Focal Adhesion Kinase Research Articles

JAK inhibitors: a new choice for diabetes mellitus?

Altered tyrosine kinase signaling is associated with a variety of diseases. Tyrosine kinases can be classified into two groups: receptor type and nonreceptor type. Nonreceptor-type tyrosine kinases are subdivided into Janus kinases (JAKs), focal adhesion kinases (FAKs) and tec protein tyrosine kinases (TECs). The beneficial effects of receptor-type tyrosine kinase inhibitors (TKIs) for the treatment of diabetes mellitus (DM) and the mechanisms involved have been previously described. Recently, several clinical cases involving the reversal of type 1 diabetes mellitus (T1DM) during treatment with JAK inhibitors have been reported, and clinical studies have described the improvement of type 2 diabetes mellitus (T2DM) during treatment with JAK inhibitors. In vivo and in vitro experimental studies have elucidated some of the mechanisms behind this effect, which seem to be based mainly on the reduction in β-cell disruption and the improvement of insulin resistance. In this review, we briefly describe the beneficial effects of JAK inhibitors among nonreceptor tyrosine kinase inhibitors for the treatment of DM and attempt to analyze the mechanisms involved.

G-protein coupled receptor GPR124 protects against podocyte senescence and injury in diabetic kidney disease.

Although emerging studies highlight the pivotal role of podocyte senescence in the pathogenesis of diabetic kidney disease (DKD) and aging-related kidney diseases, therapeutic strategies for preventing podocyte senescence are still lacking. Here, we identified a previously unrecognized role of GPR124, a novel adhesion G protein-coupled receptor, in maintaining podocyte structure and function by regulation of cellular senescence in DKD. Podocyte GPR124 was significantly reduced in db/db diabetic (a type 2 diabetic mouse model) and streptozocin-induced diabetic mice (a type 1 diabetic model), which was further confirmed in kidney biopsies from patients with DKD. The level of GPR124 in glomeruli was positively correlated with the estimated glomerular filtration rate and negatively correlated with serum creatinine levels. Podocyte-specific deficiency of GPR124 significantly aggravated podocyte injury and proteinuria in the two models of diabetic mice. Moreover, GPR124 regulated podocyte senescence in both diabetic and aged mice. Mechanistically, GPR124 directly bound with vinculin and negatively regulated focal adhesion kinase (FAK) signaling, thereby mediating podocyte senescence and function. Importantly, overexpression of GPR124 or pharmacological inhibition of FAK protected against podocyte senescence and injury under diabetic conditions. Our studies suggest that targeting GPR124 may be an innovative therapeutic strategy for patients with DKD and aging-related kidney diseases.

β1 Integrin/FAK signaling regulates interleukin-8 production in human gingival epithelial Ca9-22 cells.

Interleukin-8 (IL-8), a proinflammatory factor in human tissues, plays an important role in inflammation. Type IV collagen, a key component of the basement membrane, interacts with integrins, which are primary receptors in the extracellular matrix (ECM). Integrins are essential for the regulation of various cellular behaviors and signal transduction pathways. However, the relationship between type IV collagen, β1 integrin, and gingival epithelial cells is poorly understood. The aim in this study was to elucidate the effect of the interaction between type IV collagen and β1 integrin on IL-8 secretion in human gingival epithelial cells (Ca9-22). Ca9-22 cells were treated with or without type IV collagen, and IL-8 production was assessed using an enzyme-linked immunosorbent assay (ELISA). The role of β1 integrin was investigated using a β1 integrin-neutralizing antibody. Western blotting was performed to measure the phosphorylation levels of the relevant proteins. The effects of the focal adhesion kinase (FAK) inhibitor Y15 and the MEK inhibitor U0126 on β1 integrin/FAK and Erk1/2 MAPK pathways in IL-8 production were evaluated to explore the involvement of these signaling pathways. β1 integrin induced IL-8 secretion in the Ca9-22 cells by regulating FAK, Erk1/2, and p130Cas proteins. p130Cas was independent of FAK, whereas Erk1/2 functioned downstream of FAK. Inhibition of FAK or Erk1/2 substantially reduced IL-8 secretion, highlighting their pivotal roles in this signaling pathway. β1 integrin promotes IL-8 secretion in Ca9-22 cells via the β1 integrin/FAK/Erk1/2 signaling pathway. These findings elucidate the pathogenesis of periodontitis and provide a foundation for the development of targeted therapeutic strategies.

RGS4 promotes the progression of gastric cancer through the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition.

Regulator of G protein signaling (RGS) proteins participate in tumor formation and metastasis by acting on the α-subunit of heterotrimeric G proteins. The specific effect of RGS, particularly RGS4, on the progression of gastric cancer (GC) is not yet clear. To explore the role and underlying mechanisms of action of RGS4 in GC development. The prognostic significance of RGS4 in GC was analyzed using bioinformatics based public databases and verified by immunohistochemistry and quantitative polymerase chain reaction in 90 patients with GC. Function assays were employed to assess the carcinogenic impact of RGS4, and the mechanism of its possible influence was detected by western blot analysis. A nude mouse xenograft model was established to study the effects of RGS4 on GC growth in vitro. RGS4 was highly expressed in GC tissues compared with matched adjacent normal tissues. Elevated RGS4 expression was correlated with increased tumor-node-metastasis stage, increased tumor grade as well as poorer overall survival in patients with GC. Cell experiments demonstrated that RGS4 knockdown suppressed GC cell proliferation, migration and invasion. Similarly, xenograft experiments confirmed that RGS4 silencing significantly inhibited tumor growth. Moreover, RGS4 knockdown resulted in reduced phosphorylation levels of focal adhesion kinase, phosphatidyl-inositol-3-kinase, and protein kinase B, decreased vimentin and N-cadherin, and elevated E-cadherin. High RGS4 expression in GC indicates a worse prognosis and RGS4 is a prognostic marker. RGS4 influences tumor progression via the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition.

Anti-Migratory Activity of Brazilin Chemodiversification on Breast Cancer Cells

Breast cancer is the most common and the leading cause of cancer death in women worldwide; treating invasive breast carcinomas is challenging due to the side effects of chemotherapeutics. Compounds isolated from natural sources have been proposed as potential molecules for cancer therapy; for instance, the homoisoflavonoid brazilin has shown pharmacological properties, including anti-tumoral and anti-inflammatory activities. In this study, we isolated brazilin from the heartwood of Haematoxylum brasiletto; then, we performed a semi-synthesis by adding three methyl or acetyl groups to the core structure of brazilin. We confirmed the identity of brazilin and its derivatives by spectroscopic data (1H NMR and 13C NMR) and measured their purity by optical rotation. Then, we analyzed the effects of brazilin and its derivatives in three mammary gland-derived cell lines: the TNBC MDA-MB-231, the ERα(+) MCF7, and the non-tumorigenic MCF10A. We evaluated the cell viability by MTT assays, cell migration by wound-healing assays, and focal adhesion kinase (FAK) activation by Western blot. Regarding biological assays, the MTT assay showed that these compounds showed cytotoxic effects on the MCF7 and MDA-MB-231 breast cancer cells at 20 µM but was not toxic in non-tumorigenic MCF10A mammary epithelial cells. Specifically, the greatest effects found from treatment with the compounds were in the MDA-MB-231 cell line, where the IC50 of brazilin was 49.92 μM, and for MCF7, the brazilin-(OAc)3 was 49.97 μM. These effects were dose- and time-dependent, as well as being associated with a decrease in the levels of cell migration and FAK activation.

First Report on Microbial-Derived Polydeoxyribonucleotide: A Sustainable and Enhanced Alternative to Salmon-Based Polydeoxyribonucleotide.

Polydeoxyribonucleotide (PDRN) has emerged as a potent bioactive compound with proven efficacy in wound healing, tissue regeneration, and anti-inflammatory applications and is predominantly derived from salmonid gonads. However, this study presents a groundbreaking advancement by successfully extracting and characterizing PDRN from microbial sources, specifically Lactobacillus rhamnosus, marking the first report to utilize microbial-, biome-, or Lactobacillus-derived PDRN (L-PDRN). The findings demonstrate the enhanced biological properties of L-PDRN over traditional salmon-derived PDRN across several assays. L-PDRN exhibited superior antioxidant activity, with significantly higher SOD-like and DPPH radical scavenging activities compared to PDRN, particularly at higher concentrations. In wound-healing assays, L-PDRN demonstrated superior efficacy in promoting cell migration and wound closure, even under inflammatory conditions induced by tumor necrosis factor (TNF-α). Additionally, L-PDRN demonstrated the potential for enhanced immunostimulatory effects under non-inflammatory conditions while maintaining anti-inflammatory properties under lipopolysaccharide (LPS) stimulation. Electrophoretic analysis revealed that L-PDRN consists of smaller DNA fragments (under 100 bp) compared to salmon-derived PDRN (200-800 bp), suggesting greater bioavailability and skin absorption. Mechanistic studies confirmed that L-PDRN activates the focal adhesion kinase (FAK) and protein kinase B (AKT) signaling pathway through the A2A receptor, similar to PDRN, while also engaging alternative pathways for p38 and ERK phosphorylation, highlighting its signaling versatility. This study underscores the potential of L-PDRN as a multifunctional and sustainable alternative to salmon-derived PDRN, offering enhanced bioactivity, scalability, and environmental benefits. The novel approach of utilizing microbial-derived PDRN opens new avenues for therapeutic applications in oxidative stress management, tissue regeneration, and immune modulation, paving the way for a paradigm shift in PDRN sourcing and functionality.

Coordination of Focal Adhesion Nanoarchitecture and Dynamics in Mechanosensing for Cardiomyoblast Differentiation.

Focal adhesions (FAs) are force-bearing multiprotein complexes, whose nanoscale organization and signaling are essential for cell growth and differentiation. However, the specific organization of FA components to exert spatiotemporal activation of FA proteins for force sensing and transduction remains unclear. In this study, we unveil the intricacies of FA protein nanoarchitecture and that its dynamics are coordinated by a molecular scaffold protein, BNIP-2, to initiate downstream signal transduction for cardiomyoblast differentiation. Within the FAs, BNIP-2 regulates the nano-organization of focal adhesion kinase (FAK), and the dynamics of FAK, paxillin, and vinculin. Depletion of BNIP-2 resulted in altered focal adhesion numbers and sizes per cell, reduced traction force, and decreased FA sensitivity for mechanosensing. At the molecular level, the loss of BNIP-2 disrupted the FAK-paxillin signaling axis, where FAK inhibition reproduces the effects of BNIP-2 loss by impairing the phosphorylation of both FAK and paxillin. Mechanistically, BNIP-2 preferentially binds to constitutively active FAK and acts as a molecular scaffold to mediate interactions between FAK and paxillin and between paxillin and vinculin. We have validated BNIP-2's role in the FAK-paxillin signaling axis in human embryonic stem cells (hESC). Furthermore, we showed that depletion of BNIP-2 resulted in changes in signature gene targets at the cardiac progenitor stage of differentiation. In summary, we showed that the intricate interplay of FA nanoarchitecture and dynamics, governed by BNIP-2, is crucial for force transduction and biochemical signaling in driving cardiomyoblast differentiation.

Point contact-restricted cAMP signaling controls ephrin-A5-induced axon repulsion.

Signal transduction downstream of axon guidance molecules is essential to steer developing axons. Second messengers including cAMP are key molecules shared by a multitude of signaling pathways and are required for a wide range of cellular processes including axon pathfinding. Yet, how these signaling molecules achieve specificity for each of their downstream pathways remains elusive. Subcellular compartmentation emerged as a flexible strategy to reach such a specificity. Here, we show that point contact-restricted cAMP signals control ephrin-A5-evoked axon repulsion in vitro by modulating Focal Adhesion Kinase phosphorylation and the assembly and disassembly rate of point contacts. Consistently, preventing point contact-specific cAMP signals, in developing retinal ganglion cells in vivo alters the refinement of their terminal axonal arbor in the brain. Altogether, our study identifies point contacts as a compartment containing a local cAMP signal required for ephrin-A5-dependent axon guidance and highlights the crucial role of such subcellularly restricted second messenger signals in the wiring of neuronal circuits.

A Novel In Vitro Model of the Bone Marrow Microenvironment in Acute Myeloid Leukemia Identifies CD44 and Focal Adhesion Kinase as Therapeutic Targets to Reverse Cell Adhesion-Mediated Drug Resistance.

Acute myeloid leukemia (AML) is an aggressive neoplasm. Although most patients respond to induction therapy, they commonly relapse due to recurrent disease in the bone marrow microenvironment (BMME). So, the disruption of the BMME, releasing tumor cells into the peripheral circulation, has therapeutic potential. Using both primary donor AML cells and cell lines, we developed an in vitro co-culture model of the AML BMME. We used this model to identify the most effective agent(s) to block AML cell adherence and reverse adhesion-mediated treatment resistance. We identified that anti-CD44 treatment significantly increased the efficacy of cytarabine. However, some AML cells remained adhered, and transcriptional analysis identified focal adhesion kinase (FAK) signaling as a contributing factor; the adhered cells showed elevated FAK phosphorylation that was reduced by the FAK inhibitor, defactinib. Importantly, we demonstrated that anti-CD44 and defactinib were highly synergistic at diminishing the adhesion of the most primitive CD34high AML cells in primary autologous co-cultures. Taken together, we identified anti-CD44 and defactinib as a promising therapeutic combination to release AML cells from the chemoprotective AML BMME. As anti-CD44 is already available as a recombinant humanized monoclonal antibody, the combination of this agent with defactinib could be rapidly tested in AML clinical trials.

Conformational dynamics and multi-modal interaction of Paxillin with the Focal Adhesion Targeting Domain.

Paxillin (PXN) and focal adhesion kinase (FAK) are two major components of the focal adhesion complex, a multiprotein structure linking the intracellular cytoskeleton to the cell exterior. PXN interacts directly with the C-terminal targeting domain of FAK (FAT) via its intrinsically disordered N-terminal domain. This interaction is necessary and sufficient for localizing FAK to focal adhesions. Furthermore, PXN serves as a platform for recruiting other proteins that together control the dynamic changes needed for cell migration and survival. Here, we show that the PXN disordered region undergoes large-scale conformational restriction upon binding to FAT, forming a 48-kDa multi-modal complex consisting of four major interconverting states. Although the complex is flexible, each state has unique sets of contacts involving disordered regions that are both highly represented in ensembles and conserved. Moreover, conserved intramolecular contacts from glutamine-rich regions in PXN contribute to high entropy and thus stability of the FAT bound complex. As PXN is a hub protein, the results provide a structural basis for understanding how perturbations that lead to cellular network rewiring, such as ligand binding and phosphorylation, may lead to shifts in the multi-state equilibrium and phenotypic switching.

Suppression of Dad1 induces cardiomyocyte death by weakening cell adhesion.

As cardiomyocyte loss causes heart failure, inhibition of cardiomyocyte death may be a therapeutic strategy against heart failure. In this study, we have identified defender against cell death 1 (Dad1) as a candidate regulator of cardiomyocyte death, using complementary DNA microarray and siRNA knockdown screening. Dad1 is a subunit of oligosaccharyltransferase (OST) complex that is responsible for protein N-glycosylation; however, its function in cardiomyocytes remains unknown. Importantly, the knockdown of Dad1 using siRNA reduced the viability of neonatal rat cardiomyocytes (NRCMs), accompanied by cleaved caspase3 expression, independent of endoplasmic reticulum stress. Dad1 knockdown impaired cell spreading and reduced myofibrillogenesis in NRCMs, suggesting that Dad1 knockdown induced anoikis, apoptosis by disrupting cell-matrix interactions. Consistently, knockdown of Dad1 impaired N-glycosylation of integrins α5 and β1, accompanied by inactivation of focal adhesion kinase. When cell adhesion was enhanced using adhesamine, fibronectin, or collagen type IV, cardiomyocyte death induced by Dad1 knockdown was reduced. Dad1 knockdown decreased the expression of staurosporine and temperature-sensitive 3 A (Stt3A), a catalytic subunit of OST complex. Interestingly, Stt3A knockdown using Stt3A siRNA reduced the expression of Dad1, indicating that both Dad1 and Stt3A were required for OST stabilization. In conclusion, Dad1 plays an important role in maintaining the expression of mature N-glycosylated integrins and their downstream signaling molecules to suppress cardiomyocyte anoikis.NEW & NOTEWORTHY This study found for the first time that the knockdown of Dad1 induced cardiomyocyte death, accompanied by impairment of myofibrillogenesis and cell spreading. Dad1 regulates the N-glycosylation of integrins in cooperation with Stt3A and preserves cell adhesion activity, promoting cardiomyocyte survival. This is the first demonstration that Dad1 contributes to the maintenance of cardiac homeostasis through the posttranslational modification of integrins, providing a novel insight into the biological significance of OST complex in cardiomyocytes.

Optimizing gelation time for cell shape control through active learning.

Hydrogels are popular platforms for cell encapsulation in biomedicine and tissue engineering due to their soft, porous structures, high water content, and excellent tunability. Recent studies highlight that the timing of network formation can be just as important as mechanical properties in influencing cell morphologies. Conventionally, time-dependent properties can be achieved through multi-step processes. In contrast, one-pot synthesis can improve both the efficiency and uniformity of cell encapsulation. Reaction kinetics are sensitive to temperatures and pH conditions, thus, monitoring gelation time across different conditions is essential for formulation. In this work, we choose tetra-poly(ethylene glycol) (TPEG) macromers as a model system to examine the relationship between the rate of polymer network formation and cell morphology. Previous studies of this system focused on reactions at neutral pH and room temperature, leaving much of the formulation space underexplored. We use Gaussian process regression (GPR) to minimize response surface errors by strategically selecting additional investigation points based on prior knowledge. Then we extend the knowledge from pre-trained data at neutral pH to a new surface at physiological pH. We find that the gelation time surface can effectively predict the aspect ratio of the encapsulated cells. Additionally, through focal adhesion kinase inhibition, we show that cell shape is influenced by the properties of the forming network in the initial hours as cells develop connections with the matrix. We demonstrate the utility of a high-throughput microrheology approach in enhancing fabrications of synthetic extracellular matrix and cell assemblies.

Mechanical Stretch Control of Adipocyte AKT Signaling and the Role of FAK and ROCK Mechanosensors.

Adipose tissue in vivo is physiologically exposed to compound mechanical loading due to bodyweight bearing, posture, and motion. The capability of adipocytes to sense and respond to mechanical loading milieus to influence metabolic functions may provide a new insight into obesity and metabolic diseases such as type 2 diabetes (T2D). Here, we evidenced physiological mechanical loading control of adipocyte insulin signaling cascades. We exposed differentiated 3T3-L1 adipocytes to mechanical stretching and assessed key markers of insulin signaling, AKT activation, and GLUT4 translocation, required for glucose uptake. We showed that cyclic stretch loading at 5% strain and 1 Hz frequency increases AKT phosphorylation and GLUT4 translocation to the plasma membrane by approximately two-fold increases compared to unstretched controls for both markers as assessed by immunoblotting (p < 0.05). These results indicate that cyclic stretching activates insulin signaling and GLUT4 trafficking in adipocytes. In the mechanosensing mechanism study, focal adhesion kinase (FAK) inhibitor (FAK14) and RhoA kinase (ROCK) inhibitor (Y-27632) impaired actin cytoskeleton structural formation and significantly suppressed the stretch induction of AKT phosphorylation in adipocytes (p < 0.001). This suggests the regulatory role of focal adhesion and cytoskeletal mechanosensing in adipocyte insulin signaling under stretch loading. Our finding on the impact of mechanical stretch loading on key insulin signaling effectors in differentiated adipocytes and the mediatory role of focal adhesion and cytoskeleton mechanosensors is the first of its kind to our knowledge. This may suggest a therapeutic potential of mechanical loading cue in improving conditions of obesity and T2D. For instance, cyclic mechanical stretch loading of adipose tissue could be explored as a tool to improve insulin sensitivity in patients with obesity and T2D, and the mediatory mechanosensors such as FAK and ROCK may be targeted to further invigorate stretch-induced insulin signaling activation.

Design, Synthesis, and Biochemical Evaluation of Novel MLK3 Inhibitors: A Target Hopping Example.

The human kinome has tremendous medical potential. In the past decade, mixed-lineage protein kinase 3 (MLK3) has emerged as an interesting and druggable target in oncogenic signaling. The important role of MLK3 has been demonstrated in several types of cancer. In a target hopping example we started with the focal adhesion kinase (FAK) inhibitor PF-431396 (10), which shows off-target activity toward MLK3. We were able to develop highly active compounds in the single digit nanomolar range for MLK3. Furthermore, we achieved a dramatic shift in selectivity from FAK to MLK3. Here we present a new chemical class of MLK3 inhibitors, including our lead compound 37 with an outstanding IC50 value of <1 nM in a biochemical MLK3 assay while simultaneously exhibiting kinome-wide selectivity.

MFAP2 upregulation promotes ESCC metastasis via FAK-AKT signaling pathway.

Metastasis is the leading cause of mortality from esophageal squamous cell carcinoma (ESCC). By the time of diagnosis, most ESCC tumors have already invaded the lymph nodes or distant organs; however, it has been challenging to identify and confirm genes with a crucial role in ESCC metastasis. The microfibrillar-associated protein 2 (MFAP2) is upregulated in human ESCC, and its expression level was positively associated with poor overall and disease-free survival. Consistently, upregulation of MFAP2 promoted the metastasis and invasion of ESCC cells invitro and invivo. Conversely, these processes were reduced by MFAP2 knockdown. Mechanistically, MFAP2 was shown to bind to the FERM domain of focal adhesion kinase (FAK) and to alleviate FAK intramolecular inhibition, resulting in the enhanced binding affinity between FAK and integrin beta 4 (ITGB4) and activation of the FAK-AKT signaling pathway. Treatment of ESCC cells with the FAK inhibitor PND-1186 reduced MFAP2, induced the activation of the FAK-AKT pathway invitro, and suppressed lung metastasis in a mouse model of ESCC. These findings support a major role for MFAP2 in promoting ESCC metastasis, in part via the activation of FAK-AKT signaling, and highlight the potential of MFAP2 as a promising therapeutic target for ESCC.

Neurturin gene IVSI-663 polymorphism but not RET variants is associated with increased risk for breast cancer.

Gene polymorphisms of rearranged during transfection (RET) and its ligand neurturin (NRTN) are one of the focus of studies in the investigation of cancer pathogenesis, invasion, and metastasis. In this study, we aimed to examine the possible risk of breast cancer between RET G691S, L769L, S904S, and NRTN IVSI-663 polymorphisms and to evaluate serum NRTN, brain-derived neurotrophic factor (BDNF), matrix metalloproteinase (MMP)-2, MMP-9, and focal adhesion kinase (FAK) levels. The study consists of 110 breast cancer patients and 110 controls. Polymorphisms were detected by the polymerase chain reaction method from study groups whole blood. The NRTN IVSI-663 polymorphism in G allele has been found to be 1.54 fold increased the risk of breast cancer, however AA genotype has been found 0.43 fold decreased the risk of breast cancer (P < .05, P < .05, respectively). Study groups showed a similar profile for RET G691S, L769L, S904S allele frequencies and genotype distributions (P > .05). In the patient group, significant increase in serum NRTN and FAK levels and decrease in MMP-2 and MMP-9 levels were found (P < .05, P < .05, P < .05, P < .05, respectively). In summary that increased breast cancer risk with the G allele in NRTN gene IVSI-663 polymorphism, as well as the increased serum NRTN and FAK levels, will contribute to the diagnosis, prognosis and determination of new treatment strategies.

O-GlcNAcylation of Focal Adhesion Kinase Regulates Cell Adhesion, Migration, and Proliferation via the FAK/AKT Pathway.

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase pivotal in cellular signal transduction, regulating cell adhesion, migration, growth, and survival. However, the regulatory mechanisms of FAK during tumorigenesis and progression still need to be fully understood. Our previous study demonstrated that O-GlcNAcylation regulates integrin-mediated cell adhesion. To further elucidate the underlying molecular mechanism, we focused on FAK in this study and purified it from 293T cells. Using liquid chromatography-mass spectrometry (LC-MS/MS), we identified the O-GlcNAcylation of FAK at Ser708, Thr739, and Ser886. Compared with wild-type FAK expressed in FAK-knockout 293T cells, the FAK mutant, in which Ser708, Thr739, and Ser886 were replaced with Ala, exhibited lower phosphorylation levels of Tyr397 and AKT. Cell proliferation and migration, assessed through MTT and wound healing assays, were significantly suppressed in the FAK mutant cells compared to the wild-type FAK cells. Additionally, the interaction among FAK, paxillin, and talin was enhanced, and cell adhesion was increased in the mutant cells. These data indicate that specific O-GlcNAcylation of FAK plays a critical regulatory role in integrin-mediated cell adhesion and migration. This further supports the idea that O-GlcNAcylation is essential for tumorigenesis and progression and that targeting the O-GlcNAcylation of FAK could offer a promising therapeutic strategy for cancer treatment.

FAK and p130Cas Modulate Stiffness-Mediated Early Transcription and Cellular Metabolism.

Cellular metabolism is influenced by the stiffness of the extracellular matrix. Focal adhesion kinase (FAK) and its binding partner, p130Cas, transmit biomechanical signals, such as substrate stiffness, to the cell to regulate a variety of cellular responses, but their roles in early transcriptional and metabolic responses remain largely unexplored. We cultured mouse embryonic fibroblasts with or without siRNA-mediated FAK or p130Cas knockdown and assessed the early transcriptional responses of these cells to placement on soft and stiff substrates by RNA sequencing and bioinformatics analyses. Exposure to the stiff substrate altered the expression of genes important for metabolic and biosynthetic processes, and these responses were influenced by knockdown of FAK and p130Cas. Our findings reveal that FAK-p130Cas signaling mechanotransduces substrate stiffness to early transcriptional changes that alter cellular metabolism and biosynthesis.

Pharmacokinetics and bioavailability of febrifugine in rat plasma determined by UPLC-MS/MS

AbstractFebrifugine, a potent quinazolinone compound derived from the Chinese herb Chang Shan (Dichroa febrifuga), exhibits diverse biological activities and has demonstrated anti-tumor effects by functioning as focal adhesion kinase (FAK) inhibitors. In this study, our objective was to establish a quantitative UPLC-MS/MS method and investigate the pharmacokinetic characteristics of febrifugine in rats following intravenous and oral administration routes. The rat tail vein was used for the collection of blood samples at designated time intervals following intravenous (2.0 mg kg−1) and oral (6.0 mg kg−1) administrations. Plasma samples were pretreated with acetonitrile as a protein precipitant and methylcytisine as an internal standard. Febrifugine concentration in rat plasma was determined using the UPLC-MS/MS method, and pharmacokinetic parameters were calculated using drug and statistics (DAS) software version for statistical analysis. The linear range of febrifugine in rat plasma was 1.5–1,500 ng mL−1, meeting the precision, recovery, and stability requirements for determination purposes. Febrifugine had a half-life (t1/2) of 3.2 ± 1.6 h after administered via intravenous route, while t1/2 was 2.6 ± 0.5 h after oral administration. The developed UPLC-MS/MS method is facile to operate while adhering to rigorous methodological verification standards, rendering it suitable for investigating the pharmacokinetics of febrifugine; and bioavailability was determined as 45.8%.

FAK activity exacerbates disturbed flow-mediated atherosclerosis via VEGFR2-Cbl-NF-κB signaling.

Atherosclerosis develops at predictable sites in the vasculature where branch points and curvatures create non-laminar disturbed flow. This disturbed flow causes vascular inflammation by increased endothelial cell (EC) barrier permeability and the expression of inflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1). Vascular endothelial growth factor receptor 2 (VEGFR2) is important for flow-induced EC inflammation; however, there are still some gaps in the signaling pathway. Focal adhesion kinase (FAK) is a protein tyrosine kinase whose expression has been implicated in flow-mediated signaling in ECs. However, the link between FAK and VEGFR2 in flow-mediated inflammation signaling has remained unelucidated. Here we found that priming of VEGFR2 with VEGF was critical for flow-mediated activation of FAK and NF-κB. Mechanistically, FAK activation triggers tyrosine phosphorylation of Casitas B-lineage lymphoma (CBL; an E3 ubiquitin ligase) that interacts with VEGFR2 under flow conditions. Further, Apoe-/- mice fed a western diet (WD) exhibited increased FAK activity within the atheroprone disturbed flow region of the inner aortic arch compared to the outer arch. Disturbed flow-induced FAK activation is associated with elevated VEGFR2 on the surface of ECs of the inner aortic arch, but not in the outer arch. Taken together, these data suggest that suppression of augmented FAK activity under disturbed flow may prove beneficial in reducing pro-inflammatory signaling of the endothelial layer.