GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

AMBRA1 levels predict resistance to MAPK inhibitors in melanoma.

Intrinsic and acquired resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma remains a major therapeutic challenge. Here, we show that the clinical development of resistance to MAPKi is associated with reduced tumor expression of the melanoma suppressor Autophagy and Beclin 1 Regulator 1 (AMBRA1) and that lower expression levels of AMBRA1 predict a poor response to MAPKi treatment. Functional analyses show that loss of AMBRA1 induces phenotype switching and orchestrates an extracellular signal-regulated kinase (ERK)-independent resistance mechanism by activating focal adhesion kinase 1 (FAK1). In both in vitro and in vivo settings, melanomas with low AMBRA1 expression exhibit intrinsic resistance to MAPKi therapy but higher sensitivity to FAK1 inhibition. Finally, we show that the rapid development of resistance in initially MAPKi-sensitive melanomas can be attributed to preexisting subclones characterized by low AMBRA1 expression and that cotreatment with MAPKi and FAK1 inhibitors (FAKi) effectively prevents the development of resistance in these tumors. In summary, our findings underscore the value of AMBRA1 expression for predicting melanoma response to MAPKi and supporting the therapeutic efficacy of FAKi to overcome MAPKi-induced resistance.

Mechanosensor Piezo1 mediates bimodal patterns of intracellular calcium and FAK signaling.

Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling. Laser-induced shockwaves (LIS) and its associated shear stress can mechanically activate Piezo1 to induce transient intracellular calcium (Ca[i] ) elevation, accompanied by an increase in FAK activity. Interestingly, multiple pulses of shockwave stimulation caused a more sustained calcium increase and a decrease in FAK activity. Similarly, tuning the degree of Piezo1 activation by titrating either the dosage of Piezo1 ligand Yoda1 or the expression level of Piezo1 produced a similar bimodal pattern of FAK responses. Further investigations revealed that SHP2 serves as an intermediate regulator mediating this bimodal pattern in Piezo1 sensing and signaling. These results suggest that the degrees of Piezo1 activation induced by both mechanical LIS and chemical ligand stimulation may determine downstream signaling characteristics.

ICAM-1 Deletion Using CRISPR/Cas9 Protects the Brain from Traumatic Brain Injury-Induced Inflammatory Leukocyte Adhesion and Transmigration Cascades by Attenuating the Paxillin/FAK-Dependent Rho GTPase Pathway.

Intercellular adhesion molecule-1 (ICAM-1) is identified as an initiator of neuroinflammatory responses that lead to neurodegeneration and cognitive and sensory-motor deficits in several pathophysiological conditions including traumatic brain injury (TBI). However, the underlying mechanisms of ICAM-1-mediated leukocyte adhesion and transmigration and its link with neuroinflammation and functional deficits following TBI remain elusive. Here, we hypothesize that blocking of ICAM-1 attenuates the transmigration of leukocytes to the brain and promotes functional recovery after TBI. The experimental TBI was induced in vivo by fluid percussion injury (25 psi) in male and female wild-type and ICAM-1-/- mice and in vitro by stretch injury (3 psi) in human brain microvascular endothelial cells (hBMVECs). We treated hBMVECs and animals with ICAM-1 CRISPR/Cas9 and conducted several biochemical analyses and demonstrated that CRISPR/Cas9-mediated ICAM-1 deletion mitigates blood-brain barrier (BBB) damage and leukocyte transmigration to the brain by attenuating the paxillin/focal adhesion kinase (FAK)-dependent Rho GTPase pathway. For analyzing functional outcomes, we used a cohort of behavioral tests that included sensorimotor functions, psychological stress analyses, and spatial memory and learning following TBI. In conclusion, this study could establish the significance of deletion or blocking of ICAM-1 in transforming into a novel preventive approach against the pathophysiology of TBI.

Structural basis for TNIP1 binding to FIP200 during mitophagy.

TNIP1 has been increasingly recognized as a security check to finely adjust the rate of mitophagy by disrupting the recycling of the Unc-51-like kinase complex during autophagosome formation. Through tank-binding kinase 1-mediated phosphorylation of the TNIP1 FIP200 interacting region (FIR) motif, the binding affinity of TNIP1 for FIP200, a component of the Unc-51-like kinase complex, is enhanced, allowing TNIP1 to outcompete autophagy receptors. Consequently, FIP200 is released from the autophagosome, facilitating further autophagosome expansion. However, the molecular basis by which FIP200 utilizes its claw domain to distinguish the phosphorylation status of residues in the TNIP1 FIR motif for recognition is not well understood. Here, we elucidated multiple crystal structures of the complex formed by the FIP200 claw domain and various phosphorylated TNIP1 FIR peptides. Structural and isothermal titration calorimetry analyses identified the crucial residues in the FIP200 claw domain responsible for the specific recognition of phosphorylated TNIP1 FIR peptides. Additionally, utilizing structural comparison and molecular dynamics simulation data, we demonstrated that the C-terminal tail of TNIP1 peptide affected its binding to the FIP200 claw domain. Moreover, the phosphorylation of TNIP1 Ser123 enabled the peptide to effectively compete with the peptide p-CCPG1 (the FIR motif of the autophagy receptor CCPG1) for binding with the FIP200 claw domain. Overall, our work provides a comprehensive understanding of the specific recognition of phosphorylated TNIP1 by the FIP200 claw domain, marking an initial step toward fully understanding the molecular mechanism underlying the TNIP1-dependent inhibition of mitophagy.

Focal adhesion kinase-mediated signaling controls the onset of pancreatic cell differentiation.

Signals from the endothelium play a pivotal role in pancreatic lineage commitment. As such, the fate of the epithelial cells relies heavily on the spatiotemporal recruitment of the endothelial cells to the embryonic pancreas. Although it is known that VEGFA secreted by the epithelium recruits the endothelial cells to the specific domains within the developing pancreas, the mechanism that controls the timing of such recruitment is poorly understood. Here, we have assessed the role of focal adhesion kinase (FAK) in mouse pancreatic development based on our observation that the presence of the enzymatically active form of FAK (pFAK) in the epithelial cells is inversely correlated with vessel recruitment. To study the role of FAK in the pancreas, we conditionally deleted the gene encoding focal adhesion kinase in the developing mouse pancreas. We found that homozygous deletion of Fak (Ptk2) during embryogenesis resulted in ectopic epithelial expression of VEGFA, abnormal endothelial recruitment and a delay in endocrine and acinar cell differentiation. The heterozygous mutants were born with no pancreatic phenotype but displayed gradual acinar atrophy due to cell polarity defects in exocrine cells. Together, our findings imply a role for FAK in controlling the timing of pancreatic lineage commitment and/or differentiation in the embryonic pancreas by preventing endothelial recruitment to the embryonic pancreatic epithelium.

α-Catenin promotes dermal fibroblasts proliferation and migration during wound healing via FAK/YAP activation.

Skin wound healing is a complex and organized biological process, and the dermal fibroblasts play a crucial role. α-Catenin is known to be involved in regulating various cellular signals, and its role in wound healing remains unclear. Here, we have identified the pivotal role of the α-catenin/FAK/YAP signaling axis in the proliferation and migration of dermal fibroblasts, which contributes to the process of skin wound healing. Briefly, when α-catenin was knocked down specifically in dermal fibroblasts, the wound healing rate is significantly delayed. Moreover, interfering with α-catenin can impede the proliferation and migration of dermal fibroblasts both in vitro and in vivo. Mechanistically, the overexpression of α-catenin upregulates the nuclear accumulation of YAP and transcription of downstream target genes, resulting in enhanced the proliferation and migration of dermal fibroblasts. Furthermore, the FAK Tyr397 phosphorylation inhibitor blocked the promoting effects of α-catenin on YAP activation. Importantly, the continuous phosphorylation mutation of FAK Tyr397 reversed the retardatory effects of α-catenin knockdown on wound healing, by increasing the vitality of fibroblasts. Likewise, α-catenin/FAK was validated as a therapeutic target for wound healing in the db/db chronic trauma model. In summary, our findings have revealed a novel mechanism by which α-catenin facilitates the function of fibroblasts through the activity of the FAK/YAP signaling axis. These findings define a promising therapeutic strategy for accelerating the wound healing process.

FOXS1 acts as an oncogene and induces EMT through FAK/PI3K/AKT pathway by upregulating HILPDA in prostate cancer.

Prostate cancer (PCa) is a widespread global health concern characterized by elevated rates of occurrence, and there is a need for novel therapeutic targets to enhance patient outcomes. FOXS1 is closely linked to different cancers, but its function in PCa is still unknown. The expression of FOXS1, its prognostic role, clinical significance in PCa, and the potential mechanism by which FOXS1 affects PCa progression were investigated through bioinformatics analysis utilizing public data. The levels of FOXS1 and HILPDA were evaluated in clinical PCa samples using various methods, such as western blotting, immunohistochemistry, and qRT-PCR. To examine the function and molecular mechanisms of FOXS1 in PCa, a combination of experimental techniques including CCK-8 assay, flow cytometry, wound-healing assay, Transwell assay, and Co-IP assay were employed. The FOXS1 expression levels were significantly raised in PCa, correlating strongly with tumor aggressiveness and an unfavorable prognosis. Regulating FOXS1 expression, whether upregulating or downregulating it, correspondingly enhanced or inhibited the growth, migration, and invasion capabilities of PCa cells. Mechanistically, we detected a direct interaction between FOXS1 and HILPDA, resulting in the pathway activation of FAK/PI3K/AKT and facilitation EMT in PCa cells. FOXS1 collaborates with HILPDA to initiate EMT, thereby facilitating the PCa progression through the FAK/PI3K/AKT pathway activation.

Enzymatic activity of fibroblast activation protein-α is essential for TGF-ß1-induced fibroblastic differentiation of human periodontal ligament cells.

Human periodontal ligament cells (hPDLCs) contain multipotent postnatal stem cells that can differentiate into PDL fibroblasts, osteoblasts, and cementoblasts. Interaction between the extracellular environment and stem cells is an important factor for differentiation into other progenitor cells. To identify cell surface molecules that induce PDL fibroblastic differentiation, we developed a series of monoclonal antibodies against membrane/ECM molecules. One of these antibodies, an anti-PDL25 antibody, recognizes approximately a 100 kDa protein, and this antigenic molecule accumulates in the periodontal ligament region of tooth roots. By mass spectrometric analysis, we found that the antigenic molecule recognized by the anti-PDL25 antibody is fibroblast activation protein α (FAPα). The expression level of FAPα/PDL25 increased in TGF-ß1-induced PDL fibroblasts, and this protein was localized in the cell boundaries and elongated processes of the fibroblastic cells. Ectopic expression of FAPα induced fibroblastic differentiation. In contrast, expression of representative markers for PDL differentiation was decreased by knock down and antibody blocking of FAPα/PDL25. Inhibition of dipeptidyl peptidase activity by a potent FAPα inhibitor dramatically inhibited PDL fibroblastic marker expression but did not affect in cell proliferation and migration.

DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway.

Cullin-RING ubiquitin ligase 4 (CRL4) is closely correlated with the incidence and progression of ovarian cancer. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate-recognition protein in the CRL4 E3 ubiquitin ligase complex, is involved in the occurrence and development of ovarian cancer. However, its precise function and the underlying molecular mechanism in this disease remain unclear. In this study, we confirmed that DCAF13 is highly expressed in human ovarian cancer and its expression is negatively correlated with the overall survival rate of patients with ovarian cancer. We then used CRISPR/Cas9 to knockout DCAF13 and found that its deletion significantly inhibited the proliferation, colony formation, and migration of human ovarian cancer cells. In addition, DCAF13 deficiency inhibited tumor proliferation in nude mice. Mechanistically, CRL4-DCAF13 targeted Fraser extracellular matrix complex subunit 1 (FRAS1) for polyubiquitination and proteasomal degradation. FRAS1 influenced the proliferation and migration of ovarian cancer cell through induction of the focal adhesion kinase (FAK) signaling pathway. These findings collectively show that DCAF13 is an important oncogene that promotes tumorigenesis in ovarian cancer cells by mediating FRAS1/FAK signaling. Our findings provide a foundation for the development of targeted therapeutics for ovarian cancer.

Tumor FAK orchestrates immunosuppression in ovarian cancer via the CD155/TIGIT axis.

High-grade serous ovarian cancer (HGSOC) is a lethal malignancy characterized by an immunosuppressive tumor microenvironment containing few tumor infiltrating lymphocytes (TILs) and an insensitivity to checkpoint inhibitor immunotherapies. Gains in the PTK2 gene encoding focal adhesion kinase (FAK) at Chr8 q24.3 occur in ∼70% of HGSOC tumors, and elevated FAK messenger RNA (mRNA) levels are associated with poor patient survival. Herein, we show that active FAK, phosphorylated at tyrosine-576 within catalytic domain, is significantly increased in late-stage HGSOC tumors. Active FAK costained with CD155, a checkpoint receptor ligand for TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains), in HGSOC tumors and a selective association between FAK and TIGIT checkpoint ligands were supported by patient transcriptomic database analysis. HGSOC tumors with high FAK expression were associated with low CD3 mRNA levels. Accordingly, late-stage tumors showed elevated active FAK staining and significantly lower levels of CD3+ TILs. Using the KMF (Kras, Myc, FAK) syngeneic ovarian tumor model containing spontaneous PTK2 (FAK) gene gains, the effects of tumor intrinsic genetic or oral small molecule FAK inhibitior (FAKi; VS-4718) were evaluated in vivo. Blocking FAK activity decreased tumor burden, suppressed ascites KMF-associated CD155 levels, and increased peritoneal TILs. The combination of FAKi with blocking TIGIT antibody (1B4) maintained elevated TIL levels and reduced TIGIT+ T regulatory cell levels, prolonged host survival, increased CXCL13 levels, and led to the formation of omental tertiary lymphoid structures. Collectively, our studies support FAK and TIGIT targeting as a rationale immunotherapy combination for HGSOC.

mTORC1/rpS6 and p-FAK-Y407 signaling regulate spermatogenesis: Insights from studies of the adjudin pharmaceutical/toxicant model.

In rodents and humans, the major cellular events at spermatogenesis include self-renewal of spermatogonial stem cells and undifferentiated spermatogonia via mitosis, commitment of spermatogonia to differentiation and transformation to spermatocytes, meiosis, spermiogenesis, and the release of spermatozoa at spermiation. While details of the morphological changes during these cellular events have been delineated, knowledge gap exists between the morphological changes in the seminiferous epithelium and the underlying molecular mechanism(s) that regulate these cellular events. Even though many of the regulatory proteins and biomolecules that modulate spermatogenesis are known based on studies using genetic models, the underlying regulatory mechanism(s), in particular signaling pathways/proteins, remain unexplored since much of the information regarding the signaling regulation is unknown. Studies in the past decade, however, have unequivocally demonstrated that the testis is using several signaling proteins and/or pathways to regulate multiple cellular events to modulate spermatogenesis. These include mTORC1/rpS6/Akt1/2 and p-FAK-Y407. While selective inhibitors and/or agonists and antagonists are available to examine some of these signaling proteins, their use have limitations due to their specificities and also potential systemic cytotoxicity. On the other hand, the use of genetic models has had profound implications for our understanding of the molecular regulation of spermatogenesis, and these knockout (null) models have also revealed the factors that are critical for spermatogenesis. Nonetheless, additional studies using in vitro and in vivo models are necessary to unravel the signaling pathways involved in regulating seminiferous epithelial cycle. Emerging data from studies, such as the use of the adjudin pharmaceutical/toxicant model, have illustrated that this non-hormonal male contraceptive drug is utilizing specific signaling pathways/proteins to induce specific defects in spermatogenesis, yielding mechanistic insights on the regulation of spermatogenesis. We sought to review these recent data in this article, highlighting an interesting approach that can be considered for future studies.

Whole-genome CRISPR screening identifies PI3K/AKT as a downstream component of the oncogenic GNAQ-focal adhesion kinase signaling circuitry.

G proteins and G protein-coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot-activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation via a conserved signaling circuitry mediated by FAK. Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

Maslinic acid prevented lipopolysaccharide-induced injury of IPEC-J2 cells through regulating PTEN-FAK signaling pathway.

Intestinal epithelial injury is one of the typical symptoms associated with intestinal inflammation and diarrhea, and the repair of the intestinal epithelium intricately linked to cell migration. Here, we test the hypothesis that maslinic acid (MA) regulates porcine intestinal epithelial cell migration by inhibiting focal adhesion kinase (FAK)/AKT signaling pathway. In this experiment, the optimal concentration of MA (0.5 µg/mL) on IPEC-J2 cell viability was selected to investigate the effect under low-dose lipopolysaccharide (LPS) (1 µg/mL) conditions. Transcriptome sequencing and polymerase chain reaction array results revealed that MA could alleviate LPS-induced the gene expressions decreasing in focal adhesion signaling pathway. From the pathway map analysis and western blot analysis results, MA alleviated the LPS-induced decrease in FAK protein expression mainly by promoting FAK protein phosphorylation, which in turn alleviated the decrease in cell migration and formation of cytoskeleton protein Vinculin and F-actin, the above results were verified by FAK phosphorylation inhibitors Defactinib. The molecular docking and immunoprecipitation further verified that MA could bind to PTEN protein and significantly inhibit its interaction with FAK protein, blocking the function of PTEN to inhibit FAK phosphorylation finally shown to promote the level of FAK phosphorylation, meanwhile LPS inhibited FAK protein expression and its binding to PKC and PTEN proteins. Our study revealed the role of MA and LPS in FAK protein, and increased understanding of MA anti-inflammatory mechanism.

Ankrd1 inhibits the FAK/Rho-GTPase/F-actin pathway by downregulating ITGA6 transcriptional to regulate myoblast functions.

Skeletal muscle constitutes the largest percentage of tissue in the animal body and plays a pivotal role in the development of normal life activities in the organism. However, the regulation mechanism of skeletal muscle growth and development remains largely unclear. This study investigated the effects of Ankrd1 on the proliferation and differentiation of C2C12 myoblasts. Here, we identified Ankrd1 as a potential regulator of muscle cell development, and found that Ankrd1 knockdown resulted in the proliferation ability decrease but the differentiation level increase of C2C12 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyzes as well as RNA-seq results showed that Ankrd1 knockdown activated focal adhesion kinase (FAK)/F-actin signal pathway with most genes significantly enriched in this pathway upregulated. The integrin subunit Itga6 promoter activity is increased when Ankrd1 knockdown, as demonstrated by a dual-luciferase reporter assay. This study revealed the molecular mechanism by which Ankrd1 knockdown enhanced FAK phosphorylation activity through the alteration of integrin subunit levels, thus activating FAK/Rho-GTPase/F-actin signal pathway, eventually promoting myoblast differentiation. Our data suggested that Ankrd1 might serve as a potential regulator of muscle cell development. Our findings provide new insights into skeletal muscle growth and development and valuable references for further study of human muscle-related diseases.

Upregulation of Integrin beta-3 in astrocytes upon Alzheimer's disease progression in the 5xFAD mouse model.

Integrins are receptors that have been linked to various brain disorders, including Alzheimer's disease (AD), the most prevalent neurodegenerative disorder. While Integrin beta-3 (ITGB3) is known to participate in multiple cellular processes such as adhesion, migration, and signaling, its specific role in AD remains poorly understood, particularly in astrocytes, the main glial cell type in the brain. In this study, we investigated alterations in ITGB3 gene and protein expression during aging in different brain regions of the 5xFAD mouse model of AD and assessed the interplay between ITGB3 and astrocytes. Primary cultures from adult mouse brains were used to gain further insight into the connection between ITGB3 and amyloid beta (Aß) in astrocytes. In vivo studies showed a correlation between ITGB3 and the astrocytic marker GFAP in the 5xFAD brains, indicating its association with reactive astrocytes. In vitro studies revealed increased gene expression of ITGB3 upon Aß treatment. Our findings underscore the potential significance of ITGB3 in astrocyte reactivity in the context of Alzheimer's disease.

Targeted delivery of FAK siRNA by engineered exosomes to reverse cetuximab resistance via activating paraptosis in colon cancer.

BACKGROUND: Cetuximab is extensively used in the treatment of metastatic colorectal cancer (mCRC). However, resistance poses a significant challenge to successful therapy. Recently, paraptosis, a non-classical programmed cell death, has garnered increased attention for its potential application value in antitumor treatments. We aimed to identify the essential pathways and signaling molecules involved in paraptosis inhibition and select them as therapeutic targets in cetuximab resistance. Additionally, engineered exosome technology is used as a drug delivery system with both targeted and effector properties. RESULTS: By comparing the differential expression of paraptosis-related genes between drug-resistant colon cancer cells and sensitive cells, it was observed that the paraptosis level induced by cetuximab was significantly downregulated in drug-resistant cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified the focal adhesion kinase (FAK) signaling pathway as a key pathway involved in the suppression of paraptosis. The biological function of FAK in cetuximab-resistant cells was investigated through cell morphology observation, CCK-8 assay, colony formation assay, RT-qPCR, Western Blot, and loss-of-function experiments. The results showed that the FAK signaling pathway was significantly upregulated in cetuximab-resistant colon cancer cells, and siRNA interference targeting FAK could notably inhibit cell proliferation while upregulating the paraptosis level. Based on this, engineered colon cancer cells targeted and FAK siRNA loaded exosomes (CT-Exo-siFAK1) were constructed. In vitro experiments, CT-Exo-siFAK1 could effectively activate paraptosis and inhibit the proliferation of drug-resistant colon cancer cells. In vivo experiments also confirmed that CT-Exo-siFAK1 significantly suppressed tumor growth and metastasis while upregulating the paraptosis level. CONCLUSION: This study suggests that FAK signaling pathway-mediated inhibition of paraptosis levels is crucial in the sensitivity of cetuximab targeted therapy in colon cancer, and the use of engineered exosomes to deliver FAK siRNA may be an effective strategy to reverse cetuximab resistance.

IGFBP2 induces podocyte apoptosis promoted by mitochondrial damage via integrin α5/FAK in diabetic kidney disease.

Podocyte apoptosis or loss is the pivotal pathological characteristic of diabetic kidney disease (DKD). Insulin-like growth factor-binding protein 2 (IGFBP2) have a proinflammatory and proapoptotic effect on diseases. Previous studies have shown that serum IGFBP2 level significantly increased in DKD patients, but the precise mechanisms remain unclear. Here, we found that IGFBP2 levels obviously increased under a diabetic state and high glucose stimuli. Deficiency of IGFBP2 attenuated the urine protein, renal pathological injury and glomeruli hypertrophy of DKD mice induced by STZ, and knockdown or deletion of IGFBP2 alleviated podocytes apoptosis induced by high concentration of glucose or in DKD mouse. Furthermore, IGFBP2 facilitated apoptosis, which was characterized by increase in inflammation and oxidative stress, by binding with integrin α5 (ITGA5) of podocytes, and then activating the phosphorylation of focal adhesion kinase (FAK)-mediated mitochondrial injury, including membrane potential decreasing, ROS production increasing. Moreover, ITGA5 knockdown or FAK inhibition attenuated the podocyte apoptosis caused by high glucose or IGFBP2 overexpression. Taken together, these findings unveiled the insight mechanism that IGFBP2 increased podocyte apoptosis by mitochondrial injury via ITGA5/FAK phosphorylation pathway in DKD progression, and provided the potential therapeutic strategies for diabetic kidney disease.

New insights into FAK structure and function in focal adhesions.

Focal adhesion kinase (FAK; also known as PTK2) was discovered three decades ago and is now recognised as a key player in the regulation of cell-matrix adhesion and mesenchymal cell migration. Although it is essential during development, FAK also drives invasive cancer progression and metastasis. On a structural level, the basic building blocks of FAK have been described for some time. However, a picture of how FAK integrates into larger assemblies in various cellular environments, including one of its main cellular locations, the focal adhesion (FA) complex, is only beginning to emerge. Nano-resolution data from cellular studies, as well as atomic structures from reconstituted systems, have provided first insights, but also point to challenges that remain for obtaining a full structural understanding of how FAK is integrated in the FA complex and the structural changes occurring at different stages of FA maturation. In this Review, we discuss the known structural features of FAK, the interactions with its partners within the FA environment on the cell membrane and propose how its initial assembly in nascent FAs might change during FA maturation under force.