A Mouse Model of β-Thalassemia Shows a Liver-Specific Down-Regulation of Abcc6 Expression

Abstract

␤-Thalassemia and pseudoxanthoma elasticum (PXE) are distinct genetic disorders. Yet, a dystrophic mineralization phenotype similar to PXE has frequently been associated with ␤-thalassemia or sickle cell anemia patients of Mediterranean descent. These calcifications are clinically and structurally identical to inherited PXE. As we previously excluded the presence of PXE-causing mutations in the ABCC6 gene of ␤-thalassemia patients with PXE manifestations, we hypothesized that a molecular mechanism independent of gene mutations either altered the ABCC6 gene expression or disrupted the biologic properties of its product in the liver or kidneys, which are the tissues with the highest levels of expression. To test this possibility, we investigated Abcc6 synthesis in the liver and kidneys of a ␤-thalassemia mouse model (Hbb th3/؉ ). We found a progressive liver-specific down-regulation of the Abcc6 gene expression and protein levels by quantitative PCR, Western blotting, and immunofluorescence. The levels of Abcc6 protein decreased significantly at 6 months of age and stabilized at 10 months and older ages at ϳ25% of the wild-type protein levels. We studied the transcriptional regulation of the Abcc6 gene in wild-type and Hbb th3/؉ mice, and we identified the erythroid transcription factor NF-E2 as the main cause of the transcriptional down-regulation using transcription factor arrays and chromatin immunoprecipitation. The Hbb th3/؉ mice did not develop spontaneous calcification as seen in the Abcc6 ؊/؊ mice probably because the Abcc6 protein decrease occurred late in life and was probably insufficient to promote mineralization in the Hbb th3/؉ mouse C57BL/6J genetic background. Nevertheless, our result suggested that a similar decrease of ABCC6 expression occurs in the liver of ␤-thalassemia patients and may be responsible for their frequent PXE-like manifestations.

Functional Rescue of ABCC6 Deficiency by 4-Phenylbutyrate Therapy Reduces Dystrophic Calcification in Abcc6–/– Mice

“…ABCC6 is an organic anion transporter primarily present in the basolateral plasma membrane of hepatocytes (Pomozi et al, 2013). ABCC6 deficiency is linked to several mineralization pathologies: pseudoxanthoma elasticum (PXE, OMIM 264800) is characterized by late onset and progressive calcifications in the skin leading to prominent dermal manifestations as well as in vascular and ocular tissues (Le Saux et al, 2000); β-thalassemia (OMIM 187550), which may be related to reduced levels of ABCC6 protein in the liver (Martin et al, 2011); and a subset of generalized arterial calcification of infancy (GACI, OMIM 208000), that is typically associated with ENPP1 mutations and results in early patient mortality (Nitschke et al, 2012). In several inbred strains of mice, including C3H/HeJ, ABCC6 deficiency causes an acute calcification phenotype affecting the myocardium and the media of large arteries (Aherrahrou et al, 2008; Doehring et al, 2006; Meng et al, 2007).…”

Dysregulation of gene expression in ABCC6 knockdown HepG2 cells

“…A PXE phenotype without mutations in ABCC6 has frequently been associated with β-thalassemia patients [7]. A β-thalassemia mouse model showed a significant decrease in ABCC6 protein expression, even if it occurs too late in life and is thus insufficient to promote mineralization [8]. Generalized arterial calcification of infancy (GACI) is a hereditary disorder generally associated with mutations in the ecto-nucleotide pyrophosphatase/phosphodiesterase type I (Enpp1) gene [9].…”

Is classical pseudoxanthoma elasticum a consequence of hepatic ‘intoxication’ due to ABCC6 substrate accumulation in the liver?

“…However, these patients were devoid of known PXE-causing mutations [60], and the genetic basis governing clinical findings in β-thalassemia and inherited PXE, therefore, appears distinct. It was recently demonstrated that a β-thalassemia mouse model displayed downregulated Abcc6 protein liver expression [61]. This finding raised the possibility that a reduced level of ABCC6 protein contributes to the mineralization phenotype of humans with β-thalassemia, although this needs further investigation.…”