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First published online December 5, 2007
doi: 10.1242/10.1242/jcs.03490

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Nucleotide P2Y₁ receptor regulates EGF receptor mitogenic signaling and expression in epithelial cells

¹ Centro de Regulación Celular y Patología JV Luco, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, 8330033, Santiago, Chile
² Millennium Institute for Fundamental and Applied Biology (MIFAB), 7780344 Santiago, Chile
³ Departamento de Inmunología Clínica y Reumatología, Facultad Medicina, Pontificia Universidad Católica de Chile, 8330033, Santiago, Chile
⁴ Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
⁵ INSPIRE Pharmaceuticals Inc., Durham, NC 27703, USA

View larger version (47K): [in this window] [in a new window]   Fig. 1. P2Y1R and EGFR expression in non-tumoral epithelial FRT and MDCK cells and in tumoral HeLa cells. Cell extracts analyzed by immunoblot and ECL with polyclonal antibodies against P2Y1R and the EGFR show readily detectable expression levels of both receptors in HeLa and FRT cells (lanes 1 and 2) but not in MDCK cells (lane 3). The EGFR, but not the P2Y1R, becomes apparent in MDCK extracts when the film is exposed for a longer time (lane 4). Molecular size markers (in kDa) are indicated on the left.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Nucleotides stimulate cell proliferation through P2Y1R. P2Y1R agonists, 2-MeSADP (A) and ADP (B), increase cell proliferation in a concentration-dependent manner (n=4-8). [3H]thymidine incorporation assays show that HeLa and FRT cells pretreated for 1 hour with different concentrations of the agonists increase their proliferation rate. The EC50 of the agonists is indicated. (C) [3H]thymidine incorporation after 1 hour of stimulation with 1 µM P2YR agonist. Compared with P2Y1R agonists 2-MeSADP, ATP and ADP, which increase cell proliferation, the P2Y2R agonist UTP shows no effect. The P2Y6R agonist UDP induces cell proliferation only in FRT cells. Results are normalized against control cells. Values are the means ± s.e.m. *P<0.05, **P<0.01, Dunnett's test compared with controls (n=5). (D) Cell proliferation induced by 1 µM 2-MeSADP is selectively mediated by P2Y1R, since it was abrogated by the selective P2Y1R antagonist MRS2179, which in turn does not affect the proliferative response to 10 µM UDP, as shown for FRT cells (n=3-4). Only the carrier culture medium was added for controls.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effect of EGF and FBS on FRT and HeLa cell proliferation. Serum-arrested cells were incubated with different concentrations of EGF for 1 hour (A) or with 10% FBS for either 1 hour before, or 16 hours during [3H]thymidine incubation (B). Only FRT cells show a proliferative response to EGF, indicating concentration dependency. None of the cells responded to just 1 hour of FBS, contrasting with the effect of a long incubation period, which could override the proliferative arrest (n=4-6).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Cell-released nucleotides contribute to basal cell proliferation. Steady-state concentration of extracellular ATP and its metabolites measured in the medium of FRT and HeLa cells in control conditions (A; n=10) and after a 1-hour incubation with 2 U/ml apyrase (B; n=5). ATP and ADP induce cell proliferation and both become undetectable after apyrase treatment. (C) Endogenous nucleotides acting through P2Y1Rs participate in the regulation of basal cell proliferation (n=5). Basal [3H]thymidine incorporation in FRT and HeLa cells measured after 16 hours of incubation decreased by about 15-25% in the presence of 1 µM MRS2179 (P2Y1R antagonist) or 2 U/ml apyrase. All results were normalized against the control. Values are the means ± s.e.m. *P<0.05, **P<0.01, Dunnett's test compared with control. Carrier culture medium only was added for the control.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Congruence between time dependency of P2Y1R stimulation for cell proliferation and half-life of injury-released nucleotides. (A) Stimulation time-period necessary for P2Y1R-induced cell proliferation. FRT cells were incubated with 1 µM 2-MeSADP for the indicated time periods and then the ligand was removed and the cells incubated for 16 hours with [3H]thymidine. The proliferative response requires a minimal stimulation time of 10 minutes, reaching a maximum by 120 minutes and decreases thereafter (n=4); (B) Changes in cyclin D levels detected by immunoblot. FRT cells were incubated either with carrier culture medium alone (Control, C) or with 1 µM 2-MeSADP for the indicated times. Stimulation for as little as 15 minutes leads to increased levels of cyclin D, analyzed 16 hours later. The quantification of three separate assays normalized with respect to actin is shown in the graph. (C) Half-life of nucleotides released from wounded cultured cells. Extracellular ATP and ADP concentrations show a dramatic increase after cell injury and then gradually decrease. ADP maintained higher levels for longer periods – enough to induce P2Y1R-mediated cell proliferation (n=4). Values are the means ± s.e.m. *P<0.05, **P<0.01; Dunnett's test against control conditions without agonist.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Stimulation of the P2Y1R transactivates the EGFR pathway. (A) 2-MeSADP increases the EGFR tyrosine phosphorylation in a concentration-dependent manner. Quiescent FRT and HeLa cells were treated for 5 minutes with either 1 or 10 nM 2-MeSADP or 1 nM EGF, as indicated. The EGFR was then immunoprecipitated, resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine monoclonal antibody 4G10 to detect the tyrosine phosphorylated EGFR (EGFR-P). After stripping, blotted membranes were incubated with polyclonal antibody EGFR984 for total EGFR detection. EGFR-P is clearly more intense than in control cells (`C') without agonist stimulation. (B) 2-MeSADP increases ERK1/2 activation. Quiescent cells treated for the indicated time periods either with carrier culture medium alone (Control, `C') or with 10 nM 2-MeSADP or 1 nM EGF were analyzed for ERK activation by immunoblot against total ERK or phosphorylated ERK (ERK-P). Each graph corresponds to quantification of three independent experiments, normalized with respect to the corresponding total EGFR or ERK protein in each lane. Values are the means ± s.e.m. *P<0.05, **P<0.01; Dunnett's test compared with control conditions without agonist.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Cell proliferation mediated by P2Y1R stimulation involves EGFR transactivation, PKC, Src and metalloprotease activity. (A) Induction of cell proliferation mediated by P2Y1R stimulation requires EGFR activation. The EGFR tyrosine kinase blocker AG1478 inhibits the proliferation of FRT cells induced by either 1 µM 2-MeSADP or 10 nM EGF in a concentration-dependent manner (n=4). (B) HeLa cells pre-incubated for 30 minutes in the absence or presence of 1 µM of MRS2179 (P2Y1R antagonist), Ro 318220 (non-selective PKC inhibitor), PP2 (Src kinase inhibitor), AG1478 (blocker of EGFR tyrosine-kinase activity) or Ilomastat (metalloprotease inhibitor), were then stimulated with 1 µM 2-MeSADP for 1 hour in the presence of each inhibitor. Results are normalized against control cells incubated only with the agonist. Values are the means ± s.e.m. *P<0.05, **P<0.01; Dunnett's test compared with control (n=3-6). (C) ERK1/2 activation in response to P2Y1R agonist depends on EGFR activity. The EGFR tyrosine kinase blocker AG1478 (100 nM) completely abrogates ERK1/2 activation induced by 2-MeSADP (10 nM, 5 minutes), in FRT and HeLa cells. The immunoblot is representative of three independent experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 8. P2Y1R ectopically expressed in MDCK cells lacking this receptor. (A) Measures of intracellular calcium changes show that MDCK cells do not respond to P2Y1R agonists (n=6-8). ATP and UTP, but neither ADP nor 2-MeSADP, provoke rises in intracellular Ca2+ levels in MDCK cells, further indicating that these cells do not express P2Y1R. Data are means ± s.e.m. (B) MDCK cells permanently transfected to express the P2Y1R (MDCK/P2Y1R). Cells were selected for ectopic P2Y1R expression levels lower than those of the endogenous receptor of FRT cells.

View larger version (28K): [in this window] [in a new window]   Fig. 9. MDCK cells ectopically expressing the P2Y1R display lower doubling times and EGFR-dependent increased proliferation rates. (A) Viability curves for wild-type (wt) MDCK cells, or cells stably transfected with either the P2Y1R (MDCK/P2Y1R) or -opioid receptor (MDCK/-opioid). The number of cells increases with higher slope and reaches a higher maximum for MDCK/P2Y1R cells, whereas cells expressing ectopic -opioid receptor behave like wild-type MDCK cells. MDCK P2Y1R cells also show a reduced doubling time. Values are the means ± s.e.m. (n=4-6). (B) Basal proliferation rate of MDCK P2Y1R is higher than wild-type cells and sensitive to 0.1 µM AG1478, thus indicating a dependency on EGFR activity. In MDCK P2Y1R cells, inhibition of EGFR activity significantly decreased the [3H]thymidine incorporation. Cells were treated with 100 nM AG1478 during 16 hours of [3H]thymidine incorporation. Values are the means ± s.e.m. *P<0.05, **P<0.01; Dunnett's test compared with control wild-type cells (n=6). (C) The selective P2Y1R agonist 2-MeSADP increases proliferation of MDCK/P2Y1R cells in a concentration-dependent manner. Cells were treated with exogenous 2-MeSADP for 1 hour before [3H]thymidine incorporation. Values are the means ± s.e.m. **P<0.01; Dunnett's test compared with untreated cells (n=3). (D) P2Y1R transactivates the EGFR in MDCK P2Y1R cells. The immunoblot shows an increased EGFR tyrosine phosphorylation in cells treated with either 10 nM 2-MeSADP or 1 nM EGF for 5 minutes, quantified with respect to total EGFR mass and depicted below each lane. Only the carrier culture medium was added for controls.

View larger version (21K): [in this window] [in a new window]   Fig. 10. MDCK P21R (MDCK/P21R) cells display increased expression levels of the endogenous EGFR. (A) Ectopic expression of P2Y1R increases EGFR levels. Immunoblots of total cell extracts, densitometrically quantified in four independent experiments and normalized with respect to actin show a threefold increased level of EGFR in MDCK P2Y1R cells. (B) Ectopic expression of P2Y1R increases EGFR biosynthesis. Newly synthesized EGFR was immunoprecipitated from cells metabolically labeled for 2 hours with [35S]Met/Cys (200 µCi/ml). Graph shows twofold increase in synthesis in MDCK P2Y1R cells (n=3). For A and B, values are the means ± s.e.m. ***P<0.001; paired t-test compared with wild-type cells. (C) Increased EGFR expression in MDCK P2Y1R cells depends on EGFR and PKC activity. Cells were treated with either 100 nM AG1478 or 10 nM Ro318220 for 6-18 hours before and during the metabolic labeling. Graph show the densitometric quantification of three independent experiments. Values are the means ± s.e.m. *P<0.05, **P<0.01; Dunnett's test compared with untreated cells.

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