title: Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614, USA.
authors.forename: Nishant P
authors.initials: NP
authors.lastname: Visavadiya
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Keasey
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614, USA.
authors.forename: Vladislav
authors.initials: V
authors.lastname: Razskazovskiy
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614, USA.
authors.forename: Kalpita
authors.initials: K
authors.lastname: Banerjee
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614, USA.
authors.forename: Cuihong
authors.initials: C
authors.lastname: Jia
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614, USA.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Lovins
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614, USA.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Wright
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Building 178, Maple Ave, PO Box 70582, Johnson City, TN37614, USA. haggt1@etsu.edu.
authors.forename: Theo
authors.initials: T
authors.lastname: Hagg
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016923:Cell Death; D045744:Cell Line, Tumor; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004734:Energy Metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D016023:Integrins; D017382:Reactive Oxygen Species; D050796:STAT3 Transcription Factor; D000255:Adenosine Triphosphate; D051416:Focal Adhesion Protein-Tyrosine Kinases
keywords: Bioenergetics; CRISPR; Cell death; ECM; Endothelial cell; Focal adhesion kinase; Integrin; Mitochondria; STAT3; Vitronectin