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

Cell Communication and Signaling, Dec 2016

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.

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

Visavadiya et al. Cell Communication and Signaling Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3 Nishant P. Visavadiya 0 Matthew P. Keasey 0 Vladislav Razskazovskiy Kalpita Banerjee Cuihong Jia Chiharu Lovins Gary L. Wright Theo Hagg 0 Equal contributors Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University , Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614 , USA 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 ?v?3 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. Bioenergetics; Cell death; CRISPR; ECM; Endothelial cell; Focal adhesion kinase; Integrin; Mitochondria; Vitronectin; STAT3 - Background Integrins are heterodimer transmembrane receptors which bind ECM molecules to promote cell adhesion and initiate intracellular signaling that can lead to cell survival [1, 2]. Disruption of integrin binding can cause cell death, especially for cells attached to basement membranes [3], e.g., endothelial cells in the central nervous system (CNS). Among others, endothelial cells express ?v?3 integrins which contribute to their survival [4, 5]. Integrin signaling is important for normal endothelial cell function in maintaining the blood-brain-barrier (BBB) [6, 7], whose disruption by neural injury and stroke leads to disease progression [8]. FAK is one of the major integrin signaling mediators and is activated via autophosphorylation on Y397 [9] which can suppress apoptosis in endothelial cells [10]. Mitochondria not only play a vital role in energy production, particularly in the CNS [11], but also have emerged as a key stress-signaling hub within the cell [12]. CNS endothelial cells have a very high mitochondrial mass compared to those of other organs [13], and mitochondrial function is important for maintaining the BBB and ATP-dependent trans-endothelial transport [13, 14]. Mitochondrial dysfunction after neurological insults plays a role in BBB breakdown and tissue degeneration [7, 15, 16]. Lengthy mitochondrial bioenergetic dysfunction leads to depletion of ATP, increased production of reactive oxygen/nitrogen species, calcium dysregulation, and release of pro-apoptotic proteins, leading to cell death [17, 18]. Integrins can prevent apoptosis through FAK-AKT signaling [10, 19, 20], and inhibiting mitochondria-associated bit1 [20, 21], but have not been implicated in bioenergeti (...truncated)


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Nishant Visavadiya, Matthew Keasey, Vladislav Razskazovskiy, Kalpita Banerjee, Cuihong Jia, Chiharu Lovins, Gary Wright, Theo Hagg. Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3, Cell Communication and Signaling, 2016, pp. 32, 14, DOI: 10.1186/s12964-016-0157-7