Serum apolipoprotein A 1 (apoA 1 ) concentration is inversely correlated with the risk of premature atherosclerosis. Serum apoA 1 concentrations are regulated, in part, at the transcriptional level. ApoA 1 mRNA is synthesized primarily in the liver and small intestine, under the direction of a number of signaling molecules and tissue-specific regulatory elements. Previously, we demonstrated that extracellular acidosis suppresses apoA 1 mRNA levels at the level of transcription. Here we demonstrate that intracellular acidosis, in the absence of extracellular pH changes, represses apoA 1 promoter activity. Repression occurs through a pH responsive element (pH-RE) located within the apoA 1 gene promoter. Acidosis increases the specific DNA binding activity of a putative repressor protein within the immediate 5 -flanking region of the apoA 1 gene. The cis-element that binds the putative repressor protein contains a negative thyroid hormone response element (nTRE) located 3 and adjacent to the apoA 1 TATA box. Mutation of the nTRE/ pH-RE abrogates protein binding and alters the activity of reporter genes controlled by this element. Repression by acidosis did not require de novo mRNA and protein synthesis. Inhibition of tyrosine kinase activity and diacylglycerol-stimulated protein kinase C (PKC) signaling pathways with tyrophostin A47 and phorbol myristate acetate, respectively, did not affect the repression of apoA 1 promoter activity with acidosis. These results suggest that transcriptional repression of the apoA 1 gene by alterations in ambient pH is associated with enhanced DNA binding activity of a repressor protein, through a mechanism which appears to be independent of de novo mRNA and protein synthesis, tyrosine kinase activity, or PKC activation.
To determine if ketoacidosis contributes to reduced apolipoprotein A1 (apoA1) expression in insulindeficient diabetic rats, we examined the regulation of apoA1 gene expression in response to changes in ambient pH or ketone body concentrations. Hepatic apoAI mRNA levels were reduced 42% in diabetic rats relative to nondiabetic controls (means ..; 321·8 43·7 vs 438·7 58·8 arbitrary units; P<0·03). Neither endogenous apoA1 mRNA nor transcriptional activity of the rat apoA1 gene promoter (from 474 to 7) were altered by sodium butyrate or isobutyramide (0·3 mM to 10 mM) in Hep G2 or Caco-2 cells. Rat hepatic and intestinal apoA1 mRNA levels, and plasma apoA1 concentration, were not altered 24 h after isobutyramide administration (500 mg/kg by gavage). When the effect of altering ambient pH within a wide range commonly encountered in vivo was studied, acidosis (pH 6·7), relative to alkalosis (pH 7·9), decreased apoAI mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase mRNA by 47% in Hep G2 cells (P<0·025) and by 24% in Caco-2 cells (P<0·017). Acidosis did not alter cytomegalo virus (CMV)--galactosidase activity, or the activity of the simian virus (SV40) early-region promoter, in either cell line transfected with the respective constructs. The lowering of ambient pH was associated with a graded reduction in apoAI promoter activity. At pH 6·7, apoAI promoter activity was reduced by 75% compared with promoter activity at pH 7·9. These observations indicate that acidosis, but not ketosis, contributes to the reduction in apoA1 expression during diabetic ketoacidosis by down-regulating apoAI promoter activity.
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