Focal Adhesion Kinase Research Articles

Acoustic modulation of mechanosensitive genes and adipocyte differentiation

Eukaryotic cells are equipped with multiple mechanosensory systems and perceive a wide range of mechanical stimuli from the environment. However, cell-level responses to audible range of acoustic waves, which transmit feeble yet highly frequent physical perturbations, remain largely unexplored. Here, we established a direct sound emission system with a vibrational transducer, and acoustic waves at frequency 440 Hz, 14 kHz, and white noise were transmitted to the murine C2C12 myoblasts at 100 Pa intensity. After 2 and 24 h sound emission, 42 and 145 differentially expressed genes, respectively, were identified using RNA-sequencing. Both cell- and sound-related factors important for inducing gene responses were further investigated. The activation of prostaglandin-endoperoxide synthase 2/cyclooxygenase-2 (Ptgs2/Cox-2), a high and immediate sound-responding gene, is dependent on focal adhesion kinase activation and mediates sound-triggered gene responses by activating prostaglandin E2 synthesis. Adipocyte cells exhibited prominently high sound responses, and their differentiation was significantly suppressed by continuous or periodic acoustic stimulation. Collectively, these findings redefine acoustic waves as cellular stimulators and provide new avenues for applying acoustic techniques in biosciences.

Fibulin-4 and latent-transforming growth factor beta-binding protein-4 interactions with syndecan-2 and syndecan-3 are required for elastogenesis.

Elastogenesis is a cell surface-located hierarchical process that requires the core components tropoelastin and fibrillins and several accessory proteins, including fibulin-4 (FBLN4) and latent TGF-β binding protein-4 (LTBP4). FBLN4 and LTBP4 interact with cells, but their cell receptors and associated molecular elastogenic mechanisms remain unknown. Primary skin fibroblasts and several vascular smooth muscle cells bound strongly to FBLN4 multimers and LTBP4 monomers. We identified two cell interaction epitopes on FBLN4 located in cbEGF2-3 and the C-terminal domain, whereas FBLN4 multimerization sites were mapped to cbEGF4-5 and the C-terminal domain. We also determined a novel cell interaction site in the N-terminal half of LTBP4. Cell binding to FBLN4 and LTBP4 was strongly inhibited in the presence of heparin, heparan sulfate, or after enzymatic removal of heparan sulfate, suggesting heparan sulfate proteoglycans as relevant cell surface receptors. siRNA knockdown experiments identified syndecan (SDC)2 and SDC3 as cell receptors for FBNL4 and SDC3 for LTBP4. Direct protein interactions between FBLN4 and the recombinant ectodomains of SDC2 and SDC3, and between LTBP4 and SDC3 validated these results. Interaction of the elastogenic cells with FBLN4 and LTBP4 enhanced elastogenesis, whereas SDC2 and/or SDC3 knockdowns led to reduced elastic fiber formation. The cell interactions with FBLN4 and LTBP4 significantly enhanced focal adhesion formation, induced cell contraction, and led to activation of focal adhesion kinase (FAK), Erk1/2, and RhoA. Pharmacological inhibition of these effectors markedly attenuated elastic fiber formation, and siRNA knockdown of SDC2 and SDC3 led to reduced levels of pFAK, pERK, and active RhoA. Together, these data demonstrate that FBLN4 and LTBP4 cell interactions through SDC2 and SDC3 promote elastogenesis by enhancing focal adhesion formation, leading to cell contractility through FAK, Erk1/2, and RhoA activation, underscoring the significance of these pathways in elastogenesis.

Spatial regulation of mitochondrial membrane potential by 𝛂5ꞵ1 integrin engagement in collective cell migration.

The mechanistic links between mechanical forces and bioenergetics remain elusive. We report an increase in mitochondrial membrane potential (MMP) along the leading row of collectively migrating Xenopus laevis mesendoderm cells at sites where fibronectin- a5b1 integrin substrate traction stresses are greatest. Real-time metabolic analyses reveal a5b1 integrin-dependent increases in respiration efficiency in cells on fibronectin substrates. Elevation of metabolic activity is reduced following pharmacologic inhibition of focal adhesion kinase (FAK) signaling. Attachment of mesendoderm cells to fibronectin fragments that support differing a5b1 integrin conformational and ligand-binding affinity states, increases MMP when both the Arg-Gly-Asp (RGD) and Pro-Pro-Ser-Arg-Asn (PPSRN) synergy sites of fibronectin are engaged by the receptor. Cell stretch on deformable fibronectin substrates also results in a FAK-dependent increase in MMP. Inhibition of MMP or ATP-synthase activity slows collective cell migration velocity in vivo, further suggesting that integrin-dependent adhesion and signaling contribute to metabolic changes. These data highlight an underexplored link between ECM-integrin adhesion and metabolic activity in embryonic cell migration. We propose that fibronectin-integrin adhesion and signaling help shape the metabolic landscape of collectively migrating cells.

NELL2, a novel osteoinductive factor, regulates osteoblast differentiation and bone homeostasis through fibronectin 1/integrin-mediated FAK/AKT signaling

Neural EGFL-like 2 (NELL2) is a secreted protein known for its regulatory functions in the nervous and reproductive systems, yet its role in bone biology remains unexplored. In this study, we observed that NELL2 was diminished in the bone of aged and ovariectomized (OVX) mice, as well as in the serum of osteopenia and osteoporosis patients. In vitro loss-of-function and gain-of-function studies revealed that NELL2 facilitated osteoblast differentiation and impeded adipocyte differentiation from stromal progenitor cells. In vivo studies further demonstrated that the deletion of NELL2 in preosteoblasts resulted in decreased cancellous bone mass in mice. Mechanistically, NELL2 interacted with the FNI-type domain located at the C-terminus of Fibronectin 1 (Fn1). Moreover, we found that NELL2 activated the focal adhesion kinase (FAK)/AKT signaling pathway through Fn1/integrin β1 (ITGB1), leading to the promotion of osteogenesis and the inhibition of adipogenesis. Notably, administration of NELL2-AAV was found to ameliorate bone loss in OVX mice. These findings underscore the significant role of NELL2 in osteoblast differentiation and bone homeostasis, suggesting its potential as a therapeutic target for managing osteoporosis.

FAK regulates trophoblast functions of invasion and proliferation through Rap1 pathway in early-onset preeclampsia.

FAK regulates trophoblast functions of invasion and proliferation through Rap1 pathway in early-onset preeclampsia.

Inducible FAK Loss but not FAK Inhibition in Endothelial Cells of PYK2-null Mice Activates p53 Tumor Suppressor to Prevent Tumor Growth.

Focal adhesion kinase (FAK) and the related tyrosine kinase PYK2 are signaling and scaffolding proteins in co-expressed in endothelial cells (ECs) that regulate blood vessel function and tumor growth. As FAK-PYK2 share overlapping cellular roles, we generated PYK2-/- FAKfl/fl mice with tamoxifen-inducible EC-specific Cre expression. EC-specific FAK inactivation in PYK2-/- but not PYK2+/+ mice led to increased heart and lung mass, vascular leakage, and created a tumor microenvironment that was repressive to syngeneic melanoma, breast, and lung carcinoma implanted tumor growth. Tumor suppression was associated with defective vessel sprouting, enhanced p53 tumor suppressor and p21CIP1 protein expression in ECs, elevated markers of DNA damage, and altered blood cytokine levels in tumor-bearing mice. However, EC-specific hemizygous kinase-defective (KD) FAK expression in EC FAK-/KD PYK2-/- mice was not associated with elevated p53 levels. Instead, EC FAK-/KD PYK2-/- mice supported primary tumor growth but prevented metastasis, implicating EC FAK activity in tumor spread. In vitro, combined genetic or small molecule FAK-PYK2 knockdown in ECs or tumor cells elevated p21CIP1 and prevented cell proliferation in a p53-dependent manner, highlighting a linkage between EC FAK-PYK2 loss and p53 activation in tumor regulation.

HINT1 aggravates aortic aneurysm by targeting ITGA6/FAK axis in vascular smooth muscle cells.

Aortic aneurysm is a high-risk cardiovascular disease without effective cure. Vascular Smooth Muscle Cell (VSMC) phenotypic switching is a key step in the pathogenesis of aortic aneurysm. Here, we revealed the role of histidine triad nucleotide-binding protein 1 (HINT1) in aortic aneurysm. HINT1 was upregulated both in aortic tissue from patients with aortic aneurysm and Ang II-induced aortic aneurysm mice. VSMC-specific HINT1 deletion alleviated aortic aneurysm via preventing VSMC phenotypic switching. With the stimulation of pathological factors, the increased nuclear translocation of HINT1 mediated by nucleoporin 98 (Nup98) promoted the interaction between HINT1 and transcription factor AP-2 alpha (TFAP2A) and further triggered the transcription of integrin alpha 6 (ITGA6) mediated by TFAP2A, and consequently activated the downstream focal adhesion kinase (FAK)/STAT3 signal pathway, leading to aggravation of VSMC phenotypic switching and aortic aneurysm. Importantly, Defactinib treatment was demonstrated to limit aortic aneurysm development by inhibiting the FAK signal pathway. Thus, HINT1/ITGA6/FAK axis emerges as potential therapeutic strategies in aortic aneurysm.

Peptidergic Systems and Neuroblastoma

The peptidergic systems are involved in neuroblastoma. Peptides (angiotensin II, neuropeptide Y, neurotensin, substance P) act as oncogenic agents in neuroblastoma, whereas others (adrenomedullin, corticotropin-releasing factor, urocortin, orexin) exert anticancer effects against neuroblastoma. This plethora of peptidergic systems show the functional complexity of the mechanisms regulated by peptides in neuroblastoma. Peptide receptor antagonists act as antineuroblastoma agents since these compounds counteracted neuroblastoma cell growth and migration and the angiogenesis promoted by oncogenic peptides. Other therapeutic approaches (signaling pathway inhibitors, focal adhesion kinase inhibitors, peptide receptor knockdown, acetic acid analogs) that also counteract the beneficial effects mediated by the oncogenic peptides in neuroblastoma are discussed, and future research lines to be developed in neuroblastoma (interactions between oncogenic and anticancer peptides, combination therapy using peptide receptor antagonists and chemotherapy/radiotherapy) are also suggested. Although the data regarding the involvement of the peptidergic systems in neuroblastoma are, in many cases, fragmentary or very scarce for a particular peptidergic system, taken together, they are quite promising with respect to potentiating and developing this research line with the aim of developing new therapeutic strategies to treat neuroblastoma in the future. Peptidergic systems are potential and promising targets for the diagnosis and treatment of neuroblastoma.

Obesity promotes urinary tract infection by disrupting urothelial immune defenses.

Obesity is a significant public health concern that is associated with numerous health risks. Infections are a major complication of obesity, but the mechanisms responsible for increased infection risk are poorly defined. Here, we use a diet induced obesity mouse model and investigate how obesity impacts urinary tract infection (UTI) susceptibility and bladder immune defenses. Our results show that high-fat diet fed female and male mice exhibit increased susceptibility to uropathogenic E. coli (UPEC) following experimental UTI. Transcriptomic analysis of bladder urothelial cells shows that obesity alters gene expression in a sex-specific manner, with distinct differentially expressed genes in male and female mice, but shared activation of focal adhesion and extracellular matrix signaling. Western blot and immunostaining confirm activation of focal adhesion kinase, a central component of the focal adhesion pathway, in the bladders of obese female and male mice. Mechanistically, experiments using primary human urothelial cells demonstrate that focal adhesion kinase overexpression promotes UPEC invasion. These findings demonstrate that obesity enhances UTI susceptibility by activating focal adhesion kinase and promoting bacterial invasion of the urothelium. Together, they explain how obesity promotes UTI vulnerability and identify modifiable targets for managing obesity-associated UTI.

Therapeutic potential of GNRHR analogs and SRC/FAK inhibitors to counteract tumor growth and metastasis in breast cancer.

Therapeutic potential of GNRHR analogs and SRC/FAK inhibitors to counteract tumor growth and metastasis in breast cancer.

Design, synthesis and biological evaluation of coumarin-containing 2,4-diphenylpyrimidine derivatives as novel focal adhesion kinase inhibitors for treatment of non-small cell lung cancer.

Design, synthesis and biological evaluation of coumarin-containing 2,4-diphenylpyrimidine derivatives as novel focal adhesion kinase inhibitors for treatment of non-small cell lung cancer.

Podocyte aging and diabetic kidney disease.

Podocyte aging and diabetic kidney disease.

Targeting the integrin beta 1‐focal adhesion kinase axis with artemisinin: Biophysical disruption of cell adhesion, migration, and invasion in tongue cancer

AbstractTongue cancer is a significant threat to human health due to its propensity to spread throughout the oral cavity and to other regions of the head and neck. The challenges posed by its high invasion, metastasis, and late clinical detection underscore the urgency for effective clinical interventions. In this study, we elucidate the promising anti‐cancer properties of artemisinin, an anti‐malarial drug, in inhibiting cellular interactions within a tongue cancer cell line. Our findings reveal that artemisinin treatment effectively suppresses phosphorylated focal adhesion kinase and its downstream AKT pathway, thereby enhancing apoptotic processes and inducing cell cycle arrest, consequently impeding cellular proliferation. Moreover, artemisinin treatment induces focal adhesion rearrangement and diminishes the cell's capacity to generate traction stress, consequently restraining cell migration on the matrix, as determined via traction force microscopy. Additionally, a transition from N‐cadherin to E‐cadherin expression occurs at cellular junctions, lowering intracellular stress, as measured by monolayer stress microscopy. This transition significantly curtails cellular migratory capabilities. Our in vivo studies corroborate these findings, showing a significant reduction in tumor volume following artemisinin treatment. Our study highlights the therapeutic potential of artemisinin use as a novel strategy for tongue cancer treatment, which acts via modulating both intracellular and intercellular interactions.

Role of the PI3K/AKT signaling pathway in the cellular response to Tumor Treating Fields (TTFields)

Tumor Treating Fields (TTFields) are electric fields that induce cancer cell death. Genomic analysis of glioblastoma tumors resected from TTFields-treated patients suggested a potential link between a reduced or absent response to TTFields and activating mutations in the phosphatidylinositol 3-kinase (PI3K) p110α subunit (PIK3CA). Our study aimed to investigate the role of the PI3K/AKT pathway in the response to TTFields. We tested changes in signaling pathways in control versus TTFields-treated U-87 MG glioblastoma, A2780 ovarian carcinoma, and H1299 non-small cell lung cancer (NSCLC) cells using the Luminex multiplex assay, validated by western blot analysis and inhibition assays. We also performed in vivo validation using immunohistochemistry on tumor sections from animals bearing orthotopic N1-S1 hepatocellular, MOSE-L ovarian, or LL/2 lung tumors that were treated with TTFields or sham. Finally, we examined the efficacy of concomitant treatment with TTFields and PI3K inhibitors in cell lines and mouse models. Our findings elucidate the mechanisms driving PI3K/AKT activation following TTFields treatment, revealing that the AKT signaling amplitude increases over time and is influenced by cell-surface and cell-cell interactions. Specifically, focal adhesion kinase (FAK) and N-cadherin were found to promote AKT phosphorylation, activating cell survival pathways. Furthermore, our investigation revealed that pharmacological inhibition of PI3K sensitized cancer cells to TTFields, both in vitro and in vivo. Our research suggests that the PI3K/AKT pathway is involved in cancer cell response to TTFields, and that inhibition of this pathway may serve as a potential therapeutic target for sensitizing cancer cells to TTFields.

Targeting Signaling Excitability in Cervical and Pancreatic Cancer Cells Through Combined Inhibition of FAK and PI3K.

The Ras/PI3K/ERK signaling network is frequently mutated and overactivated in various human cancers. Focal adhesion kinase (FAK) is commonly overexpressed in several cancer types and has been implicated in treatment resistance mechanisms. A positive feedback loop between Ras, PI3K, the cytoskeleton, and FAK was previously shown to drive Ras signaling excitability. In this study, we investigated the effectiveness of targeting Ras signaling excitability by concurrently inhibiting FAK and PI3K in cervical and pancreatic cancer cells, which depend on activation Ras/PI3K signaling. We found that the combination of FAK and PI3K inhibitors synergistically suppressed the growth of cervical and pancreatic cancer cell lines through increased apoptosis and decreased mitosis. PI3K inhibitors alone caused only a transient suppression of downstream AKT activity and paradoxically increased FAK signaling in cancer cells. The addition of an FAK inhibitor effectively counteracted this PI3K-inhibitor-induced FAK activation. Furthermore, PI3K inhibitors were found to activate multiple receptor tyrosine kinases (RTKs), including insulin receptor, IGF-1R, EGFR, HER2, HER3, AXL, and EphA2. Taken together, our results suggest that FAK inhibition is necessary to counteract the compensatory RTK activation induced by PI3K inhibitors, thereby achieving more effective suppression of cancer cell growth. These findings highlight the therapeutic potential of combined FAK and PI3K inhibition in cancer treatment.

Conformation-Driven Phase Separation in the Linker Domain of Focal Adhesion Kinases.

Protein tyrosine kinase 2 (Pyk2), also known as focal adhesion kinase 2, and focal adhesion kinase 1 (Fak1) are two related nonreceptor tyrosine kinases (hereafter referred to as FAKs). Here, we focused on characterizing a linker region of the FAK proteins (hereafter referred to as FAK KFL for Kinase FAT Linker), which in the case of Pyk2 has previously been shown to play a functional role in calcium sensing through its interaction with calmodulin. Using structural nuclear magnetic resonance spectroscopy, we provide chemical shift assignments for the FAK KFLs, defining their conformational properties. Analysis of the FAK KFL conformations revealed their predominantly disordered nature, except for well-defined segments with a significant tendency to form α-helices, which were modeled to form homodimeric interfaces. In addition, we showed that the FAK KFL segments form condensates in vitro under high crowding conditions. By directly comparing the conformational properties of the Pyk2 and Fak1 KFL domains and providing structural data, this study provides valuable insights into the structural basis of FAK KFL interactions. Furthermore, the results show that disordered segments in proteins within the focal adhesion complex undergo phase separation, a process of potential biological significance due to protein clustering.

Trifunctional Sialylation-Based SF-ZIF@NA Hydrogel for Selective Osteoclast Inhibition and Enhanced Bone-Vessel Regeneration in Osteoporotic Bone Defects.

Osteoporotic bone defects are challenging to repair due to imbalances in bone resorption and formation, coupled with insufficient vascularization. To address these issues, it develops a trifunctional hydrogel (SF-ZIF@NA) designed to selectively inhibit osteoclast activity and enhance vascularized bone regeneration. By enzymatically removing sialic acid, SF-ZIF@NA prevents precursor osteoclasts (pOCs) from fusing into bone-resorbing mature osteoclasts (mOCs), thereby preserving pOCs and their anabolic functions. Additionally, the hydrogel releases Zinc ion (Zn2⁺) in response to acidic conditions, promoting osteogenesis and angiogenesis. In vitro results confirmed that SF-ZIF@NA impedes osteoclast fusion, enhances platelet-derived growth factor-BB (PDGF-BB secretion from pOCs, and activates the FAK (focal adhesion kinase) signaling pathway to stimulate vascularized bone formation. In osteoporotic bone defect models, SF-ZIF@NA accelerated bone repair with increased bone density and vascularization. These findings demonstrate that SF-ZIF@NA offers a targeted and multifunctional strategy for osteoporotic bone regeneration by concurrently modulating osteoclast activity and promoting angiogenesis.

Fisetin alleviates Salmonella typhimurium-induced colitis through the TLR2/TLR4-NF-κB pathway, regulating microbiota, and repressing intracellular bacterial proliferation by focal adhesion kinase.

Salmonella typhimurium is a pathogen responsible for millions of cases of gastroenteritis in both humans and animals. This study aimed to evaluate the hypothesis that fisetin administration could mitigate colon damage and regulate the intestinal microbiota in mice treated with Salmonella typhimurium. Six-week-old male mice were orally administered with or without 100mg/kg fisetin and infected with Salmonella typhimurium. Fisetin administration ameliorated Salmonella typhimurium-induced colitis as shown by the decreased body weight loss, lowered disease activity index score, reduced colon shortening, inflammatory infiltration, and apoptosis. Meanwhile, Salmonella typhimurium exposure upregulated colonic Tnf-α, Il-6, Il-1β, Ifn-β, Ccl2, Ccl8 relative expression and MDA and H2O2 concentrations, whereas lowered Il-10, Nrf2, Nqo1, Coq10b, and Gss abundance, all of which were ameliorated by fisetin treatment. Moreover, fisetin recovered the occludin, zonula occludens-1, and zonula occludens-2 protein abundance in response to Salmonella typhimurium administration. Further investigation demonstrated that the protective role of fisetin was related to the inactivation of the TLR2/TLR4-NF-κB pathway, upregulation of Lactobacillus, and reduction of Salmonella abundance. Additionally, fisetin could limit the intracellular Salmonella typhimurium proliferation, which was likely to be attributed to the inhibition of focal adhesion kinase. Together, our study elucidated the therapeutic potential of fisetin in ameliorating key aspects of colitis, including intestinal inflammation, oxidative stress, and barrier dysfunction, and these beneficial effects were mediated through the inactivation of the TLR2/TLR4-NF-κB pathway, the regulation of microbiota, and restricting the intracellular proliferation of Salmonella typhimurium by inhibiting focal adhesion kinase.

The Role of the CXCL12/CXCR4 Signaling Pathway in Regulating Cellular Migration.

We investigated the CXCL12/CXCR4 signaling pathway as a regulator of adipose-derived stem cell (ADSC) self-assembling toroidal constructs using collagen hydrogels. ADSCs formed toroid rings when cultured on hydrogel surfaces but failed to do so when mixed within the matrix. Gene expression profiling revealed significant upregulation of the CXCL12/CXCR4 pathway in toroid-forming conditions, supported by immunofluorescence studies that confirmed CXCL12 presence in toroids but not in mixed-in cultures. Early toroid formation was marked by the emergence of CXCL12 expression, correlating with cell migration. Targeted inhibition experiments identified the PI3K pathway as a critical regulator, delaying cell migration by ∼16 h, while N-Cadherin, Ras/Raf, and ERK1/2 inhibition either reduced or halted migration over extended periods. Through Western blot analysis, altered expression of α-Smooth muscle actin and focal adhesion kinase under PI3K inhibition was highlighted thus emphasizing their roles in toroid formation. Lastly, initial coculture studies with 4T1 breast cancer cells unexpectedly showed CXCL12 localization primarily in 4T1 cells within mixed toroids, suggesting modified chemotactic signaling. Our findings establish CXCL12/CXCR4 as crucial for ADSC toroid formation and reveal the pathway's complex involvement in cellular organization and migration, presenting a robust model for exploring cell-cell and cell-matrix interactions relevant to tissue engineering and cancer research.

Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

Skeletal mesenchymal stem cells (MSCs) possess self-renewal capacities and play a leading role in the craniofacial system. However, their engagement in controlling cranial bone development and regeneration remains largely unidentified. Herein, we discovered the neurofibromin 2 (Nf2)-encoded regulator Merlin, demonstrating indispensableness in the craniofacial system. Mice lacking Nf2 in MSCs exhibit malformed cranial bones, diminished proliferation, increased apoptosis, and more severe osteogenesis impairment. Mechanically, we substantiate that Nf2 physically interacts with focal adhesion kinase (FAK) to preferentially mediate Erk1/2 and PI3K catalytic p110 subunit/Akt signaling. Meanwhile, Nf2-FAK disturbance in MSCs results in deficient migration, cytoskeletal organization and focal adhesion dynamics, and develops retarded regeneration of cranial bone defects. Collectively, our findings underscore an unrecognized scaffolding role for Nf2-FAK as upstream element in regulating PI3K/Akt and Erk1/2 action in osteoblasts, and illuminate its essentialness in coordinating cell migration, osteogenic lineage development, cranial bone ossification and regeneration.