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. 2016 Dec 15;14(1):32.
doi: 10.1186/s12964-016-0157-7.

Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3

Nishant P Visavadiya  1 , Matthew P Keasey  1 , Vladislav Razskazovskiy  1 , Kalpita Banerjee  1 , Cuihong Jia  1 , Chiharu Lovins  1 , Gary L Wright  1 , Theo Hagg  2
Affiliations

Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3

Nishant P Visavadiya et al. Cell Commun Signal. .

Erratum in

Abstract

Background: STAT3 is increasingly becoming known for its non-transcriptional regulation of mitochondrial bioenergetic function upon activation of its S727 residue (S727-STAT3). Lengthy mitochondrial dysfunction can lead to cell death. We tested whether an integrin-FAK-STAT3 signaling pathway we recently discovered regulates mitochondrial function and cell survival, and treatments thereof.

Methods: Cultured mouse brain bEnd5 endothelial cells were treated with integrin, FAK or STAT3 inhibitors, FAK siRNA, as well as integrin and STAT3 activators. STAT3 null cells were transfected with mutant STAT3 plasmids. Outcome measures included oxygen consumption rate for mitochondrial bioenergetics, Western blotting for protein phosphorylation, mitochondrial membrane potential for mitochondrial integrity, ROS production, and cell counts.

Results: Vitronectin-dependent mitochondrial basal respiration, ATP production, and maximum reserve and respiratory capacities were suppressed within 4 h by RGD and αvβ3 integrin antagonist peptides. Conversely, integrin ligands vitronectin, laminin and fibronectin stimulated mitochondrial function. Pharmacological inhibition of FAK completely abolished mitochondrial function within 4 h while FAK siRNA treatments confirmed the specificity of FAK signaling. WT, but not S727A functionally dead mutant STAT3, rescued bioenergetics in cells made null for STAT3 using CRISPR-Cas9. STAT3 inhibition with stattic in whole cells rapidly reduced mitochondrial function and mitochondrial pS727-STAT3. Stattic treatment of isolated mitochondria did not reduce pS727 whereas more was detected upon phosphatase inhibition. This suggests that S727-STAT3 is activated in the cytoplasm and is short-lived upon translocation to the mitochondria. FAK inhibition reduced pS727-STAT3 within mitochondria and reduced mitochondrial function in a non-transcriptional manner, as shown by co-treatment with actinomycin. Treatment with the small molecule bryostatin-1 or hepatocyte growth factor (HGF), which indirectly activate S727-STAT3, preserved mitochondrial function during FAK inhibition, but failed in the presence of the STAT3 inhibitor. FAK inhibition induced loss of mitochondrial membrane potential, which was counteracted by bryostatin, and increased superoxide and hydrogen peroxide production. Bryostatin and HGF reduced the substantial cell death caused by FAK inhibition over a 24 h period.

Conclusion: These data suggest that extracellular matrix molecules promote STAT3-dependent mitochondrial function and cell survival through integrin-FAK signaling. We furthermore show a new treatment strategy for cell survival using S727-STAT3 activators.

Keywords: Bioenergetics; CRISPR; Cell death; ECM; Endothelial cell; Focal adhesion kinase; Integrin; Mitochondria; STAT3; Vitronectin.

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Figures

Fig. 1
Fig. 1
RGD and αvβ3 integrin blockade suppresses mitochondrial bioenergetics. a Traces from a representative experiment show mitochondrial bioenergetic deficits in mouse brain bEnd5 cells plated on vitronectin (VTN), in the presence of RGD integrin (RGDS) or αvβ3 integrin (P11) blocking peptides, in serum free medium for 4 h, as measured by oxygen consumption rate (OCR) in an XF24 Seahorse Flux Analyzer. Data are means of 5 wells per condition. b Integrin blockade reduced all four measures of cellular respiration relative to control (no VTN) and VTN treated cells. c Cells cultured in XF24 microplates for 4 h on no substrate or on VTN, with or without integrin blockade, seemed similar in number but cells without substrate and in the presence of RGDS were more rounded. The cell number (d) and protein content (e) in such cells, determined without subsequent Seahorse analyses, were not significantly different across all conditions. Cell counts were independently verified. f Cells cultured for 4 h with VTN, laminin (LAM) or fibronectin (FN) as substrate and added to serum-free media showed increased mitochondrial bioenergetics. All data are means ± SEM of 3–4 independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001
Fig. 2
Fig. 2
FAK inhibition causes mitochondrial dysfunction. a Pharmacological inhibition of the integrin signaling molecule, FAK, with FAK14 treatment of bEnd5 cells for 4 h produced a robust and dose-dependent suppression of mitochondrial bioenergetic function as shown in a representative OCR trace. Cells were grown for 44 h without substrate before treatment. b FAK14 causes reductions in basal respiration, ATP production, and maximum reserve and respiratory capacity. The cell number (c) and protein content (d) were not different across conditions. Cell counts were independently verified. e SiRNA-mediated knockdown of FAK (siFAK) decreased ATP production, reserve capacity and respiratory capacity relative to a non-targeting siRNA control (siControl). Data are mean ± SEM from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant versus control vehicle group
Fig. 3
Fig. 3
S727-STAT3 inhibition causes mitochondrial dysfunction. a Expression plasmids for wildtype (WT), but not functionally dead S727A mutant, STAT3 increased bioenergetics function in bEnd5 cells rendered STAT3−/− by a CRISPR-Cas9 method. N = 3. b Deletion of STAT3 protein was confirmed by western blot most clearly in cells transfected with the G1A guide RNA (G1A and G1B were replicate conditions of the same Cas9-guideRNA plasmid) which was used for the bioenergetics experiment in (A). c DNA sequence data confirmed a point mutation and frame shift in exon 1. d Pharmacological inhibition of STAT3 with stattic for 2 h in bEnd5 cells reduced mitochondrial bioenergetic measures. n = 3. e pS727-STAT3 is predominantly present in the mitochondria and nucleus as shown in Western blots of protein extracts from sub-cellular fractions. Markers = H3 protein, nucleus (N), α-tubulin, cytoplasm (C), PDH, mitochondria (M). W = whole cell lysate. tSTAT3 = total STAT3. f Cells treated for 4 h with the STAT3 inhibitor, stattic (10 μM), had less pS727-STAT3 in their isolated mitochondria. The blot is representative of three experiments. g pS727-STAT3 was increased in isolated bEnd5 mitochondria treated with okadaic acid (OA: 1 μM) in respiration buffer, as detected by western blotting (3 independent experiments are shown). Stattic did not have any effect. A higher molecular weight band appeared after OA treatment in the pS727-STAT3 blot. A faint band could be seen in the same position after longer exposure of the total STAT3 blot (not shown)
Fig. 4
Fig. 4
FAK inhibits mitochondrial S727-STAT3 phosphorylation. a A 4 h FAK14 treatment of bEnd5 cells reduced pS727-STAT3 in both the mitochondrial and cytoplasmic fractions. Blots are representative for 5 experiments. b This reduction was confirmed by quantitative capillary western blotting with representative chemiluminescent spectrograms and synthetic bands (c). d Quantitation was performed of spectrograms confirmed a clear and significant decrease in pS727-STAT3 following 4 h FAK14 treatment in the mitochondrial fractions (n = 3). e Treatment with another more lipophilic FAK antagonist (PF573228: PF at 10 or 20 μM) for 4 or 8 h showed decreases in pS727-STAT3 in conjunction with decreased pFAK in whole cell lysates. f Incubation with the global transcriptional inhibitor actinomycin D (0.3 μg/ml, 4 h) did not significantly change mitochondrial bioenergetics under control or FAK14 conditions
Fig. 5
Fig. 5
pS727-STAT3 stimulation preserves mitochondrial bioenergetics against FAK inhibition. a Bryostatin and HGF preserved reserve and respiratory capacity in bEnd5 cells incubated with FAK14 (5 μM, F5) for 4 h. Data are mean ± SEM from 3 independent experiments. *p < 0.05, **p < 0.01, **p < 0.001. b Western blots of whole cell lysates showing that bryostatin (BST) and HGF preserve pS727-STAT3 in the presence of FAK14 (representative for 3 independent experiments). c Bryostatin does not affect the mitochondrial bioenergetics dysfunction caused by STAT3 inhibition with stattic. N = 3
Fig. 6
Fig. 6
Bryostatin and HGF reduce bEND5 cell death due to FAK inhibition. a Images are representative of bEnd5 cells grown for 4 h with 5 μM FAK14 +/−bryostatin (BST) in regular tissue culture wells. b to e) Cell counts show that bryostatin and HGF can promote survival despite FAK inhibition with 5 or 10 μM FAK14 for 4 or 24 h. Data are mean ± SEM from 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001
Fig. 7
Fig. 7
FAK inhibition depresses mitochondrial membrane potential and increases ROS production. a FAK inhibition with FAK14 for 4 h caused a dose-dependent reduction in mitochondrial membrane potential, as shown by confocal images of red TMRM fluorescence in bEnd5 cells. Cells with complete mitochondrial failure are indicated by arrowheads and detectable only by blue nuclear Hoechst staining. b Spectrometry of TMRM in extracts was used to quantify the effects, and shows that 10 μM FAK14 (FAK10) reduced the membrane potential to the same level as FCCP. Values were normalized to nuclear DAPI staining to account for differences in cell numbers. Data are mean ± SEM from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. c Bryostatin (BST) dose-dependently preserved mitochondrial membrane potential in the presence of FAK14. N = 3. FAK14 dose-dependently induced formation of d) superoxide (O2 .-) and e) H2O2. N = 3
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