Stimulation of α1a adrenergic receptors induces cellular proliferation or antiproliferative hypertrophy dependent solely on agonist concentration.

Stimulation of α1aAdrenergic Receptors (ARs) is known to have anti-proliferative and hypertrophic effects; however, some studies also suggests this receptor can increase cell proliferation. Surprisingly, we find the α1aAR expressed in rat-1 fibroblasts can produce either phenotype, depending exclusively on agonist concentration. Stimulation of the α1aAR by high dose phenylephrine (>10(-7) M) induces an antiproliferative, hypertrophic response accompanied by robust and extended p38 activation. Inhibition of p38 with SB203580 prevented the antiproliferative response, while inhibition of Erk or Jnk had no effect. In stark contrast, stimulation of the α1aAR with low dose phenylephrine (∼10(-8) M) induced an Erk-dependent increase in cellular proliferation. Agonist-induced Erk phosphorylation was preceded by rapid FGFR and EGFR transactivation; however, only EGFR inhibition blocked Erk activation and proliferation. The general matrix metalloprotease inhibitor, GM6001, blocked agonist induced Erk activation within seconds, strongly suggesting EGFR activation involved extracellular triple membrane pass signaling. Erk activation required little Ca(2+) release and was blocked by PLCβ or PKC inhibition but not by intracellular Ca(2+) chelation, suggesting Ca(2+) independent activation of novel PKC isoforms. In contrast, Ca(2+) release was essential for PI3K/Akt activation, which was acutely maximal at non-proliferative doses of agonist. Remarkably, our data suggests EGFR transactivation leading to Erk induced proliferation has the lowest activation threshold of any α1aAR response. The ability of α1aARs to induce proliferation are discussed in light of evidence suggesting antagonistic growth responses reflect native α1aAR function

The antiproliferative effects of high dose PE require p38 activity.

<p>α_1aAR expressing cells placed into SF media were pretreated for 30 min with vehicle (0.1% DMSO), 20 µM PD98059 (MEK/Erk inhibitor), 10 µM SB203580 (p38 inhibitor), 10 µM SP600125 (Jnk inhibitor), or 10 µM Prazosin (α₁AR) and then stimulated with 10 µM PE for 24 h prior to (<b>A</b>) cell counting or (<b>B</b>) protein assay and estimation of cellular protein content. Values are the mean±SEM (n≥3); (*), P<0.05, compared to vehicle or inhibitor alone.</p

Stimulation of α_1aAR results in strong Ca²⁺ dependent Akt activation followed by chronic Akt downregulation at high PE concentrations.

<p>α_1aAR expressing cells were placed into SF media prior to pretreatment and stimulation with PE. Representative Western Analysis showing the PE dose dependence of (<b>A</b>) acute Akt phosphorylation from 2–10 min., (<b>B</b>) intermediate Akt and Erk phosphorylation at 1 hour and (<b>C</b>) chronic Akt dephosphorylation at 24 hours. (<b>D</b>) Treatment of cells with 0.1% DMSO vehical (−) or 20 µM BAPTA-AM (+) for 10 min. prior to 5 min stimulation with various concentration of PE demonstrate an important role for intracellular Ca²⁺ release in Akt activation that contrasts with Erk behavior.</p

Western analysis of high dose α_1aAR induced MAPK activation using phospho-specific antibodies for activated kinases.

<p>α_1aAR expressing cells were placed into SF media for 3–4 hours, treated with PE for the indicated time and collected by direct addition of SDS sample buffer. (<b>A</b>) Time course following treatment with 10⁻⁵ M PE shows strong p38 activation, transient Jnk activation and acute inhibition of already low basal ERK activity followed by transient partial recovery. (<b>B</b>) Dose response of PE and MAPK phosphorylation. Following transfer to serum free media the cells were treated for 15 or 2 min with 0 to 10⁻⁵ M PE as indicated.</p

Low dose α_1aAR stimulation activates growth-associated signaling.

<p>α_1aAR expressing cells were placed into SF media prior to pretreatment and stimulation. (<b>A</b>) Prior to counting, cells were pretreated for 30 min with vehicle (0.1% DMSO), 20 µM PD98059 (MEK/Erk inhibitor), 10 µM SB203580 (p38 inhibitor), 10 µM SP600125 (Jnk inhibitor), or 10 µM Prazosin (α₁AR) and then stimulated with 10 µM PE for 24 h. Values are the mean±SEM (n≥3); (*), P<0.05, compared to vehicle or inhibitor alone. (<b>B</b>) 20 µM PD98059 blocks PE-induced Erk phosphorylation. (<b>C</b>) Erk is acutely activated within 2 min of 10 µM PE addition and remains robustly phosphorylated for 15–30 minutes. Prior to Erk activation, stimulation induces sustained phosphorylation of EGFR and FGFR and acute transient phosphorylation of Akt at T308 downstream of PI3K/Akt signaling. Total Akt provides the load control for this multiply reprobed blot. In the lower panel, differences in temporal activation patterns are shown following semi-quantitative analysis of Erk, FGFR and Akt phosphorylation. Results represent the fold increase in phosphorylation relative to basal signal (Erk, FGFR3) or for Akt, background (arbitrary) signal.</p

EGFR transactivation through a TMP mechanism is required for low dose α_1aAR-induced proliferation and Erk activation.

<p>α_1aAR expressing cells were placed into SF media prior to pretreament and addition of PE. (<b>A</b>) Prior to counting, cells were pretreated for 30 min with 0.1% DMSO (Veh), 20 µM Genestein, 1 µM PD173704 (PD17), 1 µM AG1478 (AG), 1 µM PD173704+1 µM AG1478 prior to growth without (control) or with 10⁻⁸ M PE. Values are the mean±SEM; (*), P<0.05 compared to vehicle or inhibitor alone (n≥3); (**), P<0.05 compared to vehicle+PE. (<b>B</b>) Representative Western analysis of Erk phosphorylation in cells pretreated for 30 min with vehicle 0.1% DMSO (Veh), 1 µM PD17, 1 µM AG, 1 µM PD +1 µM AG or 20 µM genistein (Gen) prior to stimulation without (−) or with (+) PE at 2×10⁻⁸ M for 10 minutes. Lower panel shows semi-quantitative analysis of P-Erk band intensity from CCD images. Values are the mean±SEM (n = 4); (*), P<0.05 compared to vehicle or inhibitor alone. (<b>C</b>) Inhibition of Erk phosphorylation by a dilution series of the EGFR inhibitors, AG1478 and Erlotinib, added 5 minutes prior to stimulation with 3×10⁻⁸ M PE for 5 minutes. To maintain relative intensity, panels were concordantly adjusted. (<b>D</b>) Quantitation of P-Erk band intensity at the indicated AG1478 and Erlotinib concentrations relative to levels in untreated cells. (<b>E</b>) Top 3 panels shows western analysis of Erk phosphorylation in cells pretreated for 30 min with 0.1% DMSO (Veh) or GM6001 (GM) prior to incubation for 5 minutes without (−) or with (+) PE. The bottom panel shows a similar experiment in which the GM6001 preincubation was ∼15 seconds. Panel adjusted independently to emphasis inhibition pattern. (•) Indicates an artifactual band.</p