Patients with chronic hepatitis C (CHC), the leading cause of cirrhosis in the United States and Western Europe, accumulate fibrosis at highly variable rates. A gene-centric functional genome single nucleotide polymorphism (SNP) scan that sought to characterize the genetic risk factors of fibrosis progression identified a SNP in the Antizyme Inhibitor (AZIN1
) gene. The SNP in this gene had the most robust cross-validation score in the first step in the development of the multi-SNP score, as well as the highest odds ratio amongst the predictive SNPs correlating with delayed fibrosis progression (1
). However, like several of the risk-associated SNPs that emerged from this and other genetic association studies, the mechanism underlying this gene’s involvement and the specific protective effect of the minor allelic SNP variant remain to be clarified. Recently, studies from our laboratory have established mechanisms underlying effects of additional SNP variants of the TLR4 (29
), another gene included in the cirrhosis risk score, and DDX5 (30
), a separately reported gene on fibrogenic activities of HSCs, both of which were identified from the same genetic scan.
The AZIN 1 SNP encodes a silent polymorphism that does not alter the primary protein sequence. However, the SNP’s proximity to an intron-exon boundary led us to hypothesize that the SNP altered the splicing of AZIN1 pre-mRNA, and computational analysis of the exonic sequence surrounding the SNP predicted decreased binding of an exonic splicing enhancer. Indeed, we have identified eight AZIN1 splice variants that are proximal to C-terminus of the AZIN1 protein, suggesting that the SNP resides in a region of alternative splicing.
To assess the impact of the SNP variant on alternative splicing, we generated partial AZIN1
minigenes containing the two allelic SNP nucleotides. Quantitative RT-PCR demonstrated significant increases in AZIN1 SV2 and AZIN1 SV8 (Figure S1
). The increase in SV2 and SV8 was unexpected because while the SNP is located six nucleotides from the 5′ end of exon 12, it appeared to have the greatest influence on 3′ splice site recognition.
We have established the in vivo relevance of the HSC culture findings by demonstrating that the fibrosis-protective variant is correlated with the increased expression of the SV2 mRNA splice form in leukocytes from healthy patients whose genomic DNA contains at least one copy of this SNP variant. While evidence of increased splicing in liver-derived cells rather than leukocytes would be more informative, there were insufficient numbers of liver samples containing the minor allelic variant to perform such an analysis. Nonetheless, we have been able to establish that both isoforms are expressed in normal human liver. However, because HSCs, the fibrogenic cell in liver, represent only a small percentage of resident liver cells, our analysis of whole-liver mRNA likely reflects expression primarily in hepatocytes rather than in cells where the SNP regulates fibrogenic gene expression.
Prior studies have demonstrated that polyamines, whose synthesis is promoted by AZIN1, play an essential role in hepatocellular regeneration following partial hepatectomy or toxic liver injury (31
). Our study did not detect an AZIN1 splice form perturbation of polyamine content. Interestingly, AZIN1
knockout mice, which die at birth, have non-specific liver abnormalities but otherwise normal tissues, suggesting that AZIN1 is essential for normal liver development, architecture or function (9
). It is unknown whether these liver abnormalities are independent of polyamine content, but the question merits reassessment in light of the results described here.
Because the AZIN1 SNP variant correlates with a slower rate of fibrosis and leads to increased AZIN1 SV2, we characterized the effects of the two allelic variants of AZIN1 in the hepatic stellate cell, which is the cellular effector of fibrosis. Moreover, AZIN1 has not previously been characterized in any fibrogenic cell type. Over-expression of AZIN1 SV2 significantly inhibited expression of αSMA and collagen I mRNAs and proteins. While AZIN1 moderately reduced fibrogenic gene expression in stellate cells following transfection, the inhibition was markedly increased in the presence of AZIN1 SV2, suggesting that AZIN1 SV2 exerts a unique affect on stellate cell responses.
The specific inhibition of genes characteristic of activated HSCs by AZIN1 SV2 was polyamine-independent, as described above. AZIN1 SV2 does not interact with Az based on co-immunoprecipitation, and it does not interfere with the ability of AZIN1 to neutralize Az activity, excluding the possibility that AZIN1 SV2 functions as a dominant negative protein. Accordingly, ODC activity was unaffected by over-expression of AZIN1 SV2. This finding was surprising, as the AZIN1 SV2 protein conserves the known Az binding site, suggesting that the interaction between AZIN1 and Az may require inclusion of sequences from the C-terminal part of the AZIN1 protein for proper tertiary structure and interaction with Az.
Polyamine biology and biosynthetic pathways have proven a target-rich arena for therapeutic intervention with cancer as a primary and hyperproliferative disorders as a secondary disease focus (34
). Even though clinical success for these strategies has proven evasive, recruitment of the developed antagonists merit study for liver fibrosis given the implicated role of the AZIN1.
In summary, the minor allelic SNP variant in the twelfth exon of AZIN1 associated with slower rates of fibrosis progression favors the expression of a novel splice form, AZIN1 SV2, that inhibits the expression of fibrogenic genes in hepatic stellate cells via a novel, polyamine-independent pathway.