Functional expression and intracellular signaling of UTP-sensitive P2Y receptors in theca-interstitial cells

Abstract

Background: Purinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated. UTP-sensitive P2Y receptor activity may regulate cell proliferation. The aim of the present work was to study the functional expression of these receptors in theca/interstitial cells (TIC).

Methods: TIC were isolated by centrifugation in a Percoll gradient. P2Y receptors and cellular markers in TIC were detected by RT-PCR and Western blot. Intracellular calcium mobilization induced by purinergic drugs was evaluated by fluorescence microscopy, phosphorylation of MAPK p44/p42 and of cAMP response element binding protein (CREB) was determined by Western blot and proliferation was quantified by [3H]-thymidine incorporation into DNA.

Results: RT-PCR showed expression of p2y2r and p2y6r transcripts, expression of the corresponding proteins was confirmed. UTP and UDP, agonists for P2Y2 and P2Y6 receptors, induced an intracellular calcium increase with a maximum of more than 400% and 200% of basal level, respectively. The response elicited by UTP had an EC50 of 3.5 +/- 1.01 microM, while that for UDP was 3.24 +/- 0.82 microM. To explore components of the pathway activated by these receptors, we evaluated the phosphorylation induced by UTP or UDP of MAPK p44 and p42. It was found that UTP increased MAPK phosphorylation by up to 550% with an EC50 of 3.34 +/- 0.92 and 1.41 +/- 0.67 microM, for p44 and p42, respectively; these increases were blocked by suramin. UDP also induced p44/p42 phosphorylation, but at high concentrations. Phosphorylation of p44/p42 was dependent on PKC and intracellular calcium. To explore possible roles of this pathway in cell physiology, cell proliferation and hCG-induced CREB-phosphorylation assays were performed; results showed that agonists increased cell proliferation and prevented CREB-phosphorylation.

Conclusion: Here, it is shown that UTP-sensitive P2Y receptors are expressed in cultured TIC and that these receptors had the ability to activate mitogenic signaling pathways and to promote cell proliferation, as well as to prevent CREB-phosphorylation by hCG. Regulation of TIC proliferation and steroidogenesis is relevant in ovarian pathophysiology since theca hyperplasia is involved in polycystic ovarian syndrome. Purinergic receptors described might represent an important new set of molecular therapeutic targets.

Figure 1

p2y2r, p2y4r, and p2y6r expression in mouse theca cells. (A) RNA from cultured theca cells (1), whole ovary (2), heart (3), and brain (4) was reverse transcribed and amplified by PCR for p2Y2, p2Y4, p2Y6, cyp11A, cyp 17A, star, fshr, and β-actin using specific oligonucleotides to detect transcript expression. Controls in lanes (5) and (6) correspond to a PCR reaction of a theca cell RNA sample without reverse transcriptase and to the reaction mix without a cDNA template, respectively. Amplicon length is indicated in base pairs (bp). Each amplification assay was repeated in 3-5 independent cultures. (B) CREB phosphorylation evaluated by Western blot from TIC cultures in basal conditions or stimulated for 10 min by either 2 IU hCG or 1 ng/ml FSH. The 43-kDa band was analyzed by densitometry, and the result (mean ± S.E.M) of three determinations from independent cultures was plotted (*p < 0.01). PARP protein was used as gel loading control. (C) P2Y2 and P2Y6 receptors detected by Western blot (WB), from freshly isolated TIC homogenates directly (P2Y2) or after immunoprecipitation (IP) (P2Y6).

Figure 2

Dose-response (D-R) relationships and time course of intracellular Ca²⁺ responses generated by ATP, UTP, or UDP in TIC cells. (A) Images show a typical fluorescence increase of theca cells loaded with Fluo4-AM dye upon stimulation with 100 μM ATP at 0, 10, or 40 s. (B) D-R relationship (left panel) for the intracellular Ca²⁺ increase elicited by ATP, expressed as a percent of the basal level of arbitrary units of fluorescence (AUF), and time course of the response elicited by 1 mM ATP (right panel). (C) and (D) show experiments similar to those in (B) but applying UTP or UDP, respectively, instead of ATP. (E) D-R curves of the theca cell response to UTP (left) or UDP (right) in Ca²⁺-free Krebs. The stimulus was applied at the time indicated by an arrow and was maintained to the end of the recording. Data points represent the mean ± S.E.M. from 25 to 40 cells in each of 3 independent experiments.

Figure 3

D-R relationships and time course for the phosphorylation response elicited by UTP or UDP on p44 and p42 MAPK. (A) Phosphorylated isoforms of p44 (black dots) or p42 (white dots) were immunodetected (Western blot) in samples of theca cells that were stimulated with various UTP concentrations. The band intensities (OD) are expressed as a percent of the basal level, and the values were used to plot the D-R relationship. (B) Time course of the p44 and p42 phosphorylation process elicited by 10 μM UTP. (C) D-R for phosphorylation elicited by UDP in experiments similar to those in (A). (D) Time course of p44 and p42 phosphorylation elicited by 1 mM UDP. Data points are the mean ± S.E.M. of three independent experiments.

Figure 4

p44 and p42 MAPK phosphorylation induced by UTP is blocked by suramin but not by PPADS. Cultures of mouse theca cells were incubated for 30 min with suramin (A) or PPADS (B) at the indicated concentrations; subsequently, 10 μM UTP was applied for 5 min, and p44 (black dots) and p42 (white dots) MAPK phosphorylation was detected by Western blot. Data points are the mean ± S.E.M. of three independent experiments.

Figure 5

Effect of protein kinase inhibitors on p44 and p42 MAPK phosphorylation induced by UTP. (A) Western blot detection of phosphorylated p44 (white columns) and p42 (black columns) MAPK in the presence of 100 nM wortmanin or 250 nM staurosporine in theca cells stimulated or not with 10 μM UTP (5 min). In (B), down regulation of PKC was induced by an overnight (18 h) cell incubation with 1 μM PMA, and then phosphorylation of p44 and p42 MAPK was assayed for cells treated or not with 10 μM UTP (5 min). Data points represent the average optical density (± S.E.M.) of three independent experiments expressed as a percent of the basal level (*p < 0.05 vs. UTP, one-way ANOVA and Bonferroni's test).

Figure 6

Effect of intracellular BAPTA loading on p44 and p42 MAPK phosphorylation induced by UTP. Cultures of mouse theca cells were preincubated for 90 min with 10 μM BAPTA-AM; cells were then stimulated for 5 min with 10 μM UTP and assayed for phosphorylation of MAPK p44 (white columns) and p42 (black columns). Amplitude bars in the plot represent the mean ± S.E.M. of three independent experiments (*p < 0.05 vs. UTP, one-way ANOVA and Bonferroni's test).

Figure 7

Proliferative response and inhibition of hCG-induced CREB phosphorylation by P2Y agonists in mouse theca cells. Theca cells were stimulated for 48 h with UTP, UDP, or ATP using the indicated concentrations (log M); controls corresponded to cells incubated in media supplemented with 0.1% serum (basal) or with 10% FBS (S). Cell proliferation was evaluated by [³H]-thymidine incorporation, data are the mean ± S.E.M. of 4 independent determinations. B) Time-course of CREB phosphorylation in TIC induced by 2 IU hCG; the 43-kDa band was quantified as in Figure 1B showing that maximal effect was reached in 10-15 min of incubation. C) CREB-phosphorylation elicited by 2 IU hCG in TIC incubated for 10 min in basal conditions and in the presence of 0, 10, or 100 μM UTP; the 43-kDa band was quantified as in B), data are from 3 independent experiments in triplicate. (*p < 0.05 vs. basal, one-way ANOVA and Bonferroni's test).