Age-dependent regulation of the polymorphic forms of alpha 2u-globulin.

Hepatic synthesis of alpha 2u-globulin in the male rat shows a gradual decline and ultimate loss during aging and senescence. Northern blot analysis with a cloned cDNA probe showed that the decrease in alpha 2u-globulin synthesis during aging is associated with a corresponding decrease in the concentration of its hepatic mRNA. By means of two-dimensional gel electrophoresis, alpha 2u-globulin is resolved into a family of proteins. A monoclonal mouse antibody can identify at least five major isoelectric variants of alpha 2u-globulin within the total protein synthesized by rat hepatocytes. These isoelectric variants are also present in the in vitro translation products of hepatic mRNA. An examination of the hepatic synthesis of the isoelectric variants of alpha 2u-globulin during aging showed a differential regulation of the variant forms of this protein. Variant 2 (pI 6.1, the most prominent form) is the first to appear at puberty (40 days). The weakest member of the five major isoelectric forms (variant 4, pI 4.1) is the last to disappear at senescence. Although the overall decline in alpha 2u-globulin synthesis during aging seems to be due to an age-dependent decrease in the androgen responsiveness of hepatocytes, it is postulated that the differential regulation of the isoelectric variants may represent changes at the level of the genes coding for this protein.

Pretranslational regulation of alpha 2u-globulin in rat liver by growth hormone.

Hypophysectomy is known to cause complete suppression of the hepatic synthesis alpha 2u-globulin. The effect of hypophysectomy on the synthesis of alpha 2u-globulin can be reversed by multiple hormone treatment. The role of pituitary growth hormone in the multihormonal regulation of alpha 2u-globulin in rat liver was examined in the hypophysectomized male rats with and without growth hormone supplementation. Daily treatment of hypophysectomized rats with 5 alpha-dihydrotestosterone, corticosterone, thyroxine, and growth hormone for 8 days caused about 80% recovery in the hepatic content of alpha 2u-globulin and its corresponding mRNA as determined by radioimmunoassay, in vitro translation, and liquid hybridization with a cloned cDNA probe. However, omission of growth hormone from the treatment regimen failed to raise hepatic alpha 2u-globulin and its mRNA to more than 5% of the normal control. The possible effect of growth hormone on the translation of the mRNA for alpha 2u-globulin was examined with cultured hepatocytes derived from growth hormone-deficient rats. Culture of these cells in the presence of growth hormone for 24 h did not turn on the synthesis of alpha 2u-globulin. These results indicate that growth hormone regulates the synthesis of alpha 2u-globulin by acting at a step antecedent to mRNA translation.

Differential regulation of the messenger RNA for three major senescence marker proteins in male rat liver.

Changes in the mRNAs coding for specific hepatic proteins in male rats during aging were examined by in vitro translation of the liver mRNA in the rabbit reticulocyte lysate. Characterization of the [35S]methionine-labeled translation products by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by autoradiography showed major age-dependent changes in the hepatic concentrations of three mRNA species. The translation products of these three mRNAs were found to have Mr = 28,500, 26,300, and 19,500 and are called senescence marker proteins (SMP) 1, 2, 3, respectively. On the basis of its immunochemical reactivity, SMP-3 (Mr = 19,500) is identified as alpha 2u-globulin while the mRNAs for SMP-1 (Mr = 28,500) and SMP-2 (Mr = 26,300) code for two yet uncharacterized proteins. The liver of the prepubertal male rats was found to contain the mRNA for SMP-2 and showed almost complete absence of the mRNAs for SMP-1 and SMP-3. However, the mRNAs for both SMP-1 and SMP-3 were present in the postpubertal young adults while the mRNA for SMP-2 was absent. Finally, when the animals reached senescence, the mRNAs for SMP-1 and SMP-3 disappeared from the liver with the reappearance of the SMP-2 mRNA. Age-dependent regulation of the mRNAs for these three senescence marker proteins can serve as an important model for the study of differential gene expression during aging.