We have observed that, as shown previously, 15, 16 the level of phospho-ERK at rest is higher in Pkd2KO cells, compared to controls, and, more impressive, is the difference between controls and Pkd2KO cells upon acute reduction in ER [Ca2+] by exposure to thapsigargin or TPEN (Figs. 4 and 5). The advantage of TPEN as a tool to acutely decrease ER [Ca2+] in this type of experiment is that, unlike thapsigargin or agonist that LEE011 cell line produce IP3, no rise in [Ca2+]c is generated, an event that may interfere with

the observed response. Of interest, unlike Pkd2KO cells, the small increase in phospho-ERK caused by ER Ca2+ depletion in controls is not accompanied by an increase in VEGF and HIF expression. The effect on ERK phosphorylation is clearly not secondary to an ER stress response caused by thapsigargin-induced depletion of ER Ca2+, because the

expression of three well-known markers of ER stress (e.g., BiP, ATF6, and PERK) after treatment with thapsigargin was not different between Pkd2KO selleck inhibitor and WT cell (see Supporting Fig. 4). Of note, ERK1/2 phosphorylation (at rest and after ER Ca2+ depletion) was PKA dependent, as shown by the inhibition by PKI. Hofer et al. have recently demonstrated that ER [Ca2+] levels regulate cAMP production through a STIM-1-dependent process. 20, 28 This mechanism, indeed, depends on the translocation of STIM1 and its ability to stimulate unknown plasma-membrane AC isoform(s). Our data show that, in Pkd2KO cells, see more not only depletion of ER Ca2+ with TPEN (or thapsigargin) increases cAMP production and PKA-dependent ERK1/2 phosphorylation, but also that PKA-dependent ERK1/2 activation is inhibited by antagonizing STIM-1 (with ML-9 and 2-APB 20) translocation. 20 The

amount of cAMP produced by a given cell results from the highly integrated function of several isoforms of ACs that respond to different stimuli and second messengers. 41 At least seven different ACs are expressed in cholangiocytes. 22 AC6 is localized also in the cilia and is involved in shear stress-induced signaling 19 and in the formation of gap junction and [Ca2+]c regulation in endothelial cells. 42 We, here, show that AC6 mediates most, if not all, SOcAMP in Pkd2KO cholangiocytes and in WT cholangiocytes after chronic ER Ca2+ depletion. On the contrary, the Ca2+ stimulated AC8 appears not to be involved in SO-cAMP response. Altogether, these data demonstrate that PC2 plays a key role in regulating Ca2+ and cAMP homeostasis in cholangiocytes.