**7. Conclusions**

Different hypotheses were given to elucidate the physiological substrates for the ABCC6 transporter and its pathological involvement in PXE. However, mechanistic details for *ABCC6* mutations leading to ectopic mineralization were yet to be resolved [6,47,48]. In our studies, we not only investigated the structural components of ABCC6 transporter

but also lighten the mechanisms, which might be involved in ectopic mineralization. In our experiments, knockdown of *ABCC6* or its inhibition by probenecid decreased the extracellular ATP concentration and altered the expression of *NT5E* and *TNAP*. By these findings, we can propose that the lack of ABCC6 transport activity could lead to a promineralizing state through alteration of extracellular purine metabolites, which ultimately affect TNAP enzymatic activity.

Moreover, in both knockdown and probenecid-treated HepG2 cells, the expression of lamin was found decreased, which suggests that reduced ABCC6 transport activity also leads to cell senescence in PXE and can be beneficial to prevent cancer progression. Interestingly, in both the knockdown and probenecid-treated HepG2 cells, we found a decrease in migration rate, which is restored after ATP administration.

Collectively, these findings suggest that the ABCC6 transporter is not only required to maintain some important circulatory factors like PPi, but is also required to maintain proper functioning of other factors, which are involved in the conversion of extracellular nucleotides, and to feed purine pool to maintain homeostasis between Pi and PPi ratio. Moreover, our studies strengthen the idea that the ABCC6 transporter is not only involved in PXE pathophysiology, but can also be considered a target for anti-cancer therapy.

**Author Contributions:** Conceptualization, F.B. and A.O.; data curation, A.O.; writing—original draft preparation, V.A. and P.K.; writing—review and editing, P.K. and M.A.C.M.; supervision, F.B.; funding acquisition, F.B. and A.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by INBIOMED PROJECT (MIUR, ARS01\_01081).

**Conflicts of Interest:** The authors declare no conflict of interest.
