Neurite outgrowth induced by stimulation of angiotensin II AT2 receptors in SH-SY5Y neuroblastoma cells involves c-Src activation

Fig. 1. Involvement of AT2 receptor in induced neurite outgrowth in SH-SY5Y cells. (A) SH-SY5Y cells cultured in DMEM 2% FBS alone (Control) or supplemented with Ang II (0.1 μM), CGP42112A (0.01 μM, AT2 receptors agonist), RA (10 μM) or NGF (100 ng/ml) and allowed to differentiate for 3 days. Representative images of photomicrographs are shown. Arrowheads point to differentiated cells. The insets in Ang II and CGP42112A panels show a magnification to better visualize the neurite enlargement. (B) Graph showing the percentage of cells exhibiting at least one neurite longer that a cell body. Values represent means ± S.E.M (n = 4). **p < 0.01, ***p < 0.001 vs. control. Lower panel: equal amounts of cell lysates from the same treatments were separated by SDS-PAGE and immunoblotted with anti-βIII tubulin, marker of differentiation. Blot was stripped and reprobed for β tubulin to ensure equal loading and transfer. Numbers indicate relative protein levels determined by densitometry. Blots are representative of results from at least three independent experiments. (C) Graph showing the percentage of cells exhibiting 2 or more neurites. Values represent means ± S.E.M (n = 4). *p < 0.05, ***p <…

Fig. 2. Differentiation does not modulate protein levels of AT2 receptor. SH-5Y5Y cells were stimulated with Ang II, CGP42112A, RA or NGF and allowed to differentiate for 3 days. Cell lysates were prepared, equal amount of proteins were separated by SDS-PAGE and western blots were developed with anti-AT2 and anti-β-actin antibodies. A representative blot and densitometry data are shown. Values represent means ± S.E.M (n = 3).

Fig. 3. PD123319 reverts neurite induction by Ang II or CGP42112A. (A) SH-SY5Y cells were treated with Ang II (0.1 μM) or CGP42112A (0.01 μM) in the absence or the presence of PD123319 (10 μM) and allowed to differentiate for 3 days. Representative images of photomicrographs for control and different treatments are shown. Arrowheads point to differentiated cells. (B) Graph showing the percentage of cells exhibiting at least one neurite longer that a cell body. Values represent means ± S.E.M (n = 4). **p < 0.01 vs. control. (C) Graph showing the percentage of cells exhibiting 2 or more neurites. Values represent means ± S.E.M (n = 4). *p < 0.05, ***p < 0.001 vs. control.

Fig. 4. Differentiation induced by AT2 receptor stimulation is independent of PI3K, but requires ERK, SphK and c-Src. SH-SY5Y cells were pretreated for 40 min with the indicated inhibitors, stimulated with CGP42112A (0.01 μM) and allowed to differentiate during 3 days. The graphs show the percentage of cells exhibiting at least one neurite longer that a cell body. (A) Neurite outgrowth induced by CGP42112A in the absence or the presence of MEK inhibitor UO126 (10 μM). Values represent means ± S.E.M (n = 4). ***p < 0.001 vs. control. (B) Neurite outgrowth induced by CGP42112A in the absence or the presence the PI3K inhibitor LY294002 (0.5 μM). Values represent means ± S.E.M (n = 2). **p < 0.01 vs. control. (C) Neurite outgrowth induced by CGP42112A in the absence or the presence the c-Src inhibitor PP2 (2.5 μM) or its inactive analog, PP3 (2.5 μM). Values represent mean ± S.E.M (n = 5). ***p < 0.001 and **p < 0.01 vs. control. (D) Neurite outgrowth induced by CGP42112A in the absence or the presence of SphK inhibitor SKI-II (5 μM). Values represent means ± S.E.M (n = 3). ***p < 0.001 vs. control.

Fig. 5. CGP42112A stimulates c-Src phosphorylation in SH-SY5Y dells. (A) SH-SY5Y cells were stimulated with CGP42112A (0.01 μM) for the indicated times. Cell lysates were prepared, proteins separated by SDS-PAGE and immunoblotted with anti-pY416Src or anti-pY527Src antibodies. Membranes were stripped and reprobed with anti-β tubulin for loading control. (B) Densitometry of western blots for pY416Src (green bars) and pSrcY527 (blue bars) normalized to β tubulin. The data are shown as the mean ± S.E.M (n = 3), **p < 0.01, ***p < 0.001 vs. controls (time = 0).

Fig. 6. Tyrphostin AG879, a TrkA inhibitor, prevents neurite outgrowth induced by Ang II and CGP42112A. (A) SH-SY5Y cells were pre-treated with AG879 (0.6 μM, 40 min), stimulated with CGP42112A, allowed to differentiate for 3 days and neurite outgrowth was quantified. Values represent mean ± S.E.M (n = 3), ***p < 0.001 vs. control. (B) Graph showing the percentage of cells exhibiting 2 or more neurites. Values represent means ± S.E.M (n = 3). *p < 0.05, ***p < 0.001 vs. control.

Fig. 7. Proposed model of AT2 receptor mediated neurite outgrowth. The figure summarizes the present and some past findings related to the role of Ang II AT2 receptors in the mechanism of neurite elongation. We explored the participation of three different pathways in the neurite outgrowth induced by Ang II or CGP42112A by using specific inhibitors of c-Src (PP2), MEK (UO126), TrkA (AG879) and SphK (SKI-II). Besides, we evaluated the phosphorylation and thus activation of c-Src. Once activated, c-Src may enhance neurite outgrowth either directly or through the transactivation of TrkA receptor (this work and [17,27]). Moreover, activation of SphK that increases S1P levels (this work and [28]) and induction of the MEK/ERK pathway are also important for neurite elongation (this work and [27,37]). The role of SHP-1 in the AT2 receptor signaling pathway is also displayed since it has been demonstrated by our group and others [21,23,24].