Dysregulation of gene expression in ABCC6 knockdown HepG2 cells

ABCC6 protein is an ATP-dependent transporter that is mainly found in the basolateral plasma membrane of hepatocytes. ABCC6 deficiency is the primary cause of several forms of ectopic mineralization syndrome. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues. Mutations in the mouse ABCC6 gene were also associated with dystrophic cardiac calcification. Reduced levels of ABCC6 protein were found in a β-thalassemic mouse model. Moreover, some cases of generalized arterial calcification in infancy are due to ABCC6 mutations. In order to study the role of ABCC6 in the pathogenesis of ectopic mineralization, the expressions of genes involved in this process were evaluated in HepG2 cells upon stable knockdown of ABCC6 by small hairpin RNA (shRNA) technology. ABCC6 knockdown in HepG2 cells causes a significant upregulation of the genes promoting mineralization, such as TNAP, and a parallel downregulation of genes with anti-mineralization activity, such as NT5E, Fetuin A and Osteopontin. Although the absence of ABCC6 has been already associated with ectopic mineralization syndromes, this study is the first to show a direct relationship between reduced ABCC6 levels and the expression of pro-mineralization genes in hepatocytes.


INTRODUCTION
Biomineralization is the process by which minerals are deposited within or outside the cells in a variety of organisms. It is subject to complex regulatory networks. Physiological mineralization occurs in hard tissues, whereas pathological calcification occurs in soft tissues [1]. Various proteins and intracellular pathways are involved in these processes [2][3][4]. Several lines of evidence suggest that the ABCC6 transporter, encoded by the ABCC6 gene and mainly expressed in the liver and kidneys, is implicated in several ectopic mineralization defects in humans and mice [5]. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by a progressive ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues [6]. 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]. It was recently reported that some cases of GACI are due to ABCC6 mutations and show characteristic PXE clinical manifestations [10,11]. In addition, deficiency in CD73 (encoded by the NT5E gene) causes a human disease called arterial calcifications due to deficiency in CD73 (ACDC), which is characterized by a spontaneous and premature onset of arterial calcification, closely related but phenotypically different from GACI and PXE [12]. Finally, mutations in the mouse ABCC6 gene have been associated with dystrophic cardiac calcification (DCC), a disease characterized by hydroxyapatite deposition in necrotic myocytes [13,14]. ABCC6 is expressed in the basolateral membrane of hepatocytes and in the proximal kidney tubules [15,16]. The inability of this transporter to secrete its unknown substrate(s) in systemic circulation could be the primary cause of the ectopic calcification phenotype of PXE, β-thalassemia, GACI and DCC [17]. In this study, we analyzed the role of ABCC6 in the mineralization process using HepG2 cells, a suitable in vitro model system for the study of polarized human hepatocytes in which ABCC6 protein is endogenously expressed [18]. We observed a set of genes involved in hydroxyapatite deposition that are differentially expressed in ABCC6 knockdown HepG2 cells, suggesting that the molecules transported by ABCC6 in the liver are involved in the regulation of genes directly engaged in the mineralization processes.

MATERIALS AND METHODS
HepG2 cell culture and isolation of HepG2 clones stably expressing ABCC6-shRNA Human hepatocellular carcinoma cell line (HepG2) was maintained in Dulbecco's modified essential medium (DMEM) containing 4.5 g/l glucose, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37ºC, in an atmosphere of 5% CO 2 . Four short-hairpin RNAs (shRNAs) targeting different sequences in the coding regions of the ABCC6 gene (ABCC6-shRNA) and a scrambled shRNA (scr-shRNA) cloned into the pRS vector were purchased from OriGene Technologies. After selection in a medium containing puromycin (2 μg/ml) for 2 weeks, individual resistant clones were expanded in medium without puromycin and evaluated via real-time PCR and western blot analysis. The ABCC6-shRNA targeting sequence 5'-AGCTTAGACGCGAGAGGTCCATCAAGTCA-3' from base pairs 2688 to 2716 yielded significantly effective ABCC6 knockdown compared with HepG2 cells that had been stably transfected with scr-shRNA. The corresponding knockdown clone was used for further studies.

RT-PCR and real-time PCR
Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer's instructions. The RNA was transcribed to cDNA using oligo (dT) primers and the GeneAmp RNA PCR Core Kit (Applied Biosystems). cDNA was amplified via real-time PCR using PowerSYBR Green PCR Master Mix (Promega) on the 7500 Fast Real-Time PCR System (Applied Biosystems). The ABCC6 primers used in the real-time PCR experiments and the shRNA sequence used to knock down the ABCC6 gene do not map to the ABCC6 pseudogene sequence. All of the real-time PCR experiments were based on the use of primers spanning exon-exon junctions to eliminate any undesirable genomic DNA amplification. They were designed following these guidelines: GC content of 40-60%; product size of 80-150 bp; Tm of 60ºC, no secondary structures, no primer dimers (Table 1). To confirm PCR specificity, the PCR products were subjected to a melting-curve analysis. ABCC6 mRNA quantization was determined using the ΔΔCt method, The results were expressed as percentages of the values for the scr-shRNA knockdown sample.

Measurement of TNAP activity
Cells (1 x 10 6 ) were lysed with 0.1% Triton X-100 aqueous buffer, sonicated and centrifuged at 13,000 x rpm for 15 min at 4ºC to pellet the cellular debris. A volume of 160 μl assay buffer consisting of 50 mM Tris-HCl, 100 mM glycine and 2 mM MgCl 2 (pH 10.3) was added into each well of a 96-well plate, followed by the addition of 20 μl of cell extract and 20 μl of p-nitrophenylphosphate solution (5 mM in assay buffer). The reaction mixture was incubated at 37ºC for 30 min and stopped by adding 50 µl of 3 M NaOH. The OD 405nm was measured using a Multiscan Go (Thermo) plate reader. Alkaline phosphatase activity was expressed as micromoles of p-nitrophenol produced per minute and was normalized to the protein content of each sample.

Statistical analysis
Data were expressed as the means ± SD. The difference between any two groups was determined by ANOVA, using software R version 2.8.1 [19]. p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION
To investigate the possible mechanism by which ABCC6 could promote the ectopic mineralization process, several studies on ABCC6 -/transgenic mice [20,21] and on fibroblasts from PXE patients were previously performed [22]. However, these studies did not provide any insight into the pathways linking the ectopic mineralization processes to the absence of ABCC6 in the liver. In this study, the ABCC6 gene was stably knocked down in HepG2 cells and the effect on the RNA transcription level of a set of genes involved in tissue calcification was evaluated. The knockdown occurred at both the transcriptional and translational levels, as confirmed via real-time PCR (Fig. 1A) and by western blot analysis (Fig. 1B), respectively.  ABCC6 knockdown affected the expression of TNAP, a membrane-bound enzyme facing the extracellular space that promotes the mineralization process, altering the extracellular inorganic phosphate/pyrophosphate ratio toward the production of inorganic phosphate [23]. TNAP transcript levels in ABCC6 knockdown cells were significantly higher than in scr-shRNA-transfected cells (Fig. 2A). The protein level increased accordingly (Fig. 2B). Finally, TNAP activity also increased in ABCC6 knockdown cells by about 40-fold (Fig. 2C), indicating that the overexpressed protein is functionally active. Interestingly, the expression of the ecto-5'-nucleotidase (NT5E), also known as CD73, decreased in ABCC6 knockdown cells compared to the control at both the transcriptional and translational levels (Fig. 3). NT5E catalyzes the conversion of AMP to adenosine and inorganic phosphate [24] and is involved in ACDC [25]. This enzyme resides on the outer side of the plasma membrane of several cell types, supplying adenosine to receptors on the cell surface. Adenosine receptors trigger an intracellular signaling cascade that results in inhibition of TNAP activity. Thus, NT5E deficiency lowers pericellular adenosine concentrations, increasing alkaline phosphatase activity in the extracellular space and allowing calcification to occur [25,26]. The synergic and opposite regulation of TNAP and NT5E, described in ACDC, is consistent with our findings showing that TNAP overexpression is associated with NT5E downregulation in ABCC6 knockdown HepG2 cells. Indeed, we provided the first evidence that reduced levels of ABCC6 activity in liver cells can upregulate TNAP and downregulate NT5E expression in the same cells, providing the possible molecular mechanism underlying the overlap between PXE and ACDC clinical phenotypes.   4 shows the mRNA quantification of other genes involved in the mineralization processes. The Enpp1 gene encodes the enzyme hydrolyzing nucleoside 5' triphosphates to their corresponding monophosphates and pyrophosphate, which in turn inhibit the mineralization processes. Interestingly, the Enpp1 mRNA level did not significantly change in knockdown vs. control cells (data not shown). By contrast, the expression of OPN and Fetuin A genes, which encode two hepatic circulating proteins that are able to prevent deposition of hydroxyapatite, was downregulated in ABCC6 knockdown cells. This finding concurs with the decreased levels of Fetuin A observed in ABCC6 -/mouse serum [3] and PXE patients [27]. Moreover, it is evidence of a direct correlation between reduced levels of ABCC6 and OPN gene downregulation in liver cells.
A recent study has emphasized the correlation in expression between ABCC6 and OPN in a different system [28]. ABCC6 is localized at the basolateral surface of hepatocytes and, in analogy with other better-characterized members of the multidrug resistance protein (MRP) family, it is likely to serve as an efflux pump transporting substrates from hepatocytes to the circulation. It is conceivable that in the absence of functional ABCC6, serum becomes deficient in certain factors that inhibit ectopic mineralization [16]. The novelty of our data is that ABCC6 substrates also act on the liver cells, themselves modulating the expression of genes involved in the mineralization pathways, suggesting the molecular basis of the observations made in ABCC6 -/mice and PXE patients. It is indeed possible that, once secreted in the extracellular space, the ABCC6 substrates may act as autocrine factors promoting the expression of anti-mineralization genes and downregulating the expression of pro-mineralization genes. In agreement with this hypothesis, it was recently reported that cells overexpressing ABCC6 secrete an unidentified substrate that in turn induces a large efflux of nucleotide triphosphates providing the extracellular pyrophosphate, a potent inhibitor of mineralization [29]. Alternatively, it can be hypothesized that ABCC6 deficiency leads to an intracellular increase of substrates that might modulate the expression of genes involved in mineralization processes. Importantly, the pro-mineralization genes dysregulated in ABCC6-lacking hepatocytes encode both the proteins expressed on the extracellular face of the hepatocyte plasma membrane (TNAP, NT5E) and those released into the blood stream (OPN, Fetuin A). The two types of protein can exert their actions in the extracellular space and in extra-liver tissues respectively in the absence of clinically evident modifications in hepatocyte metabolism or function.

CONCLUSION
Our findings represent a significant step forward in the understanding of the pathogenic mechanism of ectopic mineralization processes associated with ABCC6. We have clarified that reduced levels of ABCC6 activity have a direct effect on the cells that normally express it. Moreover, our experiments demonstrate that ABCC6 knockdown HepG2 cells are a suitable model for recapitulating in vitro the ABCC6-lacking liver of PXE patients, thus shedding light on the complex pathogenesis of PXE.