**4. The Decrease or Loss of ABCC6 Functionality Changes Extracellular Environment**

− − In early pathological investigations of PXE, Iliás et al. in 2002 proposed that ABCC6 is a transporter of organic anions and actively transports glutathione conjugates, including leukotriene C4 and N-ethylmaleimide S-glutathione (NEM-GS), and their abolishment due to missense mutation in ABCC6 gene is responsible for PXE [26,27]. In the same line of thinking, Borst and co-workers in 2008 proposed that ABCC6 transporter mediates the efflux of vitamin-K as a glutathione or glucuronide conjugate and is involved to prevent calcification of soft tissues [28]. However, in the further investigations, it was found that ABCC6 is not a transporter of vitamin-K, as its administration in PXE mouse model (ABCC6−/−) was not able to prevent the mineralization [29,30]. As discussed above, Jansen et al. in 2013 proposed that ABCC6 transporter mediates the efflux of ATP and indirectly produces PPi to prevent ectopic mineralization [2]. However, we have demonstrated that PXE is a complex metabolic disease with the reprogramming of crucial genetic factors in the absence of ABCC6 transporter activity [31–33].

In order to better understand the pathomechanism of PXE (Figure 4), we stably knocked down the *ABCC6* gene in HepG2 cells by using shRNA, and its associated transcriptional/genetic changes were studied. We first examined the production of reactive oxygen species (ROS), which are supposed to increase according to the previous PXE fibroblast studies [34]. On the contrary, in the *ABCC6* knockdown HepG2 cells, the ratio of GSH/GSSG has been found to be increased whereas a significant decrease in ROS level was observed, which means that knockdown cells resembled the reductive stress, which is also required by proliferating cells.

However, we found significant delay in G1 to S transition and slower cell growth in ABCC6 knockdown HepG2 cells (Figure 5). In addition, expression of cyclin-dependent kinase inhibitor (CDKI) p21, which negatively regulates the activity of CDK and is required for the cell entry into the different cycle phases, was found increased in knockdown cells. Moreover, the expression of lamin A/C, which is required to maintain the strength of the nucleus and is pathologically involved in aging process, was decreased in those cells [33].

**Figure 4.** Proposed pathomechanism of PXE. ABCC6 transporter mediates the efflux of ATP, which is metabolized by some ecto-nucleotidases (such as ENPP1) in AMP, which in turn is converted in adenosine and Pi by CD73. In the extracellular milieu, nucleotides regulate the activity of TNAP through the purinergic pathway and prevent the ectopic mineralization [32]. ABCC6, ATP-binding cassette, sub-family C, member 6; ENPP1, ecto-nucleotide pyrophosphatase/phosphodiesterase type I; CD73, cluster of differentiation 73; TNAP, tissue non-specific alkaline phosphatase; Pi, inorganic phosphate; PPi, inorganic pyrophosphate [35].

β β ′ β β **Figure 5.** Pictorial presentation of senescent-like phenotype in ABCC6 knockdown HepG2 cell. (**A**)—For the cell cycle analysis, the cells were synchronized at the G1 phase by serum deprivation for 24 h, restimulated with serum for 24 h, and analyzed using flow cytometry after BrDU and PI staining. The percentage of control (scr-shRNA) and ABCC6 knockdown cells (ABCC6-shRNA) in G0/G1 was recorded. (**B**)—Representative images (40 × magnification) of senescence-associated β-galactosidase staining in control and ABCC6 knockdown cells. (**C**)—Quantitative analysis of positive β-galactosidase-stained cells. Data were generated from three independent experiments performed in triplicate and are shown as means ± SD. Statistical analysis was performed using unpaired Student's *t* test: \*\* *p* < 0.01 and \*\*\* *p* < 0.001 [33].

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Interestingly, knockdown HepG2 cells were shown to have a decreased expression of the ecto-5′ -nucleotidase (*NT5E* or CD73), which regulates the conversion of AMP to adenosine and Pi (inorganic phosphate). Additionally, nonspecific alkaline phosphatase (TNAP), whose activity normally maintains Pi/iPP ratio accelerating the mineralization, was found to be increased in knockdown HepG2 cells. These results suggest that the absence of transporter activity in the hepatic cells decreases the *NT5E* expression and increases the pro-mineralizing TNAP activity, which is also clinically found in patients with arterial calcifications due to deficiency in CD73 (ACDC) [31]. It is clear from the knockdown study that the absence of transporter activity leads to alteration in gene expression, which is required to provide PPi to prevent mineralization. However, in a further study, we also found that ABCC6 plays a crucial role to activate the purinergic pathway, which is important to maintain proper cellular function. In this study, pharmacological inhibition of ABCC6 by probenecid down-regulated the expression of CD73. On the contrary, the expression of CD73 was found increased after the application of adenosine and ATP, which strengthens the idea that ABCC6 not only mediates the efflux of ATP but also regulates the purinergic system [35].
