Extracellular ATP Regulates CD73 and ABCC6 Expression in HepG2 Cells

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.

Inhibition of ABCC6 Transporter Modifies Cytoskeleton and Reduces Motility of HepG2 Cells via Purinergic Pathway

“…In probenecid-treated HepG2 cells, we observed an almost complete lack of filopodia, or, if present, they were very short ( Figure 5B, stars) compared to the control cells ( Figure 5A, arrows); as observed in the Abcc6-shRNA cells, the addition of ATP ( Figure 5C, arrows) or adenosine ( Figure 5D, arrows) restored the normal architecture of filipodia. Previously, we showed that ATP as well as adenosine reverted the effect of probenecid on the downregulation of the protein level of CD73, restoring its protein level to that of the control conditions and even above [18]. The finding that adenosine alone mimicked the effect of ATP in this experiment suggests that the ability of ATP to revert the effect of probenecid on cytoskeleton architecture is likely dependent on its ability to be finally transformed in adenosine by CD73.…”

“…CD73 is considered a key regulator in some cancer processes such as drug resistance, tumor metastasis, and tumor angiogenesis [12,13], therefore is an excellent candidate for cancer therapy [14][15][16]. In previous studies, we have reported that knockdown of Abcc6 in hepatocarcinoma cancer cells (HepG2), or the inhibition of its activity lead to the downregulation of NT5E gene, which codifies for the CD73 protein [17][18][19]. Taken together, these data suggest a close correlation between ABCC6 and CD73.…”

“…In HepG2 cells, we have already observed a correlation between a decrease in the expression levels of CD73 upon Abcc6 silencing [17] or probenecid treatment [18]. CD73 is the main source of extracellular adenosine in all tissues and is a key regulator in some tumor processes such as invasion, migration, and metastasis [12,13].…”

“…Probenecid and adenosine were dissolved in dimethyl sulfoxide (DMSO) at 30 mg/mL and 80 mg/mL, respectively. Based on our previous cytotoxicity assays, 250 μM probenecid was selected for the experiments [ 18 ]. The final concentration of DMSO did not exceed 0.25% v / v ; control cells were treated at the same final percentage of DMSO (vehicle).…”

“…There are multiple systems through which ATP can leave cells including ABCC6 [8,9]. It has also been shown that probenecid inhibits both the expression [18] and the activity of the ABCC6 transporter [33]. In order to evaluate the contribution of ABCC6 to the outflow of ATP from HepG2, we measured the amount of ATP released from the silenced and probenecid-treated cells in comparison with MDA-MB-231 cells, which, compared to HepG2, have a lower expression of Abcc6 (Figure 1).…”