Evidence for Posttranscriptional Regulation of C/EBPα and C/EBPβ Isoform Expression during the Lipopolysaccharide-Mediated Acute-Phase Response†

Abstract

The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBP␣ and C/EBP␤) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse ␣ 1 -acid glycoprotein promoter, we detected multiple forms of C/EBP␣ and C/EBP␤ proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBP␣s with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42-and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBP␤ isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBP␣ forms specific complexes with the ␣ 1 -acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP␤ forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBP␣ and C/EBP␤ isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBP␣ and C/EBP␤ isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42-kDa C/EBP␣ and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBP␣ and the third AUG codon of the 20-kDa C/EBP␤. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors.

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

“…Our studies identified oxidatively modified mouse heart mitochondrial ETC proteins whose levels of modification in some cases correlated with the decrease in complex activity as is predicted by the Free Radical Theory of Aging [9,16,17,27,33] whereas in others, modifications had no effect on functions, thus suggesting the interaction of other factors. The decrease in CI and CV enzyme functions is consistent with our hypothesis that mitochondrial dysfunction in the aging heart may be due to the accumulation of oxidatively modified proteins.…”

“…Alternatively, failure to replace the dissociated α-chain may account for the decreased ATP production, which is a hallmark of increasing mitochondrial dysfunction with aging. Previously, it has been shown that MDA modified β-chain is associated with decreased heart CV function with age [33]. Since, we did not find MDA-modified β-chain in BN-PAGE separated CV suggests that it may have dissociated from the intact complex.…”

“…However, studies of rodent heart tissue have not shown any consensus as to which functions is affected. For example, some studies show there is a decline in CI, CIV [47] and CV [33] function while others show no changes at all. We attribute these differences in part to the variation of markers used to normalize mitochondrial content.…”

“…The reaction was initiated with the addition of 40 μM oxaloacetate and the enzyme activity was recorded at 412 nm (ε = 13.6 mM −1 cm −1 ). Rotenone-sensitive Complex I (CI) activity, Malonate-sensitive Complex II (CII) activity, Antimycin A-sensitive Complex III (CIII) activity, KCN-sensitive Complex IV (CIV) activity, and Oligomycin-sensitive Complex V (CV) activities were assayed as described [9,26,33]. Briefly, CI activity was measured at 340 nm (ε = 6.81 mM −1 cm −1 ) in 1 mL reaction mixture containing reaction buffer (50 mM Potassium Phosphate, pH 7.4, 5 mM MgCl 2 and 1 mg/mL BSA), 7-8 μg of sonicated mitochondria, 2 mM KCN, 3.7 μM Antimycin A and 100 μM Q 1 .…”

“…Oxidatively damaged proteins have been detected and identified by mass spectrometry [9,26,32]. We propose to determine whether such oxidative modifications cause mitochondrial dysfunction associated with aging and age-associated diseases [9,30,[31][32][33]. The accumulation of these oxidatively modified proteins occurs in various tissues [9] and their accumulation in cardiovascular tissue may, therefore, be an important molecular marker of age-associated decline in cardiovascular function.…”