The Role of 17ß-Estrogen in Escherichia coli Adhesion on Human Vaginal Epithelial Cells via FAK Phosphorylation.

Estrogen, the predominant sex hormone, has been found to be related to the occurrence of vaginal infectious diseases. However, its role in the occurrence and development of bacterial vaginitis caused by Escherichia coli is still unclear. The objective of this study was to investigate the role of 17ß-estrogen in E. coli adhesion on human vaginal epithelial cells. The vaginal epithelial cell line VK2/E6E7 was used to study the molecular events induced by estrogen between E. coli and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation with cell adhesion. The phosphorylation status of FAK and estrogen receptor α (ERα) upon estrogen challenge was assessed by Western blotting. Specific inhibitors for ERα were used to validate the involvement of ERα-FAK signaling cascade. The results showed that, following stimulation with 1,000 nM estrogen for 48 h, transient activation of ERα and FAK was observed, as was an increased average number of E. coli cells adhering to vaginal epithelial cells. In addition, estrogen-induced activation of ERα and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10 µM concentration and acted for 1 h, and a decrease in the number of adherent E. coli cells was observed simultaneously. However, this inhibitory effect diminished as the concentration of estrogen increased. In conclusion, FAK and ERα signaling cascades were associated with the increasing E. coli adherence to vaginal epithelial cells, which was promoted by a certain concentration of estrogen.

Tyrosine kinases regulate chondrocyte hypertrophy: promising drug targets for Osteoarthritis.

Osteoarthritis (OA) is a major health problem worldwide that affects the joints and causes severe disability. It is characterized by pain and low-grade inflammation. However, the exact pathogenesis remains unknown and the therapeutic options are limited. In OA articular chondrocytes undergo a phenotypic transition becoming hypertrophic, which leads to cartilage damage, aggravating the disease. Therefore, a therapeutic agent inhibiting hypertrophy would be a promising disease-modifying drug. The therapeutic use of tyrosine kinase inhibitors has been mainly focused on oncology, but the Food and Drug Administration (FDA) approval of the Janus kinase inhibitor Tofacitinib in Rheumatoid Arthritis has broadened the applicability of these compounds to other diseases. Interestingly, tyrosine kinases have been associated with chondrocyte hypertrophy. In this review, we discuss the experimental evidence that implicates specific tyrosine kinases in signaling pathways promoting chondrocyte hypertrophy, highlighting their potential as therapeutic targets for OA.

Characterizing the cellular architecture of dynamically remodeling vascular tissue using 3-D image analysis and virtual reconstruction.

Epithelial tubules form critical structures in lung, kidney, and vascular tissues. However, the processes that control their morphogenesis and physiological expansion and contraction are not well understood. Here we examine the dynamic remodeling of epithelial tubes in vivo using a novel model system: the extracorporeal vasculature of Botryllus schlosseri, in which the disruption of the basement membrane triggers rapid, massive vascular retraction without loss of barrier function. We developed and implemented 3-D image analysis and virtual reconstruction tools to characterize the cellular morphology of the vascular wall in unmanipulated vessels and during retraction. In both control and regressed conditions, cells within the vascular wall were planar polarized, with an integrin- and curvature-dependent axial elongation of cells and a robust circumferential alignment of actin bundles. Surprisingly, we found no measurable differences in morphology between normal and retracting vessels under extracellular matrix (ECM) disruption. However, inhibition of integrin signaling through focal adhesion kinase inhibition caused disruption of cellular actin organization. Our results demonstrate that epithelial tubes can maintain tissue organization even during extreme remodeling events, but that the robust response to mechanical signals-such as the response to loss of vascular tension after ECM disruption-requires functional force sensing machinery via integrin signaling.

Specific, targetable interactions with the microenvironment influence imatinib-resistant chronic myeloid leukemia.

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin ß3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin ß3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.

Phagocyte Transcriptomic Analysis Reveals Focal Adhesion Kinase (FAK) and Heparan Sulfate Proteoglycans (HSPGs) as Major Regulators in Anti-bacterial Defense of Crassostrea hongkongensis.

Invertebrates generally lack adaptive immunity and compensate for this with highly efficient innate immune machineries such as phagocytosis by hemocytes to eradicate invading pathogens. However, how extrinsically cued hemocytes marshal internal signals to accomplish phagocytosis is not yet fully understood. To this end, we established a facile magnetic cell sorting method to enrich professional phagocytes from hemocytes of the Hong Kong oyster (Crassostrea hongkongensis), an ecologically and commercially valuable marine invertebrate. Transcriptomic analysis on presorted cells shows that phagocytes maintain a remarkable array of differentially expressed genes that distinguish them from non-phagocytes, including 352 significantly upregulated genes and 479 downregulated genes. Pathway annotations reveal that focal adhesion and extracellular matrix-receptor interactions were the most conspicuously enriched pathways in phagocytes. Phagocytosis rate dramatically declined in the presence of an FAK inhibitor, confirming importance of the focal adhesion pathway in regulating phagocytosis. In addition, we also found that heparan sulfate proteoglycan (HSPG) families were lineage-specifically expanded in C. hongkongensis and abundantly expressed in phagocytes. Efficiency of phagocytosis and hemocytes aggregation was markedly reduced upon blockage of endogenous synthesis of HSPGs, thus implicating these proteins as key surface receptors in pathogen recognition and initiation of phagocytosis.

Hic-5 regulates Src-induced invadopodia rosette formation and organization.

Fibroblasts transformed by the proto-oncogene Src form individual invadopodia that can spontaneously self-organize into large matrix-degrading superstructures called rosettes. However, the mechanisms by which the invadopodia can spatiotemporally reorganize their architecture is not well understood. Here, we show that Hic-5, a close relative of the scaffold protein paxillin, is essential for the formation and organization of rosettes in active Src-transfected NIH3T3 fibroblasts and cancer-associated fibroblasts. Live cell imaging, combined with domain-mapping analysis of Hic-5, identified critical motifs as well as phosphorylation sites that are required for the formation and dynamics of rosettes. Using pharmacological inhibition and mutant expression, we show that FAK kinase activity, along with its proximity to and potential interaction with the LD2,3 motifs of Hic-5, is necessary for rosette formation. Invadopodia dynamics and their coalescence into rosettes were also dependent on Rac1, formin, and myosin II activity. Superresolution microscopy revealed the presence of formin FHOD1 and INF2-mediated unbranched radial F-actin fibers emanating from invadopodia and rosettes, which may facilitate rosette formation. Collectively, our data highlight a novel role for Hic-5 in orchestrating the organization of invadopodia into higher-order rosettes, which may promote the localized matrix degradation necessary for tumor cell invasion.

Cells under stress: The mechanical environment shapes inflammasome responses to danger signals.

Many intracellular signals, such as host danger-associated molecules and bacterial toxins during infection, elicit inflammasome activation. However, the mechanical environment in tissues may also influence the sensitivity of various inflammasomes to activation. The cellular mechanical environment is determined by the extracellular tissue stiffness, or its inverse, tissue compliance. Tissue stiffness is sensed by the intracellular cytoskeleton through a process termed mechanotransduction. Thus, extracellular compliance and the intracellular cytoskeleton may regulate the sensitivity of inflammasome activation. Control of proinflammatory signaling by tissue compliance may contribute to the pathogenesis of diseases such as ventilator-induced lung injury during bacterial pneumonia and tissue fibrosis in inflammatory disorders. The responsible signaling cascades in inflammasome activation pathways and mechanotransduction crosstalk are not yet fully understood. This rather different immunomodulatory perspective will be reviewed and open questions discussed here.

Zerumbone Suppresses Human Colorectal Cancer Invasion and Metastasis via Modulation of FAk/PI3k/NFκB-uPA Pathway.

The current study explored the basic molecular mechanisms of zerumbone (ZER), an herbal compound, in inhibiting the migration and invasion of colorectal cancer (CRC) cells in vitro. Two types of CRC cells, namely HCT-116 and SW48, were treated with various concentrations of ZER (8, 16, and 24 µM) for 24, 48, and 72 h, respectively. In vitro assays were performed to determine alterations in proliferation ability, mRNA expression and protein levels, and migration and invasion potential of CRC cells. An SYBR Green-based quantitative polymerase chain reaction (PCR) was utilized to detect the gene expression of focal adhesion kinase (FAK), nuclear factor (NF)-κB, and urokinase-type plasminogen activator (uPA) followed by the evaluation of the level of proteins by western blotting. Migration and invasion potentials of HCT-116 and SW48 cells treated by ZER were examined using migration and invasion assay kits, respectively. We compared the results of all experiments with control groups, including FAK inhibitor, ZER + FAK inhibitor-treated cells, NF-ß inhibitor, ZER + NF-ß inhibitor, and untreated cells. The data in the present study suggest that ZER may exert its antimetastatic effects through inhibition of FAk/PI3k/NF-κB-uPA signaling pathway, thereby possibly representing a novel class of FAK inhibitors.

Role of AGAP2 in the profibrogenic effects induced by TGFß in LX-2 hepatic stellate cells.

Liver damage induces hepatic stellate cells (HSC) activation, characterised by a fibrogenic, proliferative and migratory phenotype. Activated HSC are mainly regulated by transforming growth factor ß 1 (TGFß1), which increases the production of extracellular matrix proteins (e.g. collagen-I) promoting the progression of hepatic fibrosis. AGAP2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 2) is a GTPase/GTP-activating protein involved in the actin remodelling system and receptor recycling. In the present work the role of AGAP2 in human HSC in response to TGFß1 was investigated. LX-2 HSC were transfected with AGAP2 siRNA and treated with TGFß1. AGAP2 knockdown prevented to some extent the proliferative and migratory TGFß1-induced capacities of LX-2 cells. An array focused on human fibrosis revealed that AGAP2 knockdown partially prevented TGFß1-mediated gene expression of the fibrogenic genes ACTA2, COL1A2, EDN1, INHBE, LOX, PDGFB, TGFΒ12, while favored the expression of CXCR4, IL1A, MMP1, MMP3 and MMP9 genes. Furthermore, TGFß1 induced AGAP2 promoter activation and its protein expression in LX-2. Moreover, AGAP2 protein levels were significantly increased in liver samples from rats with thioacetamide-induced fibrosis. In addition, AGAP2 silencing affected TGFß1-receptor 2 (TGFR2) trafficking in U2OS cells, blocking its effective recycling to the membrane. AGAP2 silencing in LX-2 cells prevented the TGFß1-induced increase of collagen-I protein levels, while its overexpression enhanced collagen-I protein expression in the presence or absence of the cytokine. AGAP2 overexpression also increased focal adhesion kinase (FAK) phosphorylated levels in LX-2 cells. FAK and MEK1 inhibitors prevented the increase of collagen-I expression caused by TGFß1 in LX-2 overexpressing AGAP2. In summary, the present work shows for the first time, that AGAP2 is a potential new target involved in TGFß1 signalling, contributing to the progression of hepatic fibrosis.

Scutellarin Prevents Angiogenesis in Diabetic Retinopathy by Downregulating VEGF/ERK/FAK/Src Pathway Signaling.

BACKGROUND: Diabetic retinopathy (DR) is a serious microvascular complication of diabetes. This study demonstrates the antiangiogenic effects of scutellarin (SCU) on high glucose- and hypoxia-stimulated human retinal endothelial cells (HRECs) and on a diabetic rat model by oral administration. The antiangiogenic mechanisms of SCU in vitro and in vivo were investigated. METHOD: HRECs were cultured in high glucose- (30 mM D-glucose) and hypoxia (cobalt chloride-treated)-stimulated diabetic condition to evaluate the antiangiogenic effects of SCU by CCK-8 test, cell migration experiment (wound healing and transwell), and tube formation experiment. A streptozotocin-induced type II diabetic rat model was established to measure the effects of oral administration of SCU on protecting retinal microvascular dysfunction by Doppler waveforms and HE staining. We further used western blot, luciferase reporter assay, and immunofluorescence staining to study the antiangiogenic mechanism of SCU. The protein levels of phospho-ERK, phospho-FAK, phospho-Src, VEGF, and PEDF were examined in HRECs and retina of diabetic rats. RESULT: Our results indicated that SCU attenuated diabetes-induced HREC proliferation, migration, and tube formation and decreased neovascularization and resistive index in the retina of diabetic rats by oral administration. SCU suppressed the crosstalk of phospho-ERK, phospho-FAK, phospho-Src, and VEGF in vivo and in vitro. CONCLUSIONS: These results suggested that SCU can be an oral drug to alleviate microvascular dysfunction of DR and exerts its antiangiogenic effects by inhibiting the expression of the crosstalk of VEGF, p-ERK, p-FAK, and p-Src.

Physiological stretch induced proliferation of human urothelial cells via integrin α6-FAK signaling pathway.

AIMS: To test a kind of stretch pattern which is the optimum stress parameter to promote human urothelial cells (HUCs) proliferation, and to investigate the roles of integrin subunits and their pathway in the HUCs proliferation induced by physiological stretch. METHODS: HUCs were seeded on silicone membrane, and subjected to four kinds of stretch (0,5%,10%,15% elongation) for 24 h, as controlled by a BioDynamic® bioreactor. Cell proliferation, viability and cycle distribution were examined using Cell Counting Kit-8 and flow cytometry, respectively. The gene and protein expression of integrin subunits and focal adhesion kinase (FAK) in each group were assessed by Real-time PCR(RT-PCR) and western blot, respectively. Small interfering RNAs (siRNA) were applied to knockdown integrin α6 and FAK expression in HUCs, and FAK inhibitor was used to validate the role of α6 and FAK in cell proliferation under physiological stretch. RESULTS: The proliferation of HUCs were highest in the 5% elongation group compared to static control, 10% and 15% elongation group. RT-PCR and western blot showed that 5% cyclic stretch significantly promoted the expression of integrin α6 and FAK. The stretch-induced cell proliferation and FAK expression was inhibited by siRNA of integrin α6. Further study with FAK inhibitor revealed that elongation promoted proliferation though integrin α6 and FAK signaling pathway. CONCLUSIONS: Physiological stretch induced HUCs proliferation via integrin α6-FAK signaling pathway, and 5% elongation may be the optimal stress parameter to promote the cell proliferation.

Cyclic Tensile Strain Upregulates Pro-Inflammatory Cytokine Expression Via FAK-MAPK Signaling in Chondrocytes.

Excessive mechanical stimulation is considered an important factor in the destruction of chondrocytes. Focal adhesion kinase (FAK) is non-receptor tyrosine kinase related to a number of different signaling proteins. Little is known about the function of FAK in chondrocytes under mechanical stimulation. In the present study, we investigated the function of FAK in mechanical signal transduction and the mechanism through which cyclic tensile strain (CTS) induces expression of inflammation-related factors. Mouse ATDC5 chondrogenic cells were subjected to CTS of 0.5 Hz to 10% cell elongation with an FAK inhibitor. The expression of genes encoding COX-2, IL-1ß, and TNF-α was examined using real-time RT-PCR after CTS application with FAK inhibitor. Phosphorylation of p-38, ERK, and JNK was analyzed by Western blotting. Differences in COX-2 expression following pretreatment with FAK, p-38, ERK, and JNK inhibitors were compared by Western blotting. We found that CTS increased the expression of genes encoding COX-2, IL-1ß, and TNF-α and activated the phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with an FAK inhibitor for 2 h reduced the expression of genes encoding COX-2, IL-1ß, and TNF-α induced by CTS-associated inflammation and decreased phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with FAK, p-38, ERK, and JNK inhibitors markedly suppressed COX-2 and IL-1ß protein expression. In conclusion, FAK appears to regulate inflammation in chondrocytes under CTS via MAPK pathways.

Focal adhesion kinase family is involved in matrix contraction by transdifferentiated Müller cells.

Transdifferentiated Müller cells that adopt a fibroblastic/myofibroblastic phenotype have been identified in epiretinal membranes (ERMs) in several ocular disorders, and have been implicated to play a role in the formation and/or the contraction of ERMs. We have previously demonstrated that dasatinib, a dual inhibitor of Src-family kinases and Abl kinase, can prevent matrix contraction by transdifferentiated Müller cells. In this study, we examined molecules involved in matrix contraction downstream of primary dasatinib targets. Tyrosine phosphorylation of focal adhesion kinase (FAK) family members FAK and PYK2 was significantly reduced by dasatinib, and select inhibitors for these kinases PF431396, which inhibits both FAK and PYK2, and PF573228, which only inhibits FAK and not PYK2, significantly reduced matrix contraction by transdifferentiated Müller cells. Dasatinib and PF431396 significantly reduced phosphorylation of Hic-5, a protein implicated to play a role in focal adhesions and cell signaling. Our data shows that FAK family members are involved in matrix contraction by transdifferentiated Müller cells, and also implicates that Hic-5 is situated downstream of the FAK family within the signaling pathway.

Nuclear FAK and Runx1 Cooperate to Regulate IGFBP3, Cell-Cycle Progression, and Tumor Growth.

Nuclear focal adhesion kinase (FAK) is a potentially important regulator of gene expression in cancer, impacting both cellular function and the composition of the surrounding tumor microenvironment. Here, we report in a murine model of skin squamous cell carcinoma (SCC) that nuclear FAK regulates Runx1-dependent transcription of insulin-like growth factor binding protein 3 (IGFBP3), and that this regulates SCC cell-cycle progression and tumor growth in vivo Furthermore, we identified a novel molecular complex between FAK and Runx1 in the nucleus of SCC cells and showed that FAK interacted with a number of Runx1-regulatory proteins, including Sin3a and other epigenetic modifiers known to alter Runx1 transcriptional function through posttranslational modification. These findings provide important new insights into the role of FAK as a scaffolding protein in molecular complexes that regulate gene transcription. Cancer Res; 77(19); 5301-12. ©2017 AACR.

Periostin promotes migration and invasion of renal cell carcinoma through the integrin/focal adhesion kinase/c-Jun N-terminal kinase pathway.

Periostin (POSTN) is an extracellular matrix protein which is overexpressed in a variety of cancers and has been related to tumorigenesis of renal cell carcinoma. However, the involvement of POSTN in renal cell carcinoma migration, invasion, and their underlying mechanisms has not been established. In this study, renal cell carcinoma cell lines stably overexpressing POSTN were established using a lentiviral vector, and the effects of POSTN on renal cell carcinoma cell migration and invasion were investigated. POSTN overexpression increased the migration and invasion capabilities of renal cell carcinoma cell lines as well as activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. Integrin αvß3 and αvß5 antibodies inhibited POSTN overexpression or recombinant POSTN-induced focal adhesion kinase activation, cell migration, and invasion. Furthermore, lentivirus-mediated focal adhesion kinase knockdown and c-Jun N-terminal kinase inhibitor reduced POSTN-enhanced phosphorylation of c-Jun N-terminal kinase, matrix metalloproteinase-9 and matrix metalloproteinase-2 expressions, cell migration, and invasion. Our research thus indicates that POSTN promotes renal cell carcinoma cell migration and invasion through interaction with integrins αvß3 and αvß5 and subsequent activation of the focal adhesion kinase/c-Jun N-terminal kinase pathway. These results suggest that POSTN plays a critical role in renal cell carcinoma metastasis and may represent a potential target for novel therapeutic approaches against renal cell carcinoma.

Phospholipid binding to the FAK catalytic domain impacts function.

Focal adhesion kinase is an essential nonreceptor tyrosine kinase that plays an important role in development, in homeostasis and in the progression of human disease. Multiple stimuli activate FAK, which requires a change in structure from an autoinhibited to activated conformation. In the autoinhibited conformation the FERM domain associates with the catalytic domain of FAK and PI(4,5)P2 binding to the FERM domain plays a role in the release of autoinhibition, activating the enzyme. An in silico model of FAK/PI(4,5)P2 interaction suggests that residues on the catalytic domain interact with PI(4,5)P2, in addition to the known FERM domain PI(4,5)P2 binding site. This study was undertaken to test the significance of this in silico observation. Mutations designed to disrupt the putative PI(4,5)P2 binding site were engineered into FAK. These mutants exhibited defects in phosphorylation and failed to completely rescue the phenotype associated with fak -/- phenotype fibroblasts demonstrating the importance of these residues in FAK function. The catalytic domain of FAK exhibited PI(4,5)P2 binding in vitro and binding activity was lost upon mutation of putative PI(4,5)P2 binding site basic residues. However, binding was not selective for PI(4,5)P2, and the catalytic domain bound to several phosphatidylinositol phosphorylation variants. The mutant exhibiting the most severe biological defect was defective for phosphatidylinositol phosphate binding, supporting the model that catalytic domain phospholipid binding is important for biochemical and biological function.

Focal adhesion kinase maintains, but not increases the adhesion of dental pulp cells.

Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.

Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib through the laminin-332/α3 integrin axis recovery of focal adhesion kinase ubiquitination.

In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of α3ß1 and α6ß4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both α3ß1 and α6ß4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing α6ß4 but lacking the α3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B α3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. CONCLUSION: This study unveils a novel mechanism of sorafenib resistance depending on the α3ß1/Ln-332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103-2117).

Left Ventricular Unloading After Acute Myocardial Infarction Reduces MMP/JNK Associated Apoptosis and Promotes FAK Cell-Survival Signaling.

BACKGROUND: The mechanism underlying left ventricular remodeling and reverse remodeling in the setting of mechanical support following acute myocardial infarction (MI) is unclear. We tested the hypothesis that left ventricular assist device (LVAD) unloading can decrease apoptotic signals after MI. METHODS: An MI model was created in 16 sheep by coronary artery ligation. Eight were unloaded with a LVAD during the first 2 weeks after MI and observed for 10 more weeks. Myocardial tissue was collected from the nonischemic adjacent zone and the remote zone. Proteins in the apoptotic matrix metalloproteinases (MMPs)-2/c-Jun N-terminal kinase (JNK) and prosurvival ß1D-integrin/focal adhesion kinase (FAK) pathway were quantified. RESULTS: Increased TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) positive nuclei were observed in the MI group and to a lesser extent in the LVAD group (6.18 ± 0.26 versus 0.82 ± 0.18; p < 0.05). Pro-MMP-2, MMP-2, JNK, and phosphorylated (p)-JNK were all elevated in the adjacent zone of the MI-only group but not in the adjacent zone of the LVAD-supported group. There were higher levels of prosurvival p-FAK in the LVAD-supported group than in the MI group. CONCLUSIONS: MMP-2/JNK apoptotic and ß1D-integrin/FAK survival pathways are activated in the nonischemic adjacent zone after MI in adult sheep. LVAD unloading of approximately 50% cardiac output for 2 weeks attenuates remodeling in part by its negative effect on stretch-induced apoptosis and inhibition of MMP-2 activity.