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. 2014 Jul 5;392(1-2):173-81.
doi: 10.1016/j.mce.2014.05.015. Epub 2014 May 22.

Protein Kinase C and Src Family Kinases Mediate Angiotensin II-induced Protein Kinase D Activation and Acute Aldosterone Production

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Free PMC article

Protein Kinase C and Src Family Kinases Mediate Angiotensin II-induced Protein Kinase D Activation and Acute Aldosterone Production

Lawrence O Olala et al. Mol Cell Endocrinol. .
Free PMC article

Abstract

Recent evidence has shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone secretion upon angiotensin II (AngII) stimulation. However, the mechanism by which AngII activates PKD remains unclear. In this study, using both pharmacological and molecular approaches, we demonstrate that AngII-induced PKD activation is mediated by protein kinase C (PKC) and Src family kinases in primary bovine adrenal glomerulosa cells and leads to increased aldosterone production. The pan PKC inhibitor Ro 31-8220 and the Src family kinase inhibitors PP2 and Src-1 inhibited both PKD activation and acute aldosterone production. Additionally, like the dominant-negative serine-738/742-to-alanine PKD mutant that cannot be phosphorylated by PKC, the dominant-negative tyrosine-463-to-phenylalanine PKD mutant, which is not phosphorylatable by the Src/Abl pathway, inhibited acute AngII-induced aldosterone production. Taken together, our results demonstrate that AngII activates PKD via a mechanism involving Src family kinases and PKC, to underlie increased aldosterone production.

Keywords: Adrenal glomerulosa; Aldosterone; Angiotensin II (AngII); Protein kinase C (PKC); Protein kinase D (PKD); Src family kinases.

Figures

Figure 1
Figure 1. PKD distribution in human and bovine adrenal glands
(A) PKD distribution was determined in the positive control, human tonsillar tissue (a) and in human [(b) through (d)] and bovine [(e) through (d)] adrenal gland as described in Methods. PKD is present both in the zona glomerulosa and zona fasciculata regions. Sections incubated in the absence of primary antibody served as negative controls [(b) and (e)]. Panels (c) and (f) show PKD immunoreactivity, noted as the brown staining in these tissue sections, photographed at a 10x magnification and panels (d) and (g) show a 40x magnification. (B) The intensity of PKD staining in different regions of human adrenal gland sections was assessed as described in the Methods and presented as the means ± SEM of the values obtained in three glands.
Figure 2
Figure 2. The PKC inhibitor Ro 31-8220 inhibited AngII-induced PKD activation and aldosterone secretion
Primary cultures of bovine adrenal glomerulosa cells were pretreated for 30 minutes with 3 μM Ro 31-8220 prior to a 30-minute stimulation with or without 10 nM AngII. Panel (A) illustrates a representative blot. Note that the lower band in this blot appears to be non-specific, as it is only observed with certain lot numbers of the polyclonal antibody. (B) Band intensities from multiple experiments were quantified and normalized to actin. (C) Supernatants from the PKD activation experiments were assayed for aldosterone content. Values are expressed relative to the maximal response (in the presence of AngII) and represent means ± SEM from three experiments performed in duplicate; ***p<0.001 vs. the control, †p<0.05 vs. AngII. (D) Cells were pretreated for 30 minutes with 3 μM Ro 31-8220 prior to a 30-minute incubation with or without 25 μM 22(R)-hydroxycholesterol after which supernatants were assayed for aldosterone content. Values represent the means ± SEM of three separate experiments performed in duplicate.
Figure 3
Figure 3. AngII increased tyrosine phosphorylation of PKD
Primary cultures of bovine adrenal glomerulosa cells were stimulated with or without 10 nM AngII and immunoprecipitated with total PKD antibody (Santa Cruz). Immunoblot analysis was performed using an antibody recognizing phosphotyrosine (Millipore). Panel (A) illustrates a representative blot. Immunoblot analysis with total PKD antibody was performed on one-tenth of the immunoprecipitated protein, which served as loading control (input). Panel (B) illustrates band intensities from multiple experiments quantified and normalized to the loading control and are expressed as fold over control values. Values represent means ± SEM from four experiments performed in duplicate; *p<0.05 vs. control. Note that similar results were observed when lysates were immunoprecipitated with anti-phosphotyrosine antibody and the immunoprecipitate probed with anti-PKD antibody (data not shown).
Figure 4
Figure 4. PP2 inhibited AngII-induced PKD activation and aldosterone secretion
Primary cultures of bovine adrenal glomerulosa cells were pretreated for 30 minutes with 10 μM PP2 prior to a 30-minute stimulation with or without 10 nM AngII. Panel (A) illustrates a representative blot. (B) Band intensities from multiple experiments were quantified and normalized to actin. (C) Supernatants from the PKD activation experiments were assayed for aldosterone content. Values represent means ± SEM from four experiments performed in duplicate and are expressed relative to the maximal response (in the presence of AngII); **p<0.01 vs. the control, ***p<0.001 vs. the control, p<0.05 vs. AngII. (D) Cells were pretreated for 30 minutes with 10 μM PP2 prior to a 30-minute incubation with or without 25 μM 22(R)-hydroxycholesterol, after which supernatants were assayed for aldosterone content. Values represent the means ± SEM of four separate experiments performed in duplicate.
Figure 5
Figure 5. Src-1 decreased AngII-mediated PKD activation and aldosterone secretion
Primary cultures of bovine adrenal glomerulosa cells were pretreated for 30 minutes with 0.75 μM Src-1 prior to a 30-minute stimulation with or without 10 nM AngII. Panel (A) illustrates a representative blot. (B) Band intensities from multiple experiments were quantified and normalized to actin. (C) Supernatants from the PKD activation experiments were assayed for aldosterone content. Values are expressed relative to the maximal response (in the presence of AngII) and represent means ± SEM from three experiments performed in duplicate; *p<0.05 vs. the control, ***p<0.001 vs. the control, ††p<0.01 vs. AngII. (D) Cells were pretreated for 30 minutes with 0.75 μM Src-1 prior to a 30-minute incubation with or without 25 μM 22(R)-hydroxycholesterol after which supernatants were assayed for aldosterone content. Values represent the means ± SEM of four separate experiments performed in duplicate.
Figure 6
Figure 6. The tyrosine-463-to-phenylalanine PKD mutant decreased aldosterone production
Primary cultures of bovine adrenal glomerulosa cells were infected with pAdtrackCMV (empty vector), or the tyrosine 463-to-phenylalanine PKD mutant (PKDY463F) for 4 hours, and media was replaced with serum-free media for an additional 16-20 hours before treatment with or without 10nM AngII. Panel (A) illustrates a representative experiment showing PKDY463F (and GFP) overexpression in adenovirus-infected cells 20-24 hours post-infection. (B) Supernatants from the PKD overexpression experiments were assayed for aldosterone content. Values represent means ± SEM from three experiments performed in duplicate and are expressed relative to the maximal response (in the presence of AngII); *p<0.05 vs. the control, ***p<0.001 vs. the control (vector), p<0.05 vs. vector + AngII.
Figure 7
Figure 7. The tyrosine-463-to-phenylalanine PKD mutant did not inhibit 22(R)-hydroxycholesterol-mediated aldosterone production
Primary cultures of bovine adrenal glomerulosa cells were incubated for 4 hours with adenovirus expressing pAdtrackCMV (empty vector) or tyrosine-463-to-phenylalanine PKD (PKDY463F) and media was replaced with serum-free media for an additional 16-20 hours before treatment with or without 25 μM 22(R)-hydroxycholesterol for one hour and assay of aldosterone content in the media. 22(R)-Hydroxycholesterol-mediated aldosterone secretory rates were not stat istically different. Data represent means ± SEM from three experiments performed in duplicate.
Figure 8
Figure 8. Ro 31-8820 and PP2 additively decreased AngII-mediated PKD activation and aldosterone secretion
Primary cultures of bovine adrenal glomerulosa cells were pretreated for 30 minutes with 3 μM Ro 31-8220, 10 μM PP2, or a combination of Ro 31-8220 and PP2 prior to a 30-minute stimulation with or without 10 nM AngII. Panel (A) illustrates a representative blot. (B) Band intensities from multiple experiments were quantified and normalized to actin. (C) Supernatants were assayed for aldosterone content. Values represent means ± SEM from six experiments performed in duplicate and are expressed relative to the maximal response (in the presence of AngII); *p<0.05 vs. the control, ***p<0.001 vs. the control, †p<0.05 vs. AngII, †††p<0.001 vs. AngII §§p<0.01 vs. PP2 + AngII, #p<0.05 vs. Ro 31-8220 + AngII. (D) Cells were pretreated for 30 minutes with 3 μM Ro 31-8220 and 10 μM PP2 prior to a 30-minute incubation with or without 25 μM 22(R)-hydroxycholesterol, after which supernatants were assayed for aldosterone content. Values represent the means ± SEM of three separate experiments performed in duplicate.

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