Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

Abstract

Activation of V(1a) receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. We found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V(1a) receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and beta-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways.

Figure 1. Time-course of retinoblastoma protein (Rb) phosphorylation and protein expression of early immediate genes (c-Fos and Egr-1) and cyclin D 1 after AVP stimulation

A10 cells were incubated with 100 nM AVP for different time duration and cell extract subjected to Western blotting using specific anti-phosphorylated Rb (P-Rb), anti-c-Fos, anti-Egr-1 or anti-cyclin D1 and anti-actin which was used as a house keeping gene.

Figure 2. Time-course of transcriptional up-regulation of IEG after AVP or EGF stimulation of A-10 cells

A-10 cells were incubated with 100 nM AVP (A) or 1 nM EGF (B) for the different time duration; total RNA was prepared and subjected to semi-quantitative RT-PCR using specific primers for c-Fos, Egr-1 and GAPDH which was used as a house keeping gene. Data represent the mean±SE of at least three experiments.

Figure 3. The transcriptional up-regulation of IEG is mediated the V_1a vasopressin receptor

A-10 cells were incubated with 1 µM of the V_1a selective antagonist prior to the stimulation with 100 nM AVP. Total RNA was subjected to RT-PCR and products separated by agarose eletrophoresis. Figures are representative of at least three independent experiments.

Figure 4. PKC, intracellular Ca⁺² and β-arrestin 2 are involved in the transcriptional up-regulation of IEGs by transactivation of EGFR

A-10 cells were treated for 16 h. with PMA (A) to down-regulate PKC or with the specific inhibitor of PKC; Gö6983 (B) or with 10 or 50 µM BAPTA-AM (C) for 30 min prior to the AVP stimulation. A-10 cells were also transfected with a control siRNA (ct) or a siRNA for β-arrestin 2 (Arr) and then stimulated with 100 nM AVP. Then total RNA was submitted to RT-PCR and products separated by agarose electrophoresis. The down-regulation of PKCs by PMA completely inhibited the AVP-induced up-regulation of c-Fos and Egr-1 as well as Gö6983 and BAPTA at the highest dose (10 and 50 µM, respectively). The β-arrestin 2 silencing totally blocked the AVP-induced up-regulation of both genes. Figures are representative of at least three independent experiments.

Figure 5. The AVP-induced up-regulation of the IEGs is due to the transactivation of the EGFR

A-10 cells were treated with 1 µM of the EGFR tyrosine kinase inhibitor (A) or transfected with the dominant negative mutant HERCD533 (2, 4 and 6 µg of DNA) (B) prior to the stimulation with 100 nM AVP. Total RNA was subjected to RT-PCR and products separated by agarose eletrophoresis. Figures are representative of at least three independent experiments.

Figure 6. AVP-induced EGFR transactivation and transcriptional up-regulation of c-Fos is dependent upon the metalloproteinase and Rho kinase activities

A-10 cells were treated by two different metalloproteinase inhibitor; MMP inhibitor II (A) and GM6001 (B) or with a Rho kinase inhibitor (Y27632) (C) and then stimulated with 100 nM AVP. RT-PCR showed that both metalloproteinase and the Rho kinase inhibitors completely blocked the transcriptional up-regulation of c-Fos. Figures are representative of at least three independent experiments.

Figure 7. AVP-induced EGFR transactivation and transcriptional up-regulation of Egr-1 is dependent of the c-Src kinase activity and RAS/MEK pathway

A-10 cells were incubated with the c-Src inhibitor PP1 (A) or transfected with a c-Src dominant negative mutant (B) or with the dominant negative L61S186Ras (C) or incubated with the MEK inhibitor PD98059 (D) and then stimulated with AVP. RT-PCR showed that the inhibition of c-Src blocked the AVP-induced up-regulation of Egr-1 but it has no effect on that of c-Fos (A, B)). Similarly, the inhibition of Ras or MEK activities completely abrogated the Egr-1 up-regulation (C, D). Figures are representative of at least three independent experiments.

Figure 8. Neither pertussis toxin nor CT-βGRK2 has any affect on the AVP-mediated IEG up-regulation

A-10 cells were treated with Pertussis toxin (A) or transfected with plasmids encoding the catalytic subunit S1 (PTX S1) of the Pertussis toxin (B) or the c-terminus of the β-GRK2 (C). RT-PCR showed that neither Pertussis toxin nor the over-expression of S1 subunit or CT-β-GRK2 blocked the expression of these genes. Figures are representative of three independent experiments.

Figure 9. AVP-induced association of the V_1a vasopressin receptor and the EGFR

A-10 cells were transfected with the GFP-tagged V_1a vasopressin receptor and then were or not stimulated with 100 nM AVP. Cellular extracts were immunoprecipitated with anti-GFP/Protein A-agarose and subjected to Western blotting using anti-EGFR. After activation of the vasopressin receptor, a strong band, corresponding to the EGFR, can be seen.

Figure 10. Diagrammatic representation of the pathways involved in the IEG transcriptional up-regulation by the AVP-mediated EGFR transactivation