Transcriptional activation of low density lipoprotein receptor gene by ...

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4097-4101, May 1993 Medical Sciences

Transcriptional activation of low density lipoprotein receptor gene by angiotensin-converting enzyme inhibitors and Ca2 -channel blockers involves protein kinase C isoforms LUTZ H. BLOCK*t, RADOVAN KEULt, MARYSE CRABOSt, ROLF ZIESCHE*, AND MICHAEL ROTHt *Department of Medicine, University of Vienna, A-1090 Vienna, Austria; and *Department of Research, Kantonsspital, University of Basel, CH-4031 Basel, Switzerland

Communicated by Tadeus Reichstein, January 13, 1993

Evidence suggests that angiotensin-converting enzyme (ACE) inhibitors and Ca2+-channel blockers, both of which are known to lower elevated blood pressure, are beneficial in protection of organs against cardiovascular diseases (7, 8). However, the exact mode of action underlying their protective potency remains uncertain. While Ca2+-channel blockers potentiated the rPDGF-induced transcription of genes for LDLR, HMG-CoA reductase, c-Fos, c-Jun, and cytokines (2, 3), the effect of ACE inhibitors on gene transcription has not been elucidated. In view of the potential ability of ACE inhibitors to affect the changes induced by rPDGF, their role in transcription of LDLR and HMG-CoA reductase genes was examined. In addition, their possible effect on PKC isoforms was evaluated at the protein level by determining the distribution of the isoforms and compared with the potency of Ca2+-channel blockers.

The pharmacological potency of angiotensinABSTRACT converting enzyme (ACE) inhibitors (lisinopril and enalaprilat) on the transcription of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase genes was examined in human vascular smooth muscle ceils and compared with the action of Ca2+-channel blockers (manidipine, verapamil, and diltiazem). Analogous to Ca2+-channel blockers, nanomolar concentrations of enalaprilat or lisinopril stimulated the synthesis of low density lipoprotein receptor mRNA and amplified the transcription induced by recombinant plateletderived growth factor BB. In contrast to Ca2+-channel blockers, ACE inhibitors did not alter the transcription of the 3-hydroxy-3-methylglutaryl-CoA reductase gene. Plateletderived growth factor BB stimulated the translocation of a and E isoforms of protein kinase C. Similar to Ca2+-channel blockers, ACE inhibitors reduced the translocation of a and E isoforms of protein kinase C. Furthermore, ACE inhibitors and Ca2+-channel blockers inhibited platelet-derived growth factor BB-induced transcription of c-fos and c-jun genes. The findings suggest that increased de novo synthesis of mRNA low density lipoprotein receptor apparently involves the participation of 8 and E isoforms of protein kinase C and transcription factors c-Fos and c-Jun.

METHODS Human VSMCs. Three primary human VSMC lines of different origin (human aorta, hVSMC003 and hVSMC004; human lung aorta, hLVSMC001) were established and used in all experiments. Cells were cultivated as described (3). Two days prior to experiments, 1 x 106 VSMCs were seeded into a 750-ml cell culture flask (Falcon); the culture medium was exchanged 4 hr later with RPMI medium 1640 supplemented with 0.1% fetal calf serum (low serum condition) to bring the cells to a quiescent state. Measurement of Mitogenicity. The mitogenic effect was measured by the amount of [3H]thymidine incorporated into DNA of human VSMCs by the procedure of Chesterman et al. (9) and by assessment of cell proliferation as described by Lindl and Bauer (10). The experiments were performed in triplicate in each of the three cell lines. Extraction of Total RNA and Northern Blot Analyses. The total RNA was extracted by a modified protocol of Chomczynski and Sacchi (11), as described earlier (3). The RNA was quantitated by spectrophotometry (A260/A280). The transfer of RNA to nylon membranes was accomplished by capillary blotting overnight, and then the RNA was fixed to the membranes by UV irradiation. The membranes were then prepared as described (2). The hybridization of the radioactive probes was performed at the appropriate temperatures (37°C-45°C for oligonucleotides; 65°C for cDNA probes) overnight or for at least 16 hr (12). To determine the binding of the radiolabeled probes to the corresponding mRNA band, the membranes were washed

Among the various factors involved in tissue injury, plateletderived growth factor (PDGF) has been shown to stimulate growth and proliferation of vascular smooth muscle cells (VSMCs) and fibroblasts (1). Furthermore, PDGF is capable of regulating the expression of genes encoding inflammatory mediators, such as interleukin 1,3, interleukin 6, and granulocyte/monocyte-colony-stimulating factor, in human mesangial cells (2). PDGF stimulates the transcription of low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase genes, which are primarily responsible for the regulation of cellular cholesterol biosynthesis (3). The latter effect is accompanied by the rapid and transient formation of transcription factors (c-Fos and c-Jun) (2). Protein kinase C (PKC) represents a family of 10 closely related isotypes that play a major role in the transmembranous signal transduction of hormones and growth factors leading to cellular responses in mammalian tissues (4). PDGF causes redistribution of PKC from a cytosolic to a particulate fraction (5, 6). However, it is presently unknown which of the various PKC isoforms are expressed in VSMCs and regulated by recombinant (r) PDGF. We have reported (3) that rPDGFdependent transcription of the gene encoding HMG-CoA reductase apparently involves the action of PKC; in contrast, the transcription of LDLR gene occurs independently of the action of the enzyme (3).

Abbreviations: ACE, angiotensin-converting enzyme; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA reductase; LDLR, low density lipoprotein receptor; rPDGF, recombinant platelet-derived growth factor; PKC, protein kinase C; VSMC, vascular smooth muscle cell. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4097

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under stringent conditions [for LDLR oligonucleotide, two 15-min washes in 2.5 x standard saline citrate (SSC) at 55°C and 5x SSC at 45°C; for c-fos and c-jun oligonucleotides, two 15-min washes in 2.5 x SSC at 75°C; for HMG-CoA reductase and HLA-,8 cDNAs, two 15-min washes in 2.5x SSC at 65°C]. The membranes were then exposed to x-ray film (X-Omat AR; Kodak) for 1-7 days at -70°C. The intensity of the mRNA bands was analyzed by a computerized screenscanning system (Apple, Macintosh). Oligonucleotide and cDNA Probes. The LDLR oligonucleotide was from AMS (Lugano, Switzerland), the oligonucleotides for c-fos and c-jun were from British Bio-technology Limited (Oxford), and the cDNA probes for HMG-CoA reductase and for the constitutive control gene HLA-,B were from American Type Culture Collection. Expression of LDLR. Expression of the LDLR on the cell membrane was determined by the protocol of Gherardi et al. (13) with a murine anti-human LDLR monoclonal antibody (Amersham). Translocation ofPKC Isoforms. VSMCs (1 x 107 cells) were preincubated for 3 hr with manidipine, verapamil, diltiazem, enalaprilat, or lisinopril (each at 10 nM) followed by stimulation with rPDGF-BB (10 ng/ml) for 5 min. Thereafter, cells were scraped by a rubber policeman, resuspended in an ice-cold homogenization buffer [20 mM Tris HCl, pH 7.5/1 mM EDTA/1 mM EGTA/2 mM dithiothreitol/leupeptin (25 pg/ml)/l mM phenylmethylsulfonyl fluoride/10 mM benzamidine] and sonicated for two 30-sec bursts. The membranous and cytosol fractions were separated by ultracentrifugation (100,000 x g for 10 min at 4°C), and the proteins were size-fractionated by SDS/PAGE. The proteins were transferred to a nitrocellulose membrane by Western blot (14) and the bands of the PKC-isoform proteins were identified by specific monoclonal antibodies as described by Huwiler et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

Time Course of the Effects of ACE Inhibitors on Transcription of LDLR and HMG-CoA Reductase Genes. Lisinopril and enalaprilat (each at 10 nM) induced the transcription of LDLR mRNA by 3- to 4-fold above basal level. The transcription of the LDLR mRNA started to increase at 2 hr, peaked at 4-6 hr, and declined thereafter for both drugs (Fig. 1). This effect was similar to that obtained for rPDGF-BB (Fig. 2). In contrast to their stimulatory effect on the transcription of the LDLR gene, ACE inhibitors failed to affect the synthesis of HMG-CoA reductase mRNA (Fig. 1). The effect of all substances on the LDLR gene was abolished by pretreatment ofthe VSMCs with actinomycin D (5 Mg/ml, 30 min), an inhibitor of RNA synthesis, which suggests that the changes occurred at the mRNA level (data not shown). Effect of ACE Inhibitors on the LDLR Gene Transcription and Expression Induced by rPDGF-BB. The potential effects of ACE inhibitors on the transcription of the LDLR gene induced by rPDGF-BB were examined. The preincubation (3 hr) of VSMCs with lisinopril or enalaprilat, followed by addition of rPDGF-BB (10 ng/ml), shortened the onset of transcription of the gene from 2 hr to 1 hr. Furthermore, the rPDGF-BB-dependent transcription of the LDLR gene was enhanced by 50% with enalaprilat and by 30% with lisinopril (Fig. 2a). Similar to the changes obtained at the transcriptional level, both drugs stimulated the expression of the LDLR (Fig. 2b). a

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Chemicals. Lisinopril was obtained from ICI, enalaprilat was from MSD Isotopes, manidipine was from Takeda (Kyoto, Japan), verapamil was from Knoll (Liestal, Switzerland), diltiazem was from Godecke (Freiburg, F.R.G.), curcumin was from Merck, and ketoconazole was from Janssen Pharmaceutica.

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RESULTS Effects of ACE Inhibitors and Ca2+-Channel Blockers on rPDGF-BB-Induced de Novo DNA Synthesis and Ceil Proliferation. The addition of the recombinant rPDGF-BB to VSMCs resulted in a dose-dependent increase in incorporation of [3H]thymidine into de novo-synthesized DNA and proliferation of human VSMCs. In contrast, the addition of ACE inhibitors or Ca2+-channel blockers to the VSMCs negated the effect of rPDGF-BB in stimulating de novo DNA synthesis and cell proliferation. The IC50 values of both types of drugs are summarized in Table 1. Table 1. Effect of ACE inhibitors and Ca2+-channel blockers IC50 value, M Proliferation DNA synthesis Drug 4 x 10-8 6 x 10-8 Manidipine 3 x 10-8 9 x 10-8 Verapamil 1 x 10-7 3 x 10-7 Diltiazem 8 x 10-8 1 X 10-7 Enalaprilat 2 x 10-7 5 x 10-7 Lisinopril IC50 values of the inhibitory effect of ACE inhibitors and Ca2+channel blockers on rPDGF-BB-stimulated (10 ng/ml) de novo DNA synthesis and cell proliferation in VSMCs. Data represent the mean of three experiments performed in triplicate.

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FIG. 3. Effects of ACE inhibitors (10 nM), Ca2+-channel blockers (10 nM), ketoconazole (10 ,ug/ml), and curcumin (10 ,ug/ml) on the rPDGF-BB-induced (10 ng/ml) transcription of c-fos (a), c-jun (b), and the LDLR gene (c), assessed at the time of maximal transcription (c-fos, 30 min; c-jun, 60 min; LDLR, 2 hr) by Northern blot analysis. Lanes: 1, unstimulated VSMCs; 2, VSMCs stimulated with rPDGFBB (10 ng/ml); 3-9, VSMCs were preincubated (3 hr) with various drugs before being stimulated with rPDGF-BB; 3, manidipine; 4, verapamil; 5, diltiazem; 6, enalaprilat; 7, lisinopril; 8, ketoconazole; 9, curcumin. HLA-,B was the constitutive control.

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FIG. 2. Effects of ACE inhibitors (10 nM) on the rPDGF-BBinduced (10 ng/ml) transcription (a) and translation (b) of the LDLR gene. Cells were pretreated with the drugs for 3 hr after which rPDGF-BB was added. Each data point represents the mean + SEM. A, PDGF-BB; o, PDGF-BB plus enalaprilat; o, PDGF-BB plus lisinopril. Bars: 1, control; 2, PDGF-BB; 3, enalaprilat; 4, enalaprilat plus PDGF-BB; 5, lisinopril; 6, lisinopril plus PDGF-BB.

Effect of ACE Inhibitors, Ca2 -Channel Blockers, Ketoconazole, or Curcumin on rPDGF-BB-Induced Transcription of c-fos, c-jun, and LDLR Genes. Cells were pretreated with lisinopril or enalaprilat or with one of the three Ca2+-channel blockers for 3 hr after which rPDGF-BB was added. As depicted in Fig. 3, the ACE inhibitors clearly blocked the induction of transcription of c-fos and c-jun. Similarly, the addition of Ca2+-channel blockers resulted in an inhibition of the mRNA synthesis of both genes. A comparable effect was observed using either ketoconazole, known to stimulate the transcription of the LDLR gene (16), or curcumin, shown to block the binding of AP-1/c-Jun to its DNA-binding site (17) (Fig. 3 a and b). The inhibitory effect on mRNA synthesis of the two transcription factors of all drugs tested was associated with an increased transcription of the LDLR gene (Fig. 3c). Effect of ACE Inhibitors and Ca2+-Channel Blockers on the Activation of PKC Isoforms. Changes in redistribution of PKC isoenzymes from cytosolic to particulate fractions were studied by SDS/PAGE and Western blot analysis using monoclonal antibodies (14, 15). The addition of rPDGF-BB re-

DISCUSSION At therapeutic concentrations, the ACE inhibitors enalaprilat and lisinopril inhibited the proliferative response and de novo DNA synthesis stimulated by rPDGF-BB. They induced the synthesis of LDLR mRNA and intensified the transcription of the gene induced by rPDGF-BB. In contrast, the drugs failed to significantly modulate the transcription of the HMGCoA reductase gene. The de novo synthesis of mRNA of the transcription factors c-Fos and c-Jun achieved by rPDGF-BB was inhibited by both types of drugs. Analogous to the Ca2+-channel blockers, the two ACE inhibitors reduced the rPDGF-BB-dependent translocation of the 8 and E isoforms of PKC from cytosolic to membranous pools. Accumulating evidence suggests a key role for rPDGF in the development of atherosclerosis, the hallmark being proliferation of VSMCs accompanied by the formation of a connective tissue matrix and the accumulation of lipid and lipoproteins (18). If high blood pressure is combined with an elevation of plasma lipids, atherosclerosis progresses (19). However, clinical experience indicates a protective role of ACE inhibitors and Ca2+-channel blockers against cardiovascular disorders (such as coronary atherosclerosis). This corresponds with the antiproliferative efficacy of the drugs in VSMCs as seen in our study and with their ability to block the secretion of vasoactive factors from endothelial cells (20-22). The fact that both classes of compounds are efficacious in lowering blood pressure and in stimulating the transcription of the LDLR gene may be interpreted as a protective potency against the attack of atherogenic factors (e.g., cholesterol ester) against cardiovascular tissue. Under physiological

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FIG. 4. Translocation of the PKC isoforms (a and E) from the cytosolic to membranous pools, in VSMCs, determined by Western blot analysis. Lanes: 1, unstimulated VSMCs; 2, VSMCs stimulated with PDGF-BB (10 ng/ml); 3-7, VSMCs were preincubated (3 hr) with various drugs; 3, manidipine; 4, verapamil; 5, diltiazem; 6, enalaprilat; 7, lisinopril. All drugs were at a final concentration of 10 nM. Cells were then stimulated with PDGF-BB (10 ng/ml) for 5 min. The 80-kDa bands are indicated by arrowheads.

conditions, the LDLR is downregulated (23). Thus, an upregulation induced by both types of drugs should consequently result in an increase in binding and internalization of extracellular LDL-cholesterol ester. Our observation that this effect is achieved under therapeutic concentrations (nanomolar) may indicate an important site of action of the substances. Interestingly, the HMG-CoA reductase inhibitor mevinolin, known to lower elevated cholesterol level in plasma, stimulated the expression of LDLR gene (24), which led to an increase in binding and uptake of cholesterol ester. A similar effect was reported for Ca2+-channel blockers (25, 26) corresponding to the recent observation that Ca2+channel blockers are capable of slightly reducing the concentrations of cholesterol in plasma (27). Although the cholesterol-lowering effect of Ca2+-channel blockers in the plasma may only be marginal, the drugs may correct disturbances of cholesterol metabolism at the cellular level, as has been suggested (28). A similar potency of ACE inhibitors may be responsible for the beneficial efficacy in organ protection (29). However, the fact that they failed to block transcription of HMG-CoA reductase gene excludes a relevant influence on cellular cholesterol biosynthesis. We have found that VSMCs contain 8 and E isoforms of PKC, all of which can be translocated by rPDGF-BB from a cytosolic to a particulate fraction. However, the stimulatory effect on a and E isoforms is inhibited by ACE inhibitors and Ca2+-channel blockers. It has been shown that PKC isoforms have different subcellular localizations in tissues (30, 31) and that they might differ with respect to their endogenous substrates (32, 33). Given that the isoforms of PKC differ in their capacity to regulate various target genes, an inhibition of translocation of a and E isoforms by ACE inhibitors and Ca2+-channel blockers could account for their inhibitory influence against the action of PDGF. The mechanism by which activation of PKC leads to an increase in gene transcription is a subject of considerable interest. We have shown recently that rPDGF-BB induces transcription of the early-response genes c-fos and c-jun, which encode components of the transcription factor AP-1. Because of their universal expression in mammalian tissues, the a and E isoforms of PKC may play a role in nuclear events. It has been suggested (34) that the a isoform of PKC may be integrated directly or indirectly in a protein kinase cascade that is initiated by the action of growth factors, such as PDGF. In this regard, the E isoform of PKC, which is sensitive to fatty acids, could serve a role in the mechanism of regulation of genes responsible for controlling cellular lipid metabolism. The fact that the inhibition of translocation of the two PKC isoforms correlated with an inhibition of

rPDGF-dependent induction of c-fos and c-jun indicates that ACE inhibitors and Ca2+-channel blockers may modify cholesterol biosynthesis at the level of PKC activation. PDGF has been shown to increase c-fos and c-jun mRNAs in human mesangial cells (2). Changes in c-Jun protein phosphorylation may also be induced either directly through PKC isoforms or secondarily through PKC-dependent activation of other kinases or phosphatases (35). ACE inhibitors and Ca2+-channel blockers, both of which inhibited PDGFstimulated proliferation of VSMCs, decreased transcription of c-fos and c-jun. How can an inhibition ofboth transcription factors lead to an increased expression of LDLR gene? (i) The fact that steroids, known to interfere with AP-1 activity, usually block the expression of LDLR gene (36) makes a steroid-like effect of the two drugs on gene activation unlikely. (ii) An effect analogous to ketoconazole, which stimulates LDLR mRNA synthesis by inhibition of lipoproteinmediated suppression (16) and inhibited c-fos and c-jun transcription in our study, cannot be excluded. (iii) Furthermore, a possible role of cAMP in the event of promotion of LDLR gene has to be considered due to the fact that forskolin-dependent upregulation of LDLR was maintained in the presence of oxysterol suppression (37). Similarly, elevation of intracellular cAMP level blocked PKC-mediated activation of T cells by inhibition of transcription of c-jun (38). Thus, an influence of ACE inhibitors and Ca2+-channel blockers on protein-protein interactions leading to activation of transcription could account for their efficacy on the expression of LDLR gene. This work dation.

was

supported by a grant of the Alfried Krupp Foun-

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