from patients with mevalonic aciduria appear more sensitive to simvastatin compared with cells from patients with HIDS or those from control subjects (13).
When compared with treatment with simvastatin, treatment with ZAA or other squalene synthase inhibitors seems more promising. Not only does ZAA treatment result in up-regulation of MK activity, it also leads to an increased flux toward synthesis of geranylgeranyl groups. This was particularly evident when cells were incubated with both simvastatin and ZAA, which shows that the negative effect on flux by simvastatin is counterbalanced by ZAA, leading to a reappearance of RhoA and Rac1 in the cellular membrane. Treatment of cells with ZAA in the absence of simvastatin led to minimally increased levels of RhoA and Rac1 protein in the membranes (data not shown).
Being a specific squalene synthase inhibitor, ZAA was previously reported to be a potential drug for the treatment of hypercholesterolemia (19). However, ZAA has not been developed further for this indication, nor are there any reports of this drug being tested in humans; the only reported treatments with ZAA were performed in animals (19, 21–25). In fact, of all reported squalene synthase inhibitors, only BMS-188494 (the prodrug of BMS-187745) was tested in healthy human volunteers, which led to changes in urinary farnesyl pyrophosphate metabolite (dioic acid) excretion when doses of >100 mg were given for 4 weeks, without apparent negative effects (26, 27). This drug is no longer available, however.
Although in recent years the insights into the pathogenesis underlying the symptoms of mevalonic aciduria and HIDS have increased (18), no efficacious treatment for MK-deficient patients is currently available. In individual HIDS cases, clinical improvement through treatment with corticosteroids (28), etanercept (29, 30), or leukotriene inhibitors (28) has been reported, but in the majority of patients these treatments did not have beneficial effects, and they have not led to a general treatment of MK deficiency. Our in vitro results suggest that treatment of patients with inhibitors of isoprenoid biosynthesis may provide a possible therapeutic option to alleviate or even prevent the episodes of inflammation. It should be noted here that besides using inhibitors of isoprenoid biosynthesis, culturing cells in lipoprotein-depleted medium also led to the up-regulation of MK activity in cells from patients with HIDS. This finding suggests that patients may also benefit from a cholesterol-depleted diet.