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Research Notes: HMG-CoA Pathway Impairment in PWSSee also -
Indications of HMG-CoA pathway impairment in PWS -
Rev Neurol. 2007 Sep 16-30. INTRODUCTION. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitors or statins are drugs used in the treatment of dyslipidemies. The clinical trials performed for evaluation of the efficacy observed a reduced incidence of stroke and other trials have demonstrated a better outcome after stroke and subrachnoid hemorrhage. DEVELOPMENT. In the last years, new actions of statins have been described (pleiotropics). The statins seem to originate neuroprotector effects, such as up-regulation of endothelial nitric oxide synthase; creation of a fibrinolytic profile with suppression of the intravascular stability of the clot; immunomodulation by regulation of cytokines and cellular adhesion molecules; anti-oxidation by reduction of lipidic peroxidation; induction of neuroplasticity by increment of neurotrophic factors and protection of neuroexcitotoxicity, maybe by regulation of intracellular calcium or depletion of intracellular sterols. All these actions can be explained by decreament of isoprenoids synthesis. CONCLUSION. The pleiotropic properties of the statins offer the possibility to consider them as possible neuroprotectors, which should be evaluated in pathologies where the molecular ways interfered are involved, for example head injury and stroke. DNA Cell Biol. 2007 Sep 15. Sterol regulatory element-binding proteins (SREBPs) are transcription factors governing transcription of genes related to cholesterol and fatty acid metabolism. To become active, SREBPs must undergo a proteolytic cleavage to allow an active NH(2)-terminal segment to translocate into the nucleus. SKI-1/S1P is the first protease in the proteolytic activation cascade of SREBPs. SREBP inhibition may be useful, for example, in the treatment of liver steatosis caused by homocysteine-induced lipid synthesis. Accordingly, we overexpressed inhibitory prodomains (proSKI) of SKI-1/S1P in HepG2 cells to block SREBP activation to evaluate potential of SKI-1/S1P in controlling cellular cholesterol synthesis. SKI-1/S1P inhibition resulted in reduced cholesterol synthesis and mRNA levels of the rate-limiting enzymes, HMG-CoA reductase and squalene epoxidase, in the cholesterol synthetic pathway. The inhibitory effect was maintained also in the presence of homocysteine-induced endoplasmic reticulum stress. A gene set enrichment analysis was performed to elucidate other metabolic effects caused by SKI-1/S1P inhibition. SKI-1/S1P inhibition was observed to affect a number of other metabolic pathways, including glycolysis and citric acid cycle. These results demonstrate that inhibition of SREBPs decreases cholesterol synthesis in HepG2 cells both in the absence and presence of homocysteine. SKI-1/S1P inhibition may cause widespread changes in other key metabolic pathways. Arterioscler Thromb Vasc Biol. 2005 Feb. The 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. There is growing evidence that treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation. Accordingly, statin use has been associated with impairment of several coagulant reactions catalyzed by this enzyme. Moreover, evidence indicates that statins, via increased thrombomodulin expression on endothelial cells, may enhance the activity of the protein C anticoagulant pathway. Most of the antithrombotic effects of statins are attributed to the inhibition of isoprenylation of signaling proteins. These novel properties of statins, suggesting that these drugs might act as mild anticoagulants, may explain, at least in part, the therapeutic benefits observed in a wide spectrum of patients with varying cholesterol levels, including subjects with acute coronary events. The HMG-CoA reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. Treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation. J Biol Chem. 2004 Sep 10. The vasculoprotective effects of hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors (statins) correlate with cholesterol lowering. HMG-CoA reductase inhibitors also disrupt cellular processes by the depletion of isoprenoids and dolichol. Insulin and insulin-like growth factor (IGF) signaling appear particularly prone to such disruption as intracellular receptor processing requires dolichol for correct N-glycosylation, whereas downstream signaling through Ras requires the appropriate prenylation (farnesol). We determined how HMG-CoA reductase inhibition affected the mitogenic effects of IGF-I and metabolic actions of insulin in 3T3-L1 cells and examined the respective roles of receptor glycosylation and Ras prenylation. IGF-I- and insulin-induced proliferation was significantly reduced by all statins tested, although cerivastatin (10 nm) had the greatest effect (p < 0.005). Although inhibitors of Ras prenylation induced similar results (10 microm FTI-277 89% +/- 7.4%, p < 0.01), the effect of HMG-CoA reductase inhibition could only be partially reversed by farnesyl pyrophosphate refeeding. Treatment with statins resulted in decreased membrane expression of receptors and accumulation of proreceptors, suggesting disruption of glycosylation-dependent cleavage. Glycosylation inhibitors inhibited IGF-I-induced proliferation (tunicamycin p < 0.005, castanospermine p < 0.01, deoxymannojirimycin p < 0.01). High concentrations of statin were necessary to impair insulin-mediated glucose uptake (300 nm = 33% +/- 12% p < 0.05), and this process was not effected by farnesyl transferase inhibition. Gycosylation inhibitors mimicked the effect of statin treatment (tunicamycin p < 0.001, castanospermine p < 0.05, deoxymannojirimycin p < 0.05), and there was insulin proreceptor accumulation. These data imply that HMG-CoA reductase inhibitors disrupt IGF-I signaling by combined effects on Ras prenylation and IGF receptor glycosylation, whereas insulin signaling is only affected by disrupted receptor glycosylation. From the full text article: HMG-CoA1 reductase inhibitors are widely and successfully used to treat cardiovascular disease (1). Although their benefits are largely attributed to low density lipoprotein cholesterol lowering, there is increasing interest in the pleiotropic effects of these agents. HMG-CoA reductase inhibitors (statins) reduce the formation of mevalonic acid, an early precursor in the biosynthesis of cholesterol (2). However, mevalonic acid depletion also results in decreased levels of dolichol and isoprenoids (3); dolichol is intimately involved in the process of N-linked glycosylation of membrane-targeted proteins, whereas isoprenoids are necessary for the prenylation, subsequent membrane anchoring, and activity of downstream growth factor signaling components such as Ras. Thus statins may exert additional effects through modulation of post-translational glycosylation and isoprenylation. Insulin-like growth factor (IGF)-I and -II are polypeptides with significant structural homology to insulin. Consequently, they have acute anabolic effects on carbohydrate and protein metabolism, although importantly, they are also potent regulators of cellular replication, differentiation, and survival (4). We and others have recently found that abnormally low levels of IGF-I are associated with the premature development of type 2 diabetes (5, 6). Conversely, IGF-I is also known to promote many of the processes involved in the formation of atherosclerotic lesions and cardiovascular disease since IGF-I stimulates macrophage chemotaxis; endothelial cell migration (7); and vascular smooth muscle cell proliferation and migration (8). It also primes macrophages and monocytes for cytokine release (9). These studies clearly demonstrate that the IGF and insulin signaling systems are involved in the aetiopathogenesis of cardiovascular disease and diabetes. A reduction in mevalonic acid synthesis results in the depletion of dolichyl phosphate. Dolichyl phosphate acts as a carbohydrate donor during N-linked glycosylation of membrane-targeted proteins (10). Although most cell surface receptors do not depend on N-linked glycosylation for correct processing, such glycosylation is essential in the formation of mature, functional type 1 IGF and insulin receptors since, unusually, correct -subunit glycosylation is vital for proreceptor cleavage (11). Thus deficient glycosylation causes proreceptor retention within the endoplasmic reticulum (11, 12). The depletion of farnesyl pyrophosphate may inhibit IGF and insulin actions as these molecules are required for the prenylation of key signaling intermediates. Farnesyl pyrophosphate is necessary for the prenylation of Ras, and consequently, the association of Ras with the cell membrane (13, 14), a crucial process in mitogenic signaling induced by both the type 1 IGF receptor and the structurally similar insulin receptor. Treatment with farnesyl transferase inhibitors results in cytosolic Ras and decreased signaling in response to ligand (15). In this study, we have examined and compared the roles of IGF and insulin receptor glycosylation and Ras farnesyltion in rapidly proliferating 3T3-L1 preadipocytes and terminally differentiated 3T3-L1 adipocytes under conditions of mevalonic acid depletion induced by a broad selection of currently available HMG-CoA reducatase inhibitors. ... HMG-CoA Reductase Inhibitors Alter Processing of IGF and Insulin Receptors — HMG-CoA reductase inhibitors lead to the cellular depletion of mevalonate, and consequently, dolichyl phosphate, an essential cofactor in the process of N-linked protein glycosylation. It is therefore possible that mevalonate depletion might mediate its effect on IGF and insulin actions by disruption of receptor processing and thus receptor presentation at the cell surface. Initially, Western immunoblotting was used to assess the effect of cerivastatin on IGF and insulin receptor production. Fig. 4A shows that in preadipocytes incubated with cerivastatin (0.3–30 nM) for 24 h, there is a marked increase in the proportion of IGF-I proreceptors. Similarly, cerivastatin treatment of mature adipocytes led to an increase in the amount of insulin proreceptor, and in these cells, an alternate high molecular weight form of the insulin proreceptor was also observed (Fig. 4B). These results suggest that cerivastatin-treated cells are less efficient at processing the proreceptor to the mature alpha- and beta-subunits. This should lead to decreased expression at the cell surface, and indeed, immunoprecipitation of lysates prepared from surface biotinylated NWT3b and 3T3-IR cells demonstrated reduced levels of IGF-I and insulin (respectively) receptor subunits as a result of treatment with cerivastatin (Fig. 5) HMG-CoA Reductase Inhibitors Affect Prenylation of Proteins Associated with IGF-I Signaling Pathways — Since HMG-CoA reductase inhibition reduced the cell surface expression of receptors for both IGF-I and insulin, then altered receptor glycosylation alone cannot account for the differential affect of mevalonate depletion on the mitogenic and metabolic actions of IGF-I. We therefore investigated whether a reduction in farnesyl pyrophosphate and geranyl pyrophosphate, which also occurs as a consequence of cellular mevalonate depletion, altered the isoprenylation of proteins involved in the signaling pathways activated during a mitogenic response to IGF-I. The effect of cerivastatin on the prenylation of Ras, a known IGF-I signaling intermediary, was assessed using Western immunoblotting to visualize the proportion of Ras associated with the cellular membrane. Fig. 6A shows that cerivastin-induced inhibition of IGF-I-stimulated [3H]thymidine uptake is coupled with a reduction in membrane-associated Ras. Furthermore, the effect of cerivastatin could be partially reversed by providing mevalonate-depleted cells with farnesyl pyrophosphate (Fig. 6B). ... IGF-I-stimulated proliferation of 3T3-L1 preadipocytes was dose dependently decreased by all glycosylation inhibitors (0.25 µg/ml tunicamycin, p < 0.005, 10 µg/ml castanospermine, p < 0.01, 1 mM DMJ, p < 0.01), with tunicamycin, unsurprisingly, having the most severe effect (Fig. 7A). The farnesyl transferase inhibitor FTI-277 also inhibited IGF-I-mediated mitogenesis, suggesting that the severe effect of cerivastatin on IGF-I-stimulated cellular proliferation is due to a combination of disrupted receptor glycosylation and altered prenylation of intracellular signaling molecules. Insulin-stimulated glucose uptake was also inhibited by all glycosylation inhibitors (25 µg/ml tunicamycin, p < 0.01, 500 µg/ml castanospermine, p < 0.05, 20 mM DMJ, p < 0.05) with tunicamycin again having the most severe effect (Fig. 7B). FTI-277 had no effect on insulin-stimulated glucose uptake, confirming that the insulin metabolic pathway is sensitive to changes in receptor glycosylation but is not dependent on a prenylated signaling intermediate. ... Discussion These studies clearly demonstrate that HMG-CoA reductase inhibitors are potent inhibitors of insulin- and IGF-mediated proliferation in 3T3-L1 cells. This high degree of potency is due to the combined disruption of prenylation pathways and glycosylation-dependent proreceptor cleavage of IGF receptors. In terminally differentiated adipocytes, the metabolic effects of insulin or IGF-I on glucose uptake are substantially less affected by HMG-CoA reductase inhibition; in these cells, prenylation pathways are of lesser importance with the major abnormality remaining impaired insulin and IGF receptor processing. HMG-CoA reductase inhibitors are a group of drugs suggested to have significant pleiotropic effects (20–22). HMG-CoA reductase inhibitors decrease the proliferation of a range of cells (23–27) through the depletion of cellular mevalonate. Mevalonate depletion may lead to altered glycosylation of membrane proteins (25, 28–30) or prenylation of membrane-associated proteins (31–34). We show that in 3T3-L1 cells, it is the combined effect of these mechanisms that markedly abrogate mitogenic response of the cells to IGF-I, whereas their effect on metabolic signaling, only affected by one mechanism, is less markedly reduced. IGF- and insulin-stimulated cell proliferation was significantly inhibited by all the HMG-CoA reducatse inhibitors tested. Cerivastatin was the most potent, significantly inhibiting IGF-induced proliferation at 10 nM (p < 0.005), whereas pravastatin was the least potent, requiring 10 µM before achieving substantial inhibition of IGF-mediated cell proliferation. The order of potency, at inhibiting IGF-stimulated proliferation, correlates with their degree of hydrophobicity (ceriva > simva > fluva > atorva > prava), not their potency in inhibiting HMG-CoA reductase (atorva > ceriva > simva > fluva > prava) (35). Although hepatocytes take up statins by an active mechanism, fibroblasts do not (35). Thus in cell types without an active uptake mechanism, it appears that hydrophobicity confers the greatest effect on prenylation and glycosylation pathways. The dramatic reduction in cell proliferation resulting from statin exposure occurs at concentration and exposure times far less than those necessary to induce apoptosis. Only after prolonged treatment with a high concentration of HMG-CoA reductase inhibitor were we able to demonstrate that IGF signaling had been disrupted sufficiently to induce apoptosis. Other studies have detected statin-induced apoptosis in a variety of cells (36–38). Similarly, these studies required either very high concentrations of statins over short periods (38) or prolonged exposure to lower concentrations (36, 37). One mechanism by which HMG-CoA reductase inhibitors may alter signaling through the IGF and insulin pathways is through aberrant glycosylation of the IGF and insulin receptors. HMG-CoA reductase inhibition reduces formation of dolichyl phosphate, a carbohydrate donor during N-linked glycosylation of membrane-targeted proteins (10, 39). Due to their unusual requirement for correct N-linked glycosylation prior to the appropriate pro-receptor cleavage (11), the insulin and IGF receptor systems appear particularly prone to disruption by HMG-CoA reductase inhibitors (12, 30, 40–43). We have clearly shown that HMG-CoA reductase inhibition markedly alters both the processing and the cell surface expression of both the IGF and the insulin receptors in 3T3-L1 preadipocytes and adipocytes, respectively. This concurs with studies in melanoma and Ewing's sarcoma cells in which an HMG-CoA reductase inhibitor or the glycosylation inhibitor tunicamycin down-regulated the number of IGFRs at the cell surface (30, 43). Similar changes in receptor processing were demonstrated by Hwang and Frost (12), in which glycosylation of insulin receptors was disrupted by depriving mature 3T3-L1 adipocytes of glucose. Under these conditions, increased amounts of lower molecular weight proreceptors were formed that were subsequently degraded. We have shown that selective disruption of receptor glycosylation using specific inhibitors for the presence, length, and complexity of the carbohydrate moiety profoundly abrogates IGF and insulin receptor function. These findings are atypical. Most other membrane proteins appear less sensitive to changes in their glycosylation status (44). The transport of lysosomal acid phosphatase was dependent on the presence but not the complexity of its N-linked oligosaccharides (45), whereas lipoprotein lipase requires carbohydrate presence for secretion and activity, but prevention of glycan processing with DMJ had no effect (46). Mevalonate depletion also affects the isoprenylation of membrane-associated proteins, such as Ras. The depletion of farnesyl pyrophosphate with HMG-CoA reductase inhibitors leads to decreased signaling through Ras. Mitogenic but not metabolic signaling was inhibited by the farnesyl transferase inhibitor FTI-277, and inhibition of proliferation by cerivastatin was partially reversed by co-treatment with exogenous farnesyl pyrophosphate. Disturbed farnesylation is thus in part responsible for the inhibition of proliferation caused by cerivastatin, a process confirmed by reduced Ras association with isolated cell membranes in treated cells. Similar results have been obtained with lovastatin. In SK-MEL-2 cells, mevalonate depletion by lovastatin resulted in Ras prenylation being inhibited by 50% (47). Lovastatin also induced apoptosis in malignant mesothelioma cell lines, which was partially reversed by farnesol refeeding (48). In these cells, there was a relocalization of Ras from the membrane to the cytosol. In human glioma cell lines, lovastatin decreased cellular proliferation by 80% due to decreased signaling through Ras (31). Prior exposure of cells to insulin (and possibly IGF-I) may potentiate the subsequent actions of other mitogens (49). Draznin and co-workers (50–52) determined that insulin causes the phosphorylation and activation of farnesyl transferase, which in turn leads to a greater proportion of farnesylated and membrane-associated Ras. By blunting the effect of insulin, HMG-CoA reductase inhibitors may abrogate this effect on two levels, by decreasing the insulin activation of farnesyl transferase and by depleting the cell of farnesyl pyrophosphate. In summary, mevalonate depletion affects IGF and insulin signaling via at least two distinct but related and synergistic mechanisms: reduced prenylation of signaling intermediates such as Ras and disruption of insulin and IGF proreceptor processing. IGF-induced mitogenic processes in rapidly proliferating cells are more potently affected than insulin-mediated metabolic processes in terminally differentiated cells. Although the effects on receptor processing in most terminally differentiated cells may have little influence, those cells with active uptake mechanisms for statins may be substantially affected. In such cell types, insulin and IGF resistance might be anticipated. Drugs. 2003. Recent large clinical trials have demonstrated that HMG-CoA reductase inhibitors, or statins, markedly reduce morbidity and mortality when used in the primary and secondary prevention of cardiovascular disease. It has been established that the benefits of statin therapy in cardiovascular disease can be explained not only by the lipid-lowering potential of statins but also by nonlipid-related mechanisms (so-called "pleiotropic effects") that contribute to the positive effect of statins on the incidence of cardiovascular events.The coagulation and fibrinolytic systems are two separate but reciprocally linked enzyme cascades that regulate the formation and breakdown of fibrin. Numerous studies have demonstrated that disturbances of coagulation and fibrinolysis contribute to the development and progression of atherosclerosis, and that they affect the incidence of atherosclerosis-related clinical events. High plasma levels or activities of fibrinogen, factor VII, factor VIII, von Willebrand factor (vWF), soluble thrombomodulin, tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) are thought to be associated with increased morbidity and mortality related to cardiovascular disease.Experimental studies and many clinical studies have recently shown that statins produce favourable effects on haemostatic parameters, including those that are risk factors for cardiovascular disease. Statins diminish procoagulant activity, which is observed at different stages of the coagulation cascade, including tissue factor (TF) activity, conversion of prothrombin to thrombin and thrombin activity. In some studies, statins also reduced fibrinogen levels. By altering the levels and activities of tPA and PAI-1, statins seem to stimulate fibrinolysis. The data on the effects of combined treatment with statins and other drugs on haemostasis are rather limited. They suggest that statins combined with fibric acid derivatives, omega-3 fatty acids and 17beta-estradiol are superior to statins alone. The only two clinical studies performed in patients with acute coronary syndromes showed a relatively weak effect of statins on haemostasis in those patients. Although various statins may produce different effects on individual variables, there are no convincing data showing that differences in their physicochemical and pharmacokinetic properties significantly alter their net effect on excessive procoagulant activity. Apart from the lipid-lowering effect, statins suppress the synthesis of several important nonsterol isoprenoids derived from the mevalonate pathway, especially farnesyl and geranylgeranyl pyrophosphates, which via enhanced protein prenylation, are involved in the regulation of many cellular processes. It is presumed that the inhibitory effect of statins on the mevalonate pathway is involved in the regulation of some key steps of coagulation and fibrinolysis processes. In this way they probably regulate the synthesis of TF, tPA and PAI-1, and perhaps they also control the generation and activity of thrombin.The beneficial effects of statins on coagulation and fibrinolysis may be responsible for their ability to decrease the number of cardiovascular events. The lipid-independent effects of statins on haemostasis may contribute to the marked decrease in the incidence rates of mortality, hospitalisation and revascularisation in patients treated with these drugs. Glycobiology. 1997 Jul. One or more mevalonate derivatives of non-sterol type have been proposed to be of indispensable importance for cell growth. Conceivable mevalonate-dependent mechanisms involved in growth control are farnesylation of Ras proteins, regulation of c-myc expression, and N-linked glycosylation of the IGF-1 receptor. The latter mechanism might be rate-limited by dolichyl phosphate, which acts as a donor of oligosaccharides in glycoprotein synthesis in the endoplasmic reticulum. In order to study the significance for cell proliferation of the three aforementioned mevalonate-dependent processings, their inhibitory response due to mevalonate deprivation was explored and compared with the effect on DNA synthesis in the malignant melanoma cell line SK-MEL-2. We found that mevalonate depletion due to treatment with 3 microM lovastatin for 24 h, which efficiently growth-arrested the cells, hardly at all affected the expression of c-myc, and although Ras prenylation was inhibited by 50%, the most pronounced effect of lovastatin was seen on N-linked glycosylation of IGF-1 receptors, which was inhibited by more than 95%. The order and magnitude of the decreased IGF-1 receptor glycosylation, which was followed by a decreased expression of IGF-1 receptors at the cell membrane, correlated well with the inhibition of DNA synthesis. We investigated whether dolichol, and in particular dolichyl phosphate, through its participation in N-linked glycosylation, act as regulators of IGF-1 receptor expression. First, we could confirm that exogenous dolichol became phosphorylated and in this form took part in the glycosylation processing. Secondly, we showed that dolichyl phosphate, in a dose-dependent manner, could increase the number of IGF-1 receptors at the cell membrane, simultaneously as DNA synthesis was stimulated. Taken together, our results provide direct evidence for an important role of dolichyl phosphate as a regulator of cell growth through limiting N-linked glycosylation of the IGF-1 receptor. From the full text article: Mevalonate (MVA) has been demonstrated to be required for mammalian cell growth (Brown and Goldstein, 1980, 1994; Goldstein and Brown, 1990), as well as constituting the key metabolite in the biosynthesis of cholesterol and a variety of non-sterol isoprenoid derivatives. The production of MVA from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase, and represents the principal regulatory step in the pathway (Brown and Goldstein, 1980). Increasing evidence suggests that the MVA-derived product critical for cellular growth is an isoprene of non-sterol type (Brown and Goldstein, 1980, 1994; Goldstein and Brown, 1990). One MVA-derived product that may be involved in control of cellular growth is dolichyl phosphate, which acts as a carrier of oligosaccharides in the assembly of N-linked glycoproteins in the lumen of the endoplasmic reticulum (ER) (Hirschberg and Snider, 1987; Hart, 1992). It has been shown that proliferation of breast cancer cells is blocked specifically in Gl by HMG-CoA reductase inhibition (Larsson, 1993). This inhibition also resulted in a drastic depression of N-linked glycosylation. Following addition of MVA the depression of N-linked glycosylation was overcome and the cells subsequently initiated DNA synthesis (Larsson, 1993). However, if the MVA-induced increase in protein glycosylation was prevented by the N-linked glycosylation inhibitor tunicamycin (TM) (Heifetz et al, 1979), the cells were not able to proliferate. If instead TM was added 4 h after the addition of MVA, the cells synthesized DNA normally (Larsson, 1993). Similar stage-specific dependence of N-linked glycosylation in the Gl phase was found in 3T3-cells (Larsson, 1985) and in human fibroblasts (Carlberg et al., 1994). The latter study suggested that N-linked glycosylation of 90—240 kDa proteins in the prereplicative phase may be critical for induction of DNA replication (Carlberg et al., 1994). These high-molecular weight glycoproteins may include growth factor receptors. More recent studies have in fact shown that MVA is necessary for translocation of the IGF-1 receptor (IGF-IR) to the cell surface, a mechanism in turn critical for the subsequent initiation of DNA synthesis (Carlberg and Larsson, 1996; Carlberg et al., 1996). ... ...As shown, both the rate of N-linked glycosylation, protein prenylation and DNA synthesis was rapidly decreased following treatment with 3 (JLM lovastatin. After 24 h the levels of glycosylation, protein prenylation and DNA synthesis were reduced by 75%, 55%, and >95%, respectively. After repletion with MVA all three processings were restored within 24 h. ... ... As shown in Figure 2A, N-linked glycosylation of IGF-IR was reduced by 95% within 8 h. Addition of MVA after a 24 h depletion resulted in an increase in IGF-IR glycosylation. This increase was detected as soon as after 8 h, and after an additional 16 h N-linked glycosylation of IGF-IR was completely restored (Figure 2A). A considerably lower inhibition was seen regarding Ras prenylation (50% at 24 h) following MVA depletion (Figure 2A). This inhibition was likewise completely deleted after addition of MVA. Finally, lovastatin only slightly inhibited the expression of c-myc (Figure 2B). Thus, out of the three MVA-dependent processings analyzed in SK-MEL-2 cells, the most drastic effect of MVA depletion was seen on N-linked glycosylation of IGF-IR. In comparison with Figure 1, N-linked glycosylation of IGF-IR was more drastically affected by lovastatin (95% inhibition was obtained within 8 h) than overall N-linked glycosylation (60% inhibition was obtained within 8 h). These results might reflect a shorter half-life of IGF-IR and thus a higher turnover rate of its glycosylation than of the "average" N-linked glycoprotein. By performing Western blotting using an antibody against the a-subunit of the IGF-IR (N-20), it was shown that MVA depletion abolished the expression of membrane-bound IGF-IR in SK-MEL-2 cells (Figure 2C). A similar effect was seen in another cell line, WiDr, which is derived from human colon carcinoma (Figure 2C). Furthermore, immunocytostaining using a polyclonal antibody against the IGF-1R demonstrated that inhibition of MVA synthesis due to treatment with 3 u,M lovastatin led to a depletion of membrane-bound IGF-1R in SK-MEL-2 cells whereas the amount of intracellular ones remains unchanged (data not shown). We also confirmed that the specific N-linked glycosylation inhibitor TM and the IGF-1R antibody otIR-3 decreased IGF-1 binding and DNA synthesis substantially (by 75-90%) in SKMEL-2 cells which had been given both 3 uM lovastatin and 0.77 mM MVA (Figure 3). Thus, a growth-arresting effect can be obtained both by a lowered N-linked glycosylation of the IGF-1R, leading to an inhibited translocation and membrane expression, or by an antibody-mediated block of IGF-1 binding to the IGF-1R. From these data, N-linked glycosylation of IGF-1R emerges as pivotal in the regulation of cell growth. ... Discussion We have recently demonstrated that MVA is rate-limiting for translocation of de novo synthesized IGF-1R proteins to the cell surface and that this event is necessary for cell growth (Carlberg and Larsson, 1996; Carlberg et al., 1996). Our findings presented in this investigation provide strong evidence that dolichyl phosphate is the MVA-derived isoprene critical for cell growth through limiting N-linked glycosylation of IGF-IR proteins. Firstly, MVA deprivation due to treatment with a relatively low dose (3 uM) lovastatin drastically decreased N-linked glycosylation in SK-MEL-2 cells, especially of the IGF-IR, in close correlation with decreased DNA synthesis and growth arrest. On the contrary, lovastatin had a much less pronounced inhibitory effect on Ras prenylation, equal to the inhibition of total protein prenylation (which was not surprising since we found that the majority of prenylated proteins in SK-MEL-2 cells were of the same size as Ras). Furthermore, lovastatin hardly at all affected c-myc expression in these cells. Thus, out of the three proposed known MVA-dependent mechanisms involved in growth control, i.e., N-linked glycosylation of the IGF-IR (Carlberg and Larsson, 1996; Carlberg et al, 1996), Ras prenylation (Hancock et al, 1989; Schafer et al, 1989), and c-myc expression (Barbu and Dautry, 1990), N-linked glycosylation of the IGF-IR correlated closest to cellular growth. Secondly, addition of exogenous dolichyl phosphate, which was proven functional as an oligosaccharide donor, was demonstrated to increase de novo synthesis of membrane IGF-IR proteins in MVA-depleted SK-MEL-2. Dolichyl phosphate also in a dose-dependent manner increased 125I-IGF-1 binding (as did dolichol and MVA). In addition, the up-regulated 125IIGF-1 binding induced by dolichyl phosphate and MVA closely correlated to the stimulatory effect on DNA synthesis. Thus, these findings consist compelling evidence that MVA-dependent IGF-IR expression is regulated by the amount of dolichyl phosphate molecules, acting as oligosaccharide donors in the assembly of N-linked glycoproteins. Since the membrane expression of IGF-IR was dependent on dolichyl phosphate-stimulated glycosylation of receptor proteins, dolichyl phosphate appears crucial for growth control. Also in lovastatin-treated colon carcinoma cells (line WiDr) we found a decreased amount of membrane-bound IGF-IR. These findings indicate that dolichyl phosphate-dependent N-linked glycosylation of IGF-IR, necessary for its translocation to the cell surface, constitutes a widespread mechanism by which MVA regulates cell growth. Since lovastatin inhibits both farnesylation and geranylgeranylation, the relatively undramatic inhibitory effect on Ras prenylation could not be attributed to an unmeasured effect of any specific type of Ras prenylation. In addition, the Ras antibody used for immunoprecipitation in the present study, recognizes all kinds of mammalian Ras proteins (H-, K-, and N-Ras), why it can be excluded that our results are due to a lack of immunoreactivity with any specific Ras protein. Our results appear complementary to earlier findings that specific inhibition of Ras farnesylation using newly designed inhibitors of Ras famesyl transferase, is growth-inhibitory only in Ras-transformed cells (DeClue et al, 1991; James et al, 1994; Dalton et al, 1995; Danesi et al., 1996). Regarding Ras, it has also been shown that a very strong inhibition of HMG-CoA reductase activity is necessary to block its farnesylation (Leonard et al, 1990). In fact a 500-fold increase in lovastatin dose, as compared to that required for a 50% suppression of cholesterol synthesis, was required (Leonard et al, 1990). Moreover, the suppression of HMG-CoA reductase activity following treatment with 25-hydroxycholesterol was shown to be insufficient for this purpose (Leonard et aL, 1990). Taken together with several observations showing that growth inhibition can be obtained even with low or moderate doses of competitive inhibitors (Endo, 1985), as well as with oxysterols (Larsson and Zetterberg, 1986), not affecting Ras prenylation (Leonard et al., 1990), it appears that cell cycle arrest induced by HMG-CoA reductase inhibitors in mammalian cells is not primarily a result of suppressed farnesylation of p21ras. It has recently been reported that the farnesylation inhibitor BZA-5B, despite an effective inhibition of farnesylation of P21"1* and nuclear lamins, did not affect Ras membrane localization, lamin assembly, or growth of CH0-K1 cells (Dalton et al, 1995). Neither was NGF-mediated, Ras-dependent differentiation of PC-12 cells affected by BZA-5B (Dalton et al., 1995). The latter findings suggested that Ras function is independent of farnesylation (Dalton et al., 1995). Thus, Ras prenylation does not appear to be the most critical MVA-dependent growth regulatory mechanism. Instead, dolichyl phosphate-dependent N-linked glycosylation and cell surface expression of IGF-1R proteins might constitute a widespread MVA-regulated mechanism, obtained by MVA-mediated limitation of the number of dolichyl phosphate molecules. This matter must of course be investigated in other systems to examine the generality of our results. It would be interesting to study the role of dolichyl phosphate for expression of IGF-1R in an IGF-1R knockout cell line transfected with IGF-1R cDNA, for example. MVA has also been reported to affect c-fos and c-myc (Barbu and Dautry, 1990; Vincent et al., 1991). MVA deprivation before growth stimulation of quiescent human fibroblasts by serum decreased the gene expression of c-fos and c-myc (Barbu and Dautry, 1990). These effects seemed to take place at the transcriptional level and did not depend on the type of the initial events of signal transduction (Barbu and Dautry, 1990). A quite novel study shows that the protein prenylation inhibitor phenyl acetate also induces myc suppression (Danesiet aL, 1996), a result indicating that Ras prenylation is necessary for mediation of the molecular events leading to myc expression. The moderate inhibition of Ras prenylation and the unaffected myc expression presented in this study do not contradict this finding, assuming that the action of Ras precedes the action of myc and that downregulation of myc is very slow. It is also possible that despite a moderate inhibition of Ras prenylation, the remaining amount of prenylated Ras proteins are enough to maintain the expression of myc. Growth stimulation by for example PDGF includes activation of Ras (McCormick, 1994) and HMG-CoA reductase (Habenicht et al., 1980), induction of c-fos and c-myc RNA (Paulsson etal., 1987), IGF-1R transcripts (Rubini etal., 1994) and increased IGF-1 production (Clemmons, 1984). Recently, another set of genes, involved in the early steps of the dolichol pathway, have been demonstrated to be coordinately induced as the early genes (Lennon et aL, 1995). These genes are ALG7, ALG1, ALG2, and two genes encoding oligosaccharyl transferase units (Lennon et al., 1995). ALG7 encodes the first enzyme in the dolichol pathway of N-linked glycosylation, i.e., dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT) (Mota et aL, 1994). ALG7 is regulated at two major control points in the Gl phase of the S. cerevisiae cell cycle, G0/G1 and START (Pretel et al., 1995). Since growth stimulation requires an increased glycoprotein synthesis, due to the need for, e.g., IGF-1 receptors (Carlberg and Larsson, 1996; Carlberg et al., 1996), it seems reasonable that except for an increased production of MVA and dolichyl phosphate induced by the activation of HMG-CoA reductase, activation of the dolichol pathway enzymes is also included in the growth stimulatory response. It is certainly interesting to find out whether overexpression of the IGF-1R in tumor cells can be stimulated only by an increased dolichyl phosphate production, or if other genes like ALG7 also must be coordinately up-regulated. It appears however that cell cycle control is not at all points dependent of the dolichol pamway and N-linked glycosylation. For example, the PDGF receptor still binds its ligand PDGF and subsequently undergoes autophosphorylation even if under-glycosylated (Keating et al., 1989). Thus, it is reasonable that some pathways of growth signal transduction are not affected by an inhibited N-linked glycosylation. PDGF-induced myc expression, perhaps via Ras, might represent one such pathway, which would explain why the expression of myc was not affected by lovastatin. J Biol Chem. 1996 Jul 19. Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth. From the full text article: It is well known that MVA1 is required for growth of mammalian cells (1, 2, 3, 4) as well as constituting the key metabolite in the biosynthesis of cholesterol and a variety of nonsterol isoprenoid derivatives (e.g. dolichyl-phosphate, farnesyl pyrophosphate, isopentenyladenine, and ubiquinone). The formation of MVA from HMG-CoA, which is catalyzed by HMG-CoA reductase, is the principal regulatory step in the pathway (1). Increasing evidence suggests that the MVA-derived product critical for cell growth is an isoprene of nonsterol type (1, 2, 3, 4). A possible mechanism for MVA-regulated cell growth is the involvement of dolichyl-phosphate in N-linked glycosylation. De novo synthesized dolichyl-phosphate acts as a carrier of oligosaccharides in the assembly of glycoproteins in the lumen of the endoplasmic reticulum (ER) (5, 6). In a recent study we investigated the potential regulatory role of N-linked glycosylation in initiation of DNA synthesis in human fibroblasts stimulated by serum (7). Our results suggested that N-linked glycosylation of proteins of 90-240 kDa in the prereplicative phase may be critical for induction of DNA replication. These high molecular mass glycoproteins may include growth factor receptors. This raises the possibility that MVA may regulate the expression of growth factor receptors through limiting the biosynthesis of dolichyl-phosphate. The existence of such a mechanism would constitute a substantial link between HMG-CoA reductase and cell growth. The aim of the present study was to investigate this issue in detail. Our experiments provide evidence that MVA is critical for the translocation of insulin-like growth factor-1 receptor (IGF-1R) to the cell surface. Exp Cell Res. 1994 Jun. Human diploid fibroblasts, arrested following serum or mevalonate depletion, were restimulated to a maximal rate of DNA synthesis within 24 h after the addition of serum or mevalonate, respectively. In both cases the initiation of DNA synthesis was preceded by a 12-h prereplicative phase. Upon the stimulation with serum there was a rapid increase in HMG-CoA reductase activity, reflecting an elevated formation of mevalonate, which reached its maximal value 4 h after serum replenishment. If this serum-induced increase in mevalonate synthesis was inhibited, the subsequent initiation of DNA synthesis was prevented. Serum stimulation also increased the level of N-linked glycosylation, an event that was dependent on the increase in HMG-CoA reductase activity. After treatment of the cells with tunicamycin, an inhibitor of N-linked glycosylation, they failed to enter the S-phase. However, an increased level of N-linked glycosylation was not required during the whole of the period after serum stimulation. Instead, it seemed to be of critical importance only during the mid stage of the prereplicative phase (i.e., 4-8 h after stimulation). Our data suggest that the N-linked glycosylation required for initiation of DNA synthesis is of high-molecular-weight (90-240 kDa) proteins. These high-molecular-weight glycoproteins may include growth factor receptors. Indirect evidence raises the possibility that the expression of growth factor receptors may play a regulatory role in the mevalonate-dependent growth activation of human fibroblasts. |