Role of transcription, translation, and protein turnover in controlling the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the lens.
OBJECTIVE
To determine the principle site (epithelium or superficial cortex) of gene transcription and mRNA translation for the regulatory enzyme of lens cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). To evaluate the contribution of waning enzyme synthesis versus enzyme turnover by proteolysis in accounting for the disappearance of HMGR protein from elongated fiber cells.


METHODS
Young rats were treated with lovastatin, a drug that increases transcripts of the HMGR gene and translation of HMGR mRNA in lens secondary to inhibiting cholesterol biosynthesis. The relative concentration of HMGR mRNA in lens epithelium and superficial cortex was estimated by a competitive reverse transcriptase-polymerase chain reaction system. Relative HMGR protein levels were estimated by Western blot analysis. Because lovastatin is cleared rapidly from the lens, the half-life of HMGR protein in epithelium and cortex was estimated by following the disappearance of the increased pool of enzyme protein from each compartment with time after halting drug treatment.


RESULTS
Between 75% and 90% of the total content of HMGR mRNA and protein in the epithelium and the superficial cortex of control rat lens was located in the cortex. Treatment with lovastatin increased the content of the mRNA in epithelium and cortex by approximately 0.4-fold and HMGR protein content approximately 5-fold. Although the concentration of HMGR mRNA and protein was similarly increased in epithelium and superficial cortex, approximately 85% to 90% of the total increase in mRNA and protein content was located in the cortex because of that area's greater mass. The half-life for the disappearance of the increased pool of HMGR protein from epithelium and cortex was similar at approximately 14 to 17 hours.


CONCLUSIONS
The bulk of HMGR gene transcription and mRNA translation apparently is confined to elongating fiber cells. The 10-fold greater increase in enzyme protein than mRNA levels after lovastatin treatment indicates that enzyme concentration in lens is controlled mainly by effects on HMGR mRNA translation or rates of HMGR proteolysis. The observed rapid turnover of enzyme protein in the epithelium and the superficial cortex, if applicable to the deeper cortex and the homeostatic state (absence of drug exposure), suggests that the gradual disappearance of HMGR protein from the lens could be caused by waning of enzyme synthesis rather than to proteolysis in the absence of continuing enzyme synthesis.
Cenedella
R J
RJ
Department of Biochemistry, Kirksville College of Osteopathic Medicine, Missouri 63501, USA.
D000818:Animals; D002784:Cholesterol / Q000096:biosynthesis; D004848:Epithelium / Q000201:enzymology; D005136:Eye Proteins / Q000096:biosynthesis*; D005260:Female; D006207:Half-Life; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008148:Lovastatin / Q000494:pharmacology; D008297:Male; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic
D005136:Eye Proteins; D012333:RNA, Messenger; D002784:Cholesterol; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases; D012194:RNA-Directed DNA Polymerase