Angiotensin II and cAMP signaling pathways regulate mitochondrial biogenesis and activity in human adrenocortical cells

Figures

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  Figure 1. Ang II modulates mitochondrial biogenesis factors expression. H295R cells were serum-starved for 24 hours and subsequently stimulated with angiotensin II (Ang II, 100 nM) for the times indicated (hours, h). Total RNA was isolated and subjected to real-time qPCR using specific primers. Cyclophilin was used as an internal control. qPCR data were analyzed using the 2−ΔΔCt method (comparative Ct method) for each experimental sample. (A) Relative NRF-1 expression levels (fold change): ***P < .001 vs control cells (Ang II 0 hours). (B) Nuclear and (C) mitochondrial proteins were analyzed by Western blot. Membranes were sequentially incubated with (B) anti-NRF-1 and anti-PCNA and (C) anti-TFAM and anti-SDHA antibodies. Representative images are shown from 3 independent experiments (n = 3). Optical density for each band was taken, and control (without Ang II) sample intensity was arbitrarily defined as 1. Protein levels were relativized to respective controls (fold change): ***P < .001; **P < .01; *P < .05 vs respective control (Ang II 0 hours). Results are expressed as mean ± SD of three independent experiments. ANOVA followed by Tukey's post hoc test.

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  Figure 2. Ang II modulates Nrf2 mRNA levels in a time-dependent manner. H295R cells were serum-starved for 24 hours and subsequently stimulated with angiotensin II (Ang II, 100 nM) for the times indicated (hours, h). Total RNA was isolated, and real-time qPCR was performed using specific primers. Cyclophilin was used as an internal control. qPCR data were analyzed using the 2−ΔΔCt method (comparative Ct method) for each experimental sample. Representative images are shown from 3 independent experiments (n = 3). Relative Nrf2 expression levels (fold change): *P < .05; **P < .01 vs control cells (Ang II 0 hours). Results are expressed as mean ± SD of three independent experiments. ANOVA followed by Tukey's post hoc test.

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  Figure 3. UCP2 and ANT1 expression is regulated by Ang II. H295R cells were serum-starved for 24 hours and subsequently stimulated with angiotensin II (Ang II, 100 nM) for the times indicated (hours, h). Total RNA was isolated and subjected to real-time qPCR using specific primers. Cyclophilin was used as an internal control. qPCR data were analyzed using the 2−ΔΔCt method (comparative Ct method) for each experimental sample. Relative (A) UCP2 and (B) ANT1 expression levels (fold change): *P < .05; **P < .01; ***P < .001 vs control cells (Ang II 0 hours); ###P < .001 vs 0.5, 1, and 2 hours Ang II. Data were analyzed by ANOVA followed by Tukey's post hoc test.

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  Figure 4. Ang II stimulation promotes mitochondrial activity. H295R cells were grown on poly-L-lysine glass coverslips, serum-starved for 24 hours, and subsequently incubated with CCCP (5 μM) and/or DMSO (0.1%) as vehicle in the corresponding wells (F), followed by stimulation with or without angiotensin II (Ang II, 100 nM) for the times indicated (hours, h) (A-F). Afterwards, cells were incubated with 150 nM MitoTracker Red (MTR) at 37 °C in fresh medium for 45 minutes and samples were processed to be analyzed using a Nikon fluorescence microscope (40× magnification). (G) In the bar graph, differences in signal intensity (mitochondrial signal) are expressed as fluorescence intensity (FIJI software) relative to the number of cells within the same ROI (containing 15-20 cells/ROI in all cases). Red (MTR) and ultraviolet (DAPI) channels were studied. Representative images are shown of 10 images analyzed from 3 independent experiments (n = 3). Images were obtained in all cases with the same exposure, the background was discarded, and the intensity of the red fluorescent signal was analyzed with FIJI software. **P < .01; ***P < .001 vs control (Ang II 0 hours). Scale bar: 20 µm for every image.…

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  Figure 5. Ang II induces an increase in mitochondrial DNA content, OCR, and respiratory parameters. (A) H295R cells were treated with angiotensin II (Ang II, 100 nM) for the times indicated, followed by total DNA isolation. Mitochondrial DNA (mtDNA) content (3 different genes) was quantified by real-time qPCR and normalized to genomic DNA levels (β-actin expression). qPCR data were analyzed using the 2−ΔΔCt method (comparative Ct method) for each experimental sample. Relative genes expression levels (fold change): **P < .01; ***P < .001 vs control cells (Ang II 0 hours). For OCR and respiratory parameters measurements, 100 000 cells/well were seeded in 96-well plates in complete medium 48 hours prior to the assay. H295R cells were stimulated with angiotensin II (Ang II, 100 nM) for the times indicated. On the day of the assay, the medium was replaced with a specialized Mito Stress Test medium. ATP synthase inhibitor Oligomycin A (2.5 µM) was added to derive ATP-linked OCR, FCCP (0.5 µM) to uncouple the mitochondria for maximal OCR, and Rotenone/Antimycin A (0.5 µM) to inhibit electron transport chain. (B) OCR representative trace in H295R control cells (black circle) and Ang II-stimulated…

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  Figure 6. Mitochondrial membrane depolarization modulates the expression of NRF1, Nrf2, UCP2, and ANT1 under both basal and hormone-stimulated conditions. H295R cells were serum-starved for 24 hours, preincubated for 30 minutes with CCCP (5 μM) or DMSO (0.1%) as vehicle in the corresponding wells, and subsequently stimulated with angiotensin II (Ang II, 100 nM) for 2 hours. Total RNA was isolated and subjected to real-time qPCR using specific primers. Cyclophilin was used as an internal control. qPCR data were analyzed using the 2−ΔΔCt method (comparative Ct method) for each experimental sample. Relative (A) NRF-1, (B) Nrf2, (C) UCP2 and (D) ANT1 expression levels (fold change): ns, nonsignificant; **P < .01; ***P < .001 vs control (Ang II 0 hours); ##P < .01; ###P < .001 without CCCP. Results are expressed as the mean ± SD of three independent experiments. Data were analyzed by ANOVA followed by Tukey's post hoc test.

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  Figure 7. 8Br-cAMP modulates NRF-1 and Nrf2 mRNA levels in a time-dependent manner. H295R cells were serum-starved for 24 hours and subsequently stimulated with 8Br-cAMP (1 mM) for the times indicated (hours, h). Total RNA was isolated, and real-time qPCR was performed using specific primers. Cyclophilin was used as an internal control. qPCR data were analyzed using the 2−ΔΔC method (comparative Ct method) for each experimental sample. Relative (A) NRF-1 and (B) Nrf2 expression levels (fold change): **P < .01; ***P < .001 vs control cells (Ang II 0 hours). Results are expressed as the mean ± SD of three independent experiments. Data were analyzed by ANOVA followed by Tukey's post hoc test.

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  Figure 8. UCP2 and ANT1 expression is regulated by 8Br-cAMP. H295R cells were serum-starved for 24 hours and subsequently stimulated with 8Br-cAMP (1 mM) for the times indicated (hours, h). Total RNA was isolated and subjected to real-time qPCR using specific primers. Cyclophilin was used as an internal control. qPCR data were analyzed using the 2−ΔΔCt method (comparative Ct method) for each experimental sample. Relative (A) UCP2 and (B) ANT1 expression levels (fold change): **P < .01; ***P < .001 vs control cells (8Br-cAMP 0 hours). Results are expressed as the mean ± SD of three independent experiments. Data were analyzed by ANOVA followed by Tukey's post hoc test.

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  Figure 9. 8Br-cAMP stimulation promotes mitochondrial activity. H295R cells were grown on poly-L-lysine glass coverslips, serum-starved for 24 hours, and subsequently incubated with CCCP (5 μM) and/or DMSO (0.1%) as vehicle in the corresponding wells (F), followed by stimulation with or without 8Br-cAMP (1 mM) for the times indicated (hours, h) (A-F) (A-D). Afterwards, cells were incubated with 150 nM MitoTracker Red (MTR) at 37 °C in fresh medium for 45 minutes, and samples were processed for analysis using a Nikon fluorescence microscope (40× magnification). (G) In the bar graph, differences in signal intensity (mitochondrial signal) are expressed as fluorescence intensity (FIJI software) relative to the number of cells within the same ROI (containing 15-20 cells/ROI in all cases). Red (MTR), and ultraviolet (DAPI) channels were studied. Representative images are shown of 10 images analyzed from 3 independent experiments (n = 3). Images were obtained in all cases with the same exposure, the background was discarded, and the intensity of the red fluorescent signal was analyzed with FIJI software. ***P < .001 vs control (Ang II 0 hours). Scale bar: 20 µm for every image. Data were analyzed…

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  Figure 10. Mitochondrial membrane depolarization modulates the expression of NRF1, Nrf2, UCP2, and ANT1 under 8Br-cAMP stimulation. H295R cells were serum-starved for 24 hours, preincubated for 30 minutes with CCCP (5 μM) or DMSO (0.1%) as vehicle in the corresponding wells and subsequently stimulated with 8Br-cAMP (1 mM) for 2 hours (indicated as 8Br). Total RNA was isolated and subjected to real-time qPCR using specific primers. Cyclophilin was used as an internal control. qPCR data were analyzed using the 2−ΔΔCt method (comparative Ct method) for each experimental sample. Relative (A) NRF-1, (B) Nrf2, (C) UCP2 and (D) ANT1 expression levels (fold change): **P < .01; ***P < .001 vs control (Ang II 0 hours); ##P < .01; ###P < .001 without CCCP. Results are expressed as the mean ± SD of three independent experiments. Data were analyzed by ANOVA followed by Tukey's post hoc test.

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  Figure 11. Hormonal regulation of mitochondrial function, biogenesis, and redox homeostasis in H295R adrenocortical cells. Schematic representation of the signaling pathways activated by Ang II and ACTH in H295R human adrenocortical cells and their impact on mitochondrial function. Ang II signaling via Gq promotes Ca2+ mobilization and DAG/IP3 production, leading to PKC activation, whereas ACTH signaling via Gs stimulates cAMP/PKA pathways. These cascades modulate mitochondrial activity, steroidogenesis, and ROS production, while also influencing mitochondrial biogenesis through nuclear transcriptional regulators (NRF-1, TFAM) and mitochondrial activity through mitochondrial factors, as UCP2 and ANT1. These hormonal transduction pathways modulate the mitochondria-nucleus retrograde signaling triggered by mitochondrial membrane potential disruptors. The coordinated response integrates mitochondrial dynamics and antioxidant defenses to maintain cellular homeostasis. Created in BioRender. https://BioRender.com/6cr7t65.