Abstract
We observed and compared alterations in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase at the transcriptional level in unsynchronized, three-passage cultures of smooth-muscle cells from the aorta of chicks fed on a control diet (C-SMC) and those of chicks fed on a similar diet plus cholesterol (Ch-SMC). Alterations in reductase mRNA concentrations in senescent cultures were much lower. We used a modification of the competitive (c) reverse transcription polymerase chain reaction method, using a Thermus thermophilus DNA polymerase (Tth pol) to quantify the very scarce species of HMG-CoA reductase mRNA in samples of cytoplasmic SMC mRNA. We cloned and sequenced a 199 bp cDNA fragment of chicken HMG-CoA reductase, which encoded a region of 66 amino acids belonging to the catalytic domain of the enzyme. HMG-CoA reductase mRNA concentrations from young C-SMC cultures rose 3.89-fold 4 h after the change of medium and returned to base levels between 8 to 12 h afterward. Concentrations in Ch-SMC cultures increased less (2.36-fold) 8 h after the change to fresh medium. Increases in reductase mRNA in senescent cultures of Ch-SMC and C-SMC measured under similar conditions were only 1.28- and 1.39-fold, respectively.
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Abbreviations
- (c) RT-PCR:
-
competitive reverse transcription-polymerase chain reaction
- DMEM:
-
Dulbecco's modification of Eagle's medium
- dNTP:
-
deoxy nucleotide 5′-triphosphate
- FBS:
-
fetal bovine serum
- HMG-CoA:
-
3-hydroxy-3-methylglutaryl-CoA
- PBS:
-
phosphate-buffered saline
- SMC:
-
smooth muscle cell
- UV:
-
ultraviolet
References
Rodwell, V.W., Nordstrom, J.L., and Mitschelen, J.J. (1976) Regulation of HMG-CoA Reductase, Adv. Lipid Res. 14, 1–74.
Chin, D.J., Gil, G., Faust, J.R., Goldstein, J.L., Browun, M.S., and Luskey, K.L. (1985) Sterols Accelerate Degradation of Hamster 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase Encoded by Constitutively Expressed cDNA, Mol. Cell Biol. 5, 634–641.
Choi, J.W., and Peffley, D.M. (1995) 3′-Untranslated Sequences Mediate Post-transcriptional Regulation of 3-Hydroxy-3-methylglutaryl-CoA Reductase mRNA by 25-Hydroxycholesterol, Biochem. J. 307, 233–238.
Simonet, W.S., and Ness, G.C. (1989) Post-transcriptional Regulation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase mRNA in Rat Liver, J. Biol. Chem. 264, 569–573.
Mitropoulos, K.A. (1983) Molecular Control of HMG-CoA Reductase: The Role of Nonesterified Cholesterol, in 3-Hydroxy-3-methylglutaryl CoA Reductase (Sabine, J.R., ed.) pp. 107–127, CRC Press, Boca Raton.
García-Gonzalez, M., Segovia, J.L., and Alejandre, M.J. (1992) Homeostatic Restoration of Microsomal Lipids and Cholesterol Acyltransferase in Chick Liver, Mol. Cell. Biochem. 115, 173–178.
Kumagai, H., Chun, M.T., and Simoni, R.D. (1995) Molecular Disection of the Role of the Membrane Domain in the Regulated Degradation of 3-Hydroxy-3-methylglutary Coenzyme A Reductase, J. Biol. Chem. 270, 19107–19113.
Beg, Z., and Brewer, H.B. (1981) Regulation of Liver 3-Hydroxy-3-methylglutaryl-CoA Reductase, Curr. Top. Cell Regul. 20, 139–184.
Clarke, P.R., and Hardie, D.G. (1990) Regulation of HMG-CoA Reductase: Identification of the Site Phosphorylated by the AMP-Activated Protein Kinase in vitro and in Intact Rat Liver, EMBO J. 9, 2439–2446.
Goldstein, J.L., and Brown, M.S. (1990) Regulation of Mevalonate Pathway, Nature 343, 425–430.
Vallet, S.M., Sanchez, H.B., Rosenfeld, J.M., and Osborne, T.F. (1996) A Direct Role for Sterol Regulatory Element Binding Protein in Activation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Gene, J. Biol. Chem. 271, 12247–12253.
Tavangar, K., and Kraemer, F.B. (1988) The Regulation of Hydroxymethylglutaryl-CoA Reductase in Cultured Cells, Biochim. Biophys. Acta 970, 251–261.
Quesney-Huneeus, V., Wiley, M.H., and Siperstein, M.D. (1979) Essential Role for Mevalonate Synthesis in DNA Replication, Proc. Natl. Acad. Sci. USA 76, 5056–5060.
Quesney-Huneeus, V., Galick, H.A., Siperstein, M.D., Erickson, S.K., Spencer, T.A., and Nelson, J.A. (1983) The Dual Role of Mevalonate in the Cell Cycle, J. Biol. Chem. 258, 378–385.
Carazo, A., Alejandre, M.J., Diaz, R., Rios, A., Castillo, M., and Linares, A. (1998) Changes in Cultured Arterial Smooth Muscle Cells Isolated from Chicks Upon Cholesterol Feeding, Lipids 33, 181–190.
Bolton, M.C., Dudhja, J., and Bayliss, M.T. (1996) Quantification of Aggrecan and Link-Protein mRNA in Human Articular Cartilage of Different Ages by Competitve Reverse Transcriptase-PCR, Biochem. J. 319, 489–498.
Habenicht, A.J.R., Glonset, J.A., and Ross, R. (1980) Relation of Cholesterol and Mevalonic Acid to the Cell Cycle in Smooth Muscle and Swiss 3T3 Cells Stimulated to Divide by Platelet-derived Growth Factor, J. Biol. Chem. 255, 5134–5140.
Ross, R. (1971) The Smooth Muscle Cell II. Growth of Smooth Muscle in Culture and Formation of Elastic Fibers, J. Cell Biol. 50, 172–186.
Chamley-Campbell, J.H., Campbell, G.R., and Ross, R. (1979) The Smooth Muscle Cell in Culture, Physiol. Rev. 58, 1–61.
Hanon, E., Vanderplasschen, A., and Pastoret, P.P. (1996) The Use of Flow Cytometry for Concomitant Detection of Apoptosis and Cycle Analysis, Biochem. 2, 25–27.
Chomczinski, P., and Sacchi, N. (1987) Single-Step Methods of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction, Anal. Biochem. 162, 156–159.
Jiang, Y.H., Davidson, L.A., Lupton, J.R., and Chapkin, R.S. (1996) Rapid Competitive PCR Determination of Relative Gene Expression in Limiting Tissue Samples, Clin. Chem. 42, 227–231.
Sanger, F., Nicklen, S., and Couldson, A.R. (1977) DNA Sequencing with Chain-Terminating Inhibitors, Proc. Natl. Acad. Sci. USA 74, 5463–5467.
Myers, T.W., and Geldfand, D.H. (1991) Reverse Transcription and DNA Amplification by a Thermus thermophilus DNA Polymerase, Biochemistry 30, 7661–7666.
Gebhardt, A., Peters, A., Gerding, D., and Niendorf, A. (1994) Rapid Quantitation of mRNA Species in Ethidium Bromide-stained Gels of Competitive RT-PCR Products, J. Lipid Res. 35, 977–981.
Raeymaekers, L. (1993) Quantitative PCR: Theoretical Considerations with Practical Implications, Anal. Biochem. 214, 582–585.
Brown, M.S., Dana, S.E., and Goldstein, J.L. (1973) Regulation of 3-Hydroxy-3-methyl-glutaryl Coenzyme A Reductase Activity in Human Fibroblasts by Lipoproteins, Proc. Natl. Acad. Sci. USA 70, 2162–2166.
Chin, D.J., Gil, G., Russell, D.W., Liscum, L., Luskey, K.L., Basu, S.K., Okayama, H., Berg, P., Golstein, J.L., and Brown, M.S. (1984) Nucleotide Sequence of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase, A Glycoprotein of Endoplasmic Reticulum, Nature 308, 613–617.
Liscum, L., Cummings, C., Anderson, R.G.W., DeMartino, G.N., Goldstein, J.L., and Brown, M.S. (1983) 3-Hydroxy-3-methylglutary Coenzyme A Reductase: A Transmembrane Glycoprotein of the Endoplasmic Reticulum with N-linked “High-mannose” Oligosaccharides, Proc. Natl. Acad. Sci. USA 80, 7165–7169.
Cohen, D.C., Massoglia, S.L., and Gospodarowicz, D. (1982) Correlation Between Two Effects of High Density Lipoproteins on Vascular Endothelial Cells. The Induction of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Activity and the Support of Cellular Proliferation, J. Biol. Chem. 257, 9429–9437.
Chen, H.W. (1981) The Activity of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase and the Rate of Sterol Synthesis Diminish in Cultures with High Cell Density, J. Cell Physiol. 108, 91–97.
Harwood, H.J., Jr., Schneider, M., and Stacpoole, P.W. (1984) Measurement of Human Leukocyte Microsomal HMG-CoA Reductase Activity, J. Lipid Res. 25, 967–978.
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Carazo, A., Alejandre, M.J., Suarez, M.D. et al. Alterations in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA concentration in cultured chick aortic smooth muscle cells. Lipids 35, 587–593 (2000). https://doi.org/10.1007/s11745-000-0560-8
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DOI: https://doi.org/10.1007/s11745-000-0560-8