Expression and localization of augmenter of liver regeneration in human muscle tissue

Abstract

Mitochondrial DNA (mt-DNA) disorders and abnormal regulation of nuclear-derived proteins devoted to the cross-talk between the two cellular genomes have recently interested researchers in the field of neuromuscular diseases. We have identified, isolated and sequenced a new gene, augmenter of liver regeneration (ALR) that stimulates in vivo hepatocyte proliferation and up-regulates mt-DNA expression and ATP production. ALR protein (Alrp) is mainly located, in rat, in the mitochondrial inter-membrane space and its mRNA is particularly abundant in brain, muscle, testis and liver, tissues whose activity is mostly dependent on mitochondrial metabolism. Studies on rat Alrp sequence revealed the presence of homologous amino-acid sections into proteins derived from mouse, human, Drosophyla, plants and even DNA viruses. In this article, we evaluated ALR expression in normal human muscular tissues, both as protein and as mRNA. The data, obtained by molecular biology, immunohistochemistry and electron microscopy, demonstrated that: (i) Alrp and ALR mRNA are present in human muscular tissue; (ii) Alrp is particularly expressed in muscular fibres rich in mitochondria; (iii) Alrp is localized in the mitochondrial inter-membrane space or associated to mitochondrial cristae; and (iv) in subjects younger then 35 years of age, ALR mRNA expression is different between male and female subjects. In conclusion, the present data set Alrp, as a factor associated with mitochondria also in human tissue, call for future studies aimed at establishing Alrp as an important factor involved in the molecular events that trigger neuromuscular diseases.

Figure 1

ALR mRNA expression evaluated in tissue specimens from healthy patients, determined by real time PCR analysis. The subjects have been divided into three groups for each gender (female white columns, male hatched columns) according to their age. The data (mean ± SD) are typical of three separate experiments.

Figure 2

Alrp and mitochondrial proteins detection in human muscle tissue slides. Alrp (a), determined by a polyclonal antibody and ATPase 4.3 (b), ATPase 4.6 (c) and NADH tetrazolium reductase (d) enzymatically determined, were evaluated in serial tissue slides. The muscular fibres positive for each parameter are indicated by arrows. The data, typical of three separate experiments, were evaluated blindly by two operators.

Figure 3

Alrp immunolabelling detection by electron microscopy in normal skeletal muscle. The labelling is evident both in the mitochondrial inter-membrane space and on some mitochondrial cristae (arrows). Microphotography 44.000×.

Figure 4

Microphotography 71.000×. A stronger magnification of Alrp detection by electron microscopy is reported. Localization in the mitochondrial inter-membrane space and on some mitochondrial cristae (arrows).