©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Gene Expression in FRTL-5 Cells
II. DOWN-REGULATION BY v-K-ras ONCOGENE (*)

Bruno Perillo (1), Idolo Tedesco (1), Chiara Laezza (1), Mariarosaria Santillo (1), Alfredo Romano (1), Salvatore M. Aloj (1)(§), Maurizio Bifulco (1) (2)

From the (1)From the Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare ``L. Califano,'' Universit ``Federico II,'' 80131 Napoli, Italy and the (2)Dipartimento di Medicina Sperimentale e Clinica, Universit di Reggio Calabria, 88100 Catanzaro, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and mRNA levels were significantly reduced in FRTL-5 cells transformed with the Kirsten-Moloney sarcoma virus (KiMol); these cells have lost thyrotropin dependence and express high levels of p21. FRTL-5 cells, transformed with a temperature-sensitive mutant of the v-K-ras oncogene (Ats cells: 33 °C, permissive; 39 °C, nonpermissive), showed significant reduction of HMG-CoA reductase expression when exposed to 33 °C. In KiMol cells, as well as in Ats cells at 33 °C, the transcription driven by cAMP-responsive element was probed by measuring chloramphenicol acetyl transferase (CAT) levels after transfection with a chimeric plasmid containing the reporter gene linked to the rat reductase promoter. Basal CAT activity in KiMol cells transfected with wild-type promoter was lower than in FRTL-5 cells but was increased by forskolin to the levels attained in thyrotropin-stimulated FRTL-5 cells. Forskolin failed to increase CAT activity in KiMol cells transfected with the plasmid harboring a reductase promoter in which the cAMP-responsive element octamer was mutated to a nonpalindromic sequence.

The effect of v-K-ras could be mimicked in FRTL-5 cells by tetradecanoyl phorbol acetate and reverted in KiMol and Ats cells, expressing active Ras protein, by increasing intracellular cAMP and/or by protein kinase C inhibition.

The data are consistent with the contention that v-K-ras, through protein kinase C and depletion of intracellular cAMP, is inhibitory for the protein kinase A pathway. This is the first demonstration that active v-K-ras down-regulates HMG-CoA reductase expression.


INTRODUCTION

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)()reductase is the rate-limiting enzyme in the biosynthesis of mevalonate, the precursor of several biologically relevant isoprenoids such as cholesterol, and a number of nonsterol compounds, most notably farnesyl residues, which are covalently linked to growth-regulating proteins(1, 2) . Prenylation, a covalent post-translational modification, allows protein anchorage at the plasma membrane(2, 3) . Activation of p21 requires attachment of isoprenoid units (farnesylation)(4, 5, 6) , whose availability depends on the activity of HMG-CoA reductase. Ras proteins (H-Ras, N-Ras, K-Ras4A, and K-Ras4B) are generally considered as molecular switches in signal transduction pathways leading to cell proliferation. The involvement of ras in uncontrolled growth is relevant, since an increased expression of normal or mutated ras has been found in 40% of the human cancers(7) , including human anaplastic and follicular thyroid carcinomas(8) .

FRTL-5 cells depend on thyrotropin (TSH) and cAMP for growth and induction of housekeeping genes such as malic enzyme and HMG-CoA reductase(9, 10) , and thyroid-specific functions such as I transport and thyroglobulin synthesis(11, 12, 13) . The induction of malic enzyme and HMG-CoA reductase precedes DNA synthesis and progression of quiescent FRTL-5 cells into the cell cycle(9, 10) . Competitive inhibition of HMG-CoA reductase by lovastatin results in the arrest of cell proliferation(14) .

FRTL-5 cells transformed with the Kirsten-Moloney sarcoma virus (KiMol), express active v-K-ras protein and lose TSH-dependence for growth and cell-specific functions(15, 16) . These effects are accompanied by cAMP depletion, down-regulation of the thyroid-specific trans-acting factor TTF-1(17) , and predominance of protein kinase C(17, 18) .

Since we have identified and characterized a cAMP-responsive element (CRE) in the reductase promoter(19) , it was hypothesized that HMG-CoA reductase gene expression should be down-regulated by active ras. In the present study, we investigate this hypothesis. To this end, we have measured reductase activity, mRNA levels, and gene expression in FRTL-5 and KiMol cells and in FRTL-5 cells transformed with a Kirsten-murine sarcoma virus variant (Ats cells), which expresses a temperature-sensitive Ras protein, active at 33 °C and inactive at 39 °C(20) .

We show that v-K-ras down-regulates HMG-CoA reductase gene expression; we also demonstrate that in KiMol cells CREB binds to reductase CRE and suggest that impairment of reductase transcriptional activity, in cells expressing active ras, is the result of reduced CREB phosphorylation by protein kinase A.


EXPERIMENTAL PROCEDURES

Materials

TSH was a highly purified preparation from bovine pituitary extracts(21) . [C]Acetic acid (53 mCi/mmol) and [H]mevalonolactone (35 Ci/mmol) were purchased from DuPont NEN. Thin-layer chromatographic plates were from E. Merck, Darmstad, Germany. All other materials were obtained as described in the accompanying paper(19) .

Cells

FRTL-5 cells (ATCC CRL-8305) are a strain of rat thyroid cells grown at 37 °C in a humidified atmosphere composed of 95% air and 5% CO, in Coon's modified Ham's F-12 medium supplemented with 5% calf serum and a mixture of six hormones and growth factors (referred to as 6H)(11, 19, 22) . KiMol cells, derived from FRTL-5 cells upon infection and transformation with a wild-type strain of KiMSV-MolMuLV (15, 16) and Ats cells, derived from FRTL-5 cells transformed with a temperature-sensitive mutant (33 °C, permissive; 39 °C, nonpermissive) of Kirsten-murine sarcoma virus (20), were kindly provided by Dr. G. Vecchio, Naples, Italy. KiMol cells are grown at 37 °C, and Ats cells were grown at 39 °C, both in Coon's modified Ham's F-12 medium, supplemented with 5% calf serum. Where indicated, Ats cells were shifted to 33 °C for different length of times, in different experiments.

Total RNA Preparation and Slot-Blot Analysis

Total RNA was isolated by the guanidinium thiocyanate-acid phenol procedure(23) . Total RNA slot-blots were performed using a Schleicher & Schuell Minifold II apparatus following the manufacturer's recommendations. RNA samples were mixed with 3 volumes of a denaturing solution (50% formamide, 2.2 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 0.5 mM EDTA), heated 5 min at 65 °C and diluted 2-fold in 19 SSC(10) . The samples were then applied to a nylon membrane equilibrated in 10 SSC. Prehybridization (5 h at 42 °C), hybridization (18 h at 42 °C), and high stringency washes (15 min at 37 °C, 1 h at 60 °C, 1 h at 55 °C) were carried out as already reported(10) . A XbaI fragment of HMG-CoA reductase cDNA from plasmid pRED-227 (24) was used as radiolabeled probe. Quantitative analysis of the autoradiograms was performed by laser densitometry on a PDI laser scanner. Normalization was accomplished using radiolabeled glyceraldehyde-phosphate dehydrogenase cDNA as reference probe.

[C]Acetate and [H]Mevalonate Incorporation into Cholesterol

Proliferating (6H) FRTL-5 cells and KiMol cells were incubated for 2 h with [C]acetic acid (53 mCi/mmol) or for 18 h with [H]mevalonolactone (35 Ci/mmol). At the end of the incubation period, medium was aspirated, cells were washed with ice-cold Hanks' balanced salt solution, collected in 3 ml of Hanks' balanced salt solution, and pelleted by centrifugation for 5 min at 1,000 rpm in a Beckman J-6 centrifuge. Pellets were suspended in 1 ml of 2-propanol, sonicated at 4 °C with 5 bursts of 10 s each, and recentrifuged for 15 min at 2,500 rpm (Beckman J-6 centrifuge). The supernatant was aspirated, and the pellet was re-extracted as above. Both extracts were combined, dried under a stream of N, and dissolved in 0.5-1 ml of chloroform. Aliquots of 10 µl were applied to Silica Gel-60 thin-layer chromatographic plates and developed in hexane/diethylether/acetic acid (70:30:1). Each lipid fraction was located by comparison to high purity standards, scraped off the plate, and radioassayed(10) . Cholesterol values were normalized to cell proteins measured on the 2-propanol insoluble pellet, dissolved in 0.1 N KOH.

Nuclear Extracts

Nuclear extracts from TSH-challenged FRTL-5 cells or KiMol cells were prepared as described previously (19, 25, 26) prior to use in electrophoretic mobility shift assays.

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays were performed as described previously(19, 27) . Where indicated, antiserum to CREB or ATF-2 or preimmune serum were added to nuclear extracts in the same buffer for 20 min on ice before the addition of the labeled probe and processing as above. Gels were dried and autoradiographed.

Promoter-Chloramphenicol Acetyltransferase Chimeric Plasmids

The 765-base pair (bp) PstI fragment (from -323 to +442) containing the rat HMG-CoA reductase promoter (19) was cloned into the pEMBL-8-CAT vector (19, 28) upstream to the bacterial gene for CAT. Mutations of the CRE sequence present in the reductase promoter were created by two-step, recombinant polymerase chain reaction methods, as described previously(19, 29) . Similar to the wild-type reductase promoter, the CRE-mutated 765-bp fragment (19) was cloned into the same vector, 5` to the CAT gene. Luciferase expression vector (pRSV-Luc) was used as positive control to evaluate transfection efficiency(30) .

Transient Expression Analysis

Normal and transformed FRTL-5 cells were grown to 50-60% confluence. Transfection was by the DEAE-dextran protocol as described previously(19, 31) . Cells were co-transfected with 20 µg of pEMBL-8-CAT under the control of wild-type or CRE-mutated 765-bp PstI fragment from HMG-CoA reductase promoter and 2 µg of pRSV-Luc/dish and then incubated 48 h before harvesting. Where indicated, forskolin (10 µM) was added to the cells during the last 6 h. CAT assays were performed as described previously(32) . Luciferase activity was measured using a luminometer.

Other Assays

Protein concentration was determined using a Bio-Rad kit; recrystallized bovine serum albumin was the standard.


RESULTS

HMG-CoA Reductase mRNA and Activity

We have compared reductase mRNA levels and the rates of [C]acetate incorporation into cholesterol in normal cells and in KiMol cells, which express constitutively active Ras protein. In FRTL-5 cells, HMG-CoA reductase mRNA levels are 3-fold higher than in the virally transformed counterpart. Higher reductase message yields a higher rate of [C]acetate incorporation into cholesterol in normal cells (the relative rates varying between 15- and 20-fold in different experiments) (Fig. 1). Under the conditions detailed under ``Experimental Procedures,'' when cells were exposed to [C]acetate, the yield of [C]cholesterol was 50,000 ± 5,000 cpm/mg of cell protein in FRTL-5 cells and 3,000 ± 300 cpm/mg of cell protein in KiMol cells. When cells were incubated with [H]mevalonate, the yield of [H]cholesterol was 100,000 ± 15,000 cpm/mg of cell protein in FRTL-5 cells and 20,000 ± 3,000 cpm/mg of cell protein in KiMol cells. Thus, also the rate of [H]mevalonate incorporation into cholesterol was lower in cells expressing active Ras; this is not surprising since preliminary data show that also mevalonate kinase is down-regulated by v-K-ras (data not shown). However, the difference in the relative rates of acetate versus mevalonate incorporation into cholesterol were consistent with the magnitude of reductase down-regulation (3-fold) in cells expressing active ras.


Figure 1: [C]Acetate incorporation into cholesterol and HMG-CoA reductase mRNA levels in FRTL-5 cells and in KiMol cells. Cholesterol values are normalized to cell proteins measured on 2-propanol insoluble cell pellets after solubilization in 0.1 N KOH. Total RNAs were extracted as reported under ``Experimental Procedures,'' brought to identical concentration based on optical density at 260 nm, denatured and blotted onto nylon filters as a series of 2-fold dilutions starting at 5 µg of RNA, and hybridized to P-labeled HMG-CoA reductase or glyceraldehyde-phosphate dehydrogenase probes, as described under ``Experimental Procedures.'' Filters were exposed to x-ray film and mRNAs quantitated by densitometry. The densitometric values of reductase mRNA from KiMol cells, after normalization to glyceraldehyde-phosphate dehydrogenase mRNA values, were set arbitrarily to 1; all other values are relative to this. Data are representative of three different determinations in which the variation was less than 10%. The errorbars indicate the standard error of three separate experiments performed in duplicate.



In FRTL-5 cells transformed with a Kirsten murine sarcoma virus variant producing a temperature-sensitive mutant Ras protein (Ats), the levels of reductase mRNA and enzyme activity depend upon the incubation temperature. Thus, when exposed to 39 °C, the nonpermissive temperature for ras activation, HMG-CoA reductase mRNA levels, and [C]acetate incorporation into cholesterol are comparable with those exhibited by normal FRTL-5 cells (Fig. 2). Shifting the cells to 33 °C caused a time-dependent fall of both HMG-CoA reductase mRNA levels and rate of cholesterol synthesis. Reduction was significant after 3 days, and maximal after 7 days (Fig. 2). HMG-CoA reductase activity was also decreased after 3 days at 33 °C (data not shown). Changing temperature from 33 to 39 °C resulted in recovery of the normal phenotype within 1 day only (Fig. 2), suggesting that the rate of inactivation of the mutant Ras protein is much faster than its turnover rate. Exposure of FRTL-5 cells to either 39 or 33 °C had no appreciable effect on HMG-CoA reductase mRNA and activity (data not shown).


Figure 2: The effect of exposing FRTL-5 cells transformed with the temperature sensitive mutant v-K-ras, for the noted time, at 33 °C or 39 °C, on the incorporation of [C]acetate into cholesterol and on the levels of HMG-CoA reductase mRNA, measured and normalized as detailed in the legend to Fig. 1 and under ``Experimental Procedures.'' The reductase mRNA level present in Ats cells incubated at 33 °C for 3 days has been given the arbitrary value of 1. The data indicate the mean of three separate experiments, performed in duplicate, in which the variation was less than 15%.



The Effects of Protein Kinase A and Protein Kinase C Modulation

Since TSH and cAMP induce HMG-CoA reductase gene expression in FRTL-5 cells, we tested whether the down-regulatory effect of active ras could be reverted by raising intracellular cAMP. In the presence of forskolin (10 µM), reductase mRNA levels in Ats cells at 33 °C were increased by 2-fold (Fig. 3). Nearly identical effects were obtained by treating Ats cells exposed to 33 °C with sphingosine (10 µM) (Fig. 3). The combination of forskolin and sphingosine did not cause any additive effect (Fig. 3). These observations strongly suggest that the effects of ras activation on reductase expression are mediated by cAMP depletion and protein kinase C activation. The involvement of protein kinase C was confirmed by the finding that tetradecanoyl phorbol acetate (10 ng/ml) mimicked the effect of ras activation in Ats cells at 39 °C (Fig. 3).


Figure 3: The individual and combined effects of protein kinase A and protein kinase C activation or inhibition on HMG-CoA reductase mRNA levels in Ats cells maintained either at 39 °C or shifted from 39 °C to 33 °C for 7 days. Forskolin (10 µM) and sphingosine (10 µM) were added separately or in combination for 9 h before cells harvesting. Tetradecanoyl phorbol acetate (TPA) (10 ng/ml) was added 9 h prior to RNA preparation. HMG-CoA reductase mRNA levels were estimated and normalized as detailed in the legend to Fig. 1 and under ``Experimental Procedures.'' The upperpart of the figure shows the fluorograph of one of the slot-blots performed; in the lowerpart is shown the corresponding densitometric analysis (the errorbars indicate the standard error of three separate experiments; to the mRNA level of Ats cells incubated at 33 °C has been given the arbitrary value of 1).



Transcriptional Regulation

We have investigated cAMP-dependent DNA binding factors in nuclear extracts from KiMol cells. Fig. 4shows retarded protein-DNA complexes formed with an oligonucleotide containing the CRE identified in the reductase promoter. The retardation pattern is identical to the one generated with FRTL-5 nuclear extracts, as we have characterized in the accompanying paper(19) . The KiMol protein-DNA complex, like the one from FRTL-5 cells, was supershifted by antibodies to CREB, whereas it was not affected by anti-ATF-2 antibodies or preimmune serum (Fig. 4). This observation indicates that CREB, as we have shown in the accompanying paper(19) , is the nuclear trans-acting factor that binds the reductase CRE.


Figure 4: The effect of anti CREB and anti ATF-2 antibodies and of preimmune serum on the mobility of protein-DNA complexes in EMS assays. End-labeled RED CRE (5`-CGTTCGTGACGTAGGCCG-3`) was incubated with nuclear extracts as indicated on top of each lane. Experimental details are under ``Experimental Procedures.''



Analysis of reductase promoter activity in normal FRTL-5 cells and in KiMol and Ats cells was approached by transfecting the different cell strains with a chimeric plasmid containing the rat HMG-CoA reductase promoter (the 765-bp PstI fragment, from -323 to +442) linked to the CAT reporter gene. Assays for CAT activity performed in these cells (Fig. 5) showed a significant reduction (3-fold) in KiMol and in Ats cells exposed to 33 °C, compared with FRTL-5 cells and Ats cells exposed to 39 °C.


Figure 5: The effect of 6-h incubation with forskolin (10 µM) on CAT activity in FRTL-5 cells, in KiMol cells or Ats cells at 39 and 33 °C. Each cell strain (3 dishes) was co-transfected with 20 µg of a chimeric plasmid containing either wild-type (wt) or nonpalindromic (np) CRE of HMG-CoA reductase promoter ligated to the 5` end of the bacterial CAT gene and 2 µg of pRSV-Luc. To the CAT activity of KiMol cells, transfected with the wild-type reductase promoter and not exposed to forskolin, is given the arbitrary value of 1, after normalization with pRSV-Luc for transfection efficiency. The errorbars indicate the standard error of three separate experiments.



Adenylyl cyclase stimulation gained through the incubation of the cells with forskolin (10 µM) for 6 h, was able to restore HMG-CoA reductase promoter activity, in KiMol cells, to levels comparable with those observed in TSH-stimulated FRTL-5 cells, in which forskolin failed to cause further increase. In KiMol cells transfected with a chimeric construct in which the CAT reporter gene was under the control of the CRE-mutated 765-bp fragment of the reductase promoter, forskolin failed to increase CAT activity (Fig. 5).

These findings strongly suggest that active Ras protein down-regulates HMG-CoA reductase expression by inhibiting cAMP-dependent protein kinase A activity, and by preventing protein kinase A-dependent CREB phosphorylation.


DISCUSSION

The data presented show that expression of v-K-ras in thyroid cells decreases significantly the level of HMG-CoA reductase mRNA and quenches enzyme activity. We believe that ras-dependent repression of the reductase gene is exerted at transcriptional level for two reasons. First, in normal thyroid cells cAMP increases the rate of reductase gene transcription (10, 19); second, in this study, we show that reductase promoter activity is inhibited in cells expressing active v-K-ras, and the inhibition can be released by cAMP. These data further qualify the reductase promoter as a cAMP-dependent promoter(19, 33) , a finding that has not been reported so far.

The cAMP signaling pathway is severely impaired in thyroid cells transformed by v-K-ras; indeed, constitutive levels of cAMP are drastically reduced in KiMol cells, in comparison with the parent FRTL-5 cells. This results from loss of TSH receptor(34) , reduced adenylyl cyclase(35) , and increased cAMP phosphodiesterase activities()in cells expressing active ras. Low cAMP levels are inhibitory for protein kinase A activity; under these conditions, the transfer of cytoplasmic protein kinase A to the nucleus is hampered(36) . This has been shown to impair phosphorylation of trans-acting factors such as CREB and TTF-1(36, 37) which in the nonphosphorylated state fail to bind and activate their respective targets. On the contrary, high levels of cAMP and active protein kinase A inhibit ras pathway as they interfere with Raf-1 activation (38) interrupting the link between the oncogene product (Ras-Raf) and the mitogen-activated protein kinases cascade (39).

Depletion of cAMP caused by v-K-ras in thyroid cells has also a stimulatory effect on the activity of membrane-associated protein kinase C; thus, in thyroid cells, and in other cell strains, protein kinase C activation appears to mediate the effects of ras(36, 40) . The data presented in this study are consistent with these observations, since protein kinase C stimulation in FRTL-5 cells, and in Ats cells carrying an inactive p21, mimics a v-K-ras like effect on reductase mRNA. In contrast, protein kinase C inhibition abolishes the effects of active ras.

Regulation of HMG-CoA reductase is most complex. It is exerted at different levels and affected by different factors(1, 41, 42) . We believe that the data presented in this study are consistent with the interpretation that down-regulation by active Ras protein is exerted largely, if not exclusively, at transcriptional level through depletion of intracellular cAMP, redistribution and augmentation of protein kinase C activity, and failure of CREB phosphorylation by inactive protein kinase A. It is also possible that, in addition, low cAMP may induce the uptake or reduce the efflux of cholesterol(10, 43) .

We do not know yet what is the role played by HMG-CoA reductase in ras transforming activity; therefore, the interpretation of the significance of its down-regulation remains speculative. It could be hypothesized that active ras may turn on a mechanism of negative feed-back by one of the end-products of the isoprenoid pathway. Indeed, we have found an increased level of farnesylation of p21 in v-K-ras-transformed FRTL-5 cells in comparison with normal FRTL-5 cells.()This should be clarified when the role of isoprenoid metabolism in cell transformation by ras oncogenes will be further elucidated.


FOOTNOTES

*
This study was supported in part by the Progetto Finalizzato ``Invecchiamento'' of the Consiglio Nazionale delle Ricerche. This paper was presented, in abstract form, at the annual meeting of the American Society for Biochemistry and Molecular Biology, Washington, D. C. (1994). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dip. Biol. Pat. Cell. Mol., Universit ``Federico II,'' via S. Pansini, 5 80131 Napoli, Italy. Tel.: 39-81-7463235; Fax: 39-81-7701016.

The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CAT, chloramphenicol acetyltransferase; PKA, protein kinase A; PKC, protein kinase C; TSH, thyroid-stimulating hormone; KiMol, Kirsten-Moloney sarcoma virus; MOPS, 4-morpholinepropanesulfonic acid; ATF, activating transcription factor; bp, base pair(s).

S. Obici, personal communication.

B. Perillo, I. Tedesco, C. Laezza, M. Santillo, A. Romano, S. M. Aloj, and M. Bifulco, unpublished observation.


ACKNOWLEDGEMENTS

We thank Prof. V. E. Avvedimento for critical review of the manuscript and M. Berardone, F. D'Agnello, and F. Moscato for the art work.


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