Abstract
The ATP-binding cassette sub-family C member 6 transporter (ABCC6) is an ATP dependent transporter mainly found in the basolateral plasma membrane of hepatic and kidney cells. Mutations in ABCC6 gene were associated to the Pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by a progressive ectopic calcification of elastic fibers in dermal, ocular, and vascular tissues. It is reported that the over-expression of ABCC6 in HEK293 cells results in the cellular efflux of ATP and other nucleoside triphosphates, which in turn are rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since PPi is an inhibitor of mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the ectopic mineralization, a typical feature of PXE. In the extracellular environment, ATP is converted, not only into pyrophosphate, but also into AMP by an ectonucleosidase, which in turn is transformed into adenosine and phosphate. ABCC6 protein is thus involved in the production of extracellular adenosine and therefore it could have a role in the activation of the purinergic system. In the liver, purinergic signaling has been shown to regulate key basic cellular functions. Our previous studies showed that in ABCC6 knockdown HepG2 cells the expression of some genes, related with the calcification processes, is dysregulated. In this study, experiments have been carried out in order to verify if ABCC6, besides supplying the pyrophosphate required to prevent the mineralization of soft tissues, also plays a role in the activation of the purinergic system. For this purpose, the transport activity of ABCC6 was blocked with Probenecid and the expression of ABCC6 and NT5E was analyzed with real time PCR and western blotting. The results of this study showed that both proteins are downregulated in the presence of Probenecid and upregulated in the presence of adenosine or ATP.
Highlights
The ectopic mineralization of the soft tissues represents a pathological condition characterized by the deposition of calcium phosphate complexes in the soft connective tissues
The ATP-binding cassette sub-family C member 6 transporter (ABCC6) activity in HepG2 cells was inhibited with Probenecid, a known ABC transporters inhibitor (Silverman et al, 2008; Ma et al, 2009), and the effect of adenosine and ATP was evaluated on cluster of differentiation 73 (CD73) expression
In order to verify the effective inhibition of Probenecid on ABCC6 transport activity, we evaluated its effect on the efflux of doxorubicin, a known substrate carried by ABCC6, from stably ABCC6-overexpressing Human embryonic kidney cells 293 (HEK293) cells
Summary
The ectopic mineralization of the soft tissues represents a pathological condition characterized by the deposition of calcium phosphate complexes in the soft connective tissues. Scheme 1 shows how the ATP released from cells is converted by a protein encoded by an ecto-nucleotide pyrophosphatase/phosphodiesterase 1 gene (ENPP1) into PPi and AMP, and the latter into adenosine and phosphate by an extracellular nucleotidase known as CD73. In this paper the term purinergic pathway will be used to indicate any activity related to the presence of extracellular nucleotides and purine nucleosides This scheme has been shared by various research groups and pyrophosphate has been proposed as a drug to treat PXE (Pomozi et al, 2017; Kranenburg et al, 2018), various aspects have not yet been clarified. It is not clear whether ABCC6 contributes to the activation of the purinergic system, which is able to regulate key basic cellular functions such as glucose/lipid metabolism, protein synthesis, and ionic secretion, affecting homeostatic
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