Β1 Integrin Activation Research Articles

Neutrophil DREAM Promotes Neutrophil Recruitment in Vascular Inflammation Via Nuclear Factor Kappa B-Dependent and Independent Mechanisms

The interaction between neutrophils and endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. Neutrophil recruitment to inflamed tissues is initiated by rolling on activated ECs through the interactions between P-/E-selectins and their ligands. Subsequently, activated integrins (mainly αLβ2 and αMβ2) and chemokine receptors bind to their ligands on ECs and mediate slow-rolling, adhesion, crawling, and transmigration of neutrophils. Although many neutrophil adhesion receptors have been identified, the regulation of their ligand-binding function remains not fully understood. Using real-time intravital microscopy with mice lacking downstream regulatory element antagonist modulator (DREAM) and their bone marrow chimeric mice, we demonstrated that hematopoietic cell DREAM contributes to neutrophil recruitment to sites of vascular inflammation induced by TNF-α- but not a G protein-coupled receptor ligand, MIP-2 or fMLP. Our studies using adoptive neutrophil transfers and flow chamber assays revealed that neutrophil DREAM positively regulates the neutrophil recruitment processes under TNF-α-induced inflammatory conditions. Using RNA-seq and biochemical and cell biological studies, we found that neutrophil DREAM upregulates numerous pro-inflammatory molecules and down-regulates anti-inflammatory molecules after TNF-α treatment. In particular, neutrophil DREAM repressed expression of A20, a negative regulator of NF-κB signaling, and enhanced phosphorylation of IκB kinase (IKK) in response to TNF-α, suggesting the role of neutrophil DREAM in NF-κB activity. Furthermore, we observed that DREAM deletion and IKK inhibition significantly diminishes the ligand-binding activity of β2 integrins in neutrophils after short-term treatment with TNF-α and that deletion of neutrophil DREAM does not affect the expression of other neutrophil adhesion receptors, such as PSGL-1, L-selectin, CD44, CXCR2, and CXCR4. As assessed by flow cytometry using conformation-specific reporter antibodies, knockdown of DREAM in neutrophil-like HL-60 cells decreased TNF-α-induced activation of β2 integrins. Neutrophil DREAM promoted degranulation through IKK-mediated SNAP-23 phosphorylation after short-term treatment with TNF-α, implying the role of neutrophil DREAM-IKK signaling in NF-κB-independent signaling. Using intravital microscopy with Berkeley mice (a mouse model of sickle cell disease) deficient in hematopoietic or nonhematopoietic DREAM, we demonstrated that hematopoietic cell DREAM is crucial for inducing intravascular cell-cell aggregation and vaso-occlusive events in microvessels following the TNF-α challenge. Furthermore, infusion of DREAM KO neutrophils, compared with WT neutrophils, significantly reduced neutrophil recruitment and vaso-occlusive events in TNF-α-challenged SCD mice. These results demonstrate that neutrophil DREAM positively regulates β2 integrin function and promotes neutrophil recruitment during sterile inflammation via NF-κB-dependent and independent mechanisms. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases. DisclosuresNo relevant conflicts of interest to declare.

Efficacy of Rituximab in Treatment-Resistant Focal Segmental Glomerulosclerosis With Elevated Soluble Urokinase-Type Plasminogen Activator Receptor and Activation of Podocyte β3 Integrin

IntroductionSevere, nonresponsive, primary focal segmental glomerular sclerosis (FSGS) can progress to end-stage kidney disease (ESKD) in <5 years. Soluble urokinase-type plasminogen activator receptor (suPAR) may contribute to podocyte effacement by activating podocyte β3 integrin. It has been reported as a potential permeability factor and biomarker for primary FSGS. Rituximab was found to have efficacy in case reports and small series. Whether rituximab is efficacious in patients with treatment-resistant FSGS in the context of high suPAR levels and evidence of podocyte B3 integrin activation remains unknown.MethodsIn this nonblinded, open-label pilot study, the safety and efficacy of rituximab were evaluated in treatment-resistant adult patients with primary FSGS and a suPAR level > 3500 pg/ml with evidence of β3 integrin activation. Rituximab (1 g) was given on days 1 and 15. The primary outcome was proteinuria at 12 months.ResultsOnly 13 of 38 screened patients qualified for the study, of whom 9 consented to participate. The baseline proteinuria and glomerular filtration rate (GFR) levels were 7.70 ± 4.61 g/d and 67 ± 38 ml/min, respectively. A transient response at 6 months was noted in 2 patients without a parallel change in suPAR level. At 12 months, there was no statistically significant improvement in proteinuria level with all participants remaining nephrotic (7.27 ± 7.30 g/d). GFR level marginally declined to 60 ± 38 ml/min with one patient progressing to ESKD. There were 2 serious infections, an infusion-related reaction and leucopenia attributed to rituximab.ConclusionRituximab was ineffective when administered to adult patients with treatment-resistant primary FSGS with a high suPAR and evidence of podocyte activation.

Tropoelastin improves adhesion and migration of intra-articular injected infrapatellar fat pad MSCs and reduces osteoarthritis progression

Intra-articular injection of mesenchymal stem cells (MSCs) is a promising strategy for osteoarthritis (OA) treatment. However, more and more studies reveal that the injected MSCs have poor adhesion, migration, and survival in the joint cavity. A recent study shows that tropoelastin (TE) regulates adhesion, proliferation and phenotypic maintenance of MSCs as a soluble additive, indicating that TE could promote MSCs-homing in regenerative medicine. In this study, we used TE as injection medium, and compared it with classic media in MSCs intra-articular injection such as normal saline (NS), hyaluronic acid (HA), and platelet-rich plasma (PRP). We found that TE could effectively improve adhesion, migration, chondrogenic differentiation of infrapatellar fat pad MSCs (IPFP-MSCs) and enhance matrix synthesis of osteoarthritic chondrocytes (OACs) in indirect-coculture system. Moreover, TE could significantly enhance IPFP-MSCs adhesion via activation of integrin β1, ERK1/2 and vinculin (VCL) in vitro. In addition, intra-articular injection of TE-IPFP MSCs suspension resulted in a short-term increase in survival rate of IPFP-MSCs and better histology scores of rat joint tissues. Inhibition of integrin β1 or ERK1/2 attenuated the protective effect of TE-IPFP MSCs suspension in vivo. In conclusion, TE promotes performance of IPFP-MSCs and protects knee cartilage from damage in OA through enhancement of cell adhesion and activation of integrin β1/ERK/VCL pathway. Our findings may provide new insights in MSCs intra-articular injection for OA treatment.

Mitofusin-2 regulates leukocyte adhesion and β2 integrin activation.

Neutrophils are critical for inflammation and innate immunity, and their adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 (MFN2) is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β2 -integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in MFN2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP (N-formylmethionyl-leucyl-phenylalanine) receptor FPR1 as well as the inhibited β2 integrin activation, as assessed by conformation-specific monoclonal antibodies. MFN2 deficiency also leads to decreased actin polymerization, which is important for β2 integrin activation. Mn2+ -induced cell spreading is also inhibited after MFN2 knockdown. MFN2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by CD35 and CD87 markers. MFN2 knockdown in β2-integrin activation-matured cells (CD87high population) also inhibits integrin activation, indicating that MFN2 directly affects β2 integrin activation. Our study illustrates the function of MFN2 in leukocyte adhesion and may provide new insights into the development and treatment of MFN2 deficiency-related diseases.

Integrin \u03b21 orchestrates the abnormal cell-matrix attachment and invasive behaviour of E-cadherin dysfunctional cells

BackgroundTumour progression relies on the ability of cancer cells to penetrate and invade neighbouring tissues. E-cadherin loss is associated with increased cell invasion in gastric carcinoma, and germline mutations of the E-cadherin gene are causative of hereditary diffuse gastric cancer. Although E-cadherin dysfunction impacts cell–cell adhesion, cell dissemination also requires an imbalance of adhesion to the extracellular matrix (ECM).MethodsTo identify ECM components and receptors relevant for adhesion of E-cadherin dysfunctional cells, we implemented a novel ECM microarray platform coupled with molecular interaction networks. The functional role of putative candidates was determined by combining micropattern traction microscopy, protein modulation and in vivo approaches, as well as transcriptomic data of 262 gastric carcinoma samples, retrieved from the cancer genome atlas (TCGA).ResultsHere, we show that E-cadherin mutations induce an abnormal interplay of cells with specific components of the ECM, which encompasses increased traction forces and Integrin β1 activation. Integrin β1 synergizes with E-cadherin dysfunction, promoting cell scattering and invasion. The significance of the E-cadherin-Integrin β1 crosstalk was validated in Drosophila models and found to be consistent with evidence from human gastric carcinomas, where increased tumour grade and poor survival are associated with low E-cadherin and high Integrin β1 levels.ConclusionsIntegrin β1 is a key mediator of invasion in carcinomas with E-cadherin impairment and should be regarded as a biomarker of poor prognosis in gastric cancer.

Role of β1 integrin in thrombocytopoiesis.

Thrombocytopoiesis is a complex process beginning at the level of hematopoietic stem cells, which ultimately generate megakaryocytes, large marrow cells with a distinctive morphology, and then, through a process of terminal maturation, megakaryocytes shed thousands of platelets into the circulation. This process is controlled by intrinsic and extrinsic factors. Emerging data indicate that an important intrinsic control on the late stages of thrombopoiesis is exerted by integrins, a family of transmembrane receptors composed of one α and one β subunit. One β subunit expressed by megakaryocytes is the β1 integrin, the role of which in the regulation of platelet formation is beginning to be clarified. Here, we review recent data indicating that activation of β1 integrin by outside-in and inside-out signaling regulates the interaction of megakaryocytes with the endosteal niche, which triggers their maturation, while its inactivation by galactosylation determines the migration of these cells to the perivascular niche, where they complete their terminal maturation and release platelets in the bloodstream. Furthermore, β1 integrin mediates the activation of transforming growth factor β (TGF-β), a protein produced by megakaryocytes that may act in an autocrine fashion to halt their maturation and affect the composition of their surrounding extracellular matrix. These findings suggest that β1 integrin could be a therapeutic target for inherited and acquired disorders of platelet production.

Binding of Rap1 and Riam to Talin1 Fine-Tune β2 Integrin Activity During Leukocyte Trafficking.

β2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent β2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the β2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that β2 integrin regulation primarily occurs via these two pathways.

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Polydopamine-based biofunctional substrate coating promotes mesenchymal stem cell migration

Rapid migration of mesenchymal stem cells (MSCs) on device surfaces could support in vivo tissue integration and might facilitate in vitro organoid formation. Here, polydopamine (PDA) is explored as a biofunctional coating to effectively promote MSC motility. It is hypothesized that PDA stimulates fibronectin deposition and in this way enhances integrin-mediated migration capability. The random and directional cell migration was investigated by time-lapse microscopy and gap closure assay respectively, and analysed with softwares as computational tools. A higher amount of deposited fibronectin was observed on PDA substrate, compared to the non-coated substrate. The integrin β1 activation and focal adhesion kinase (FAK) phosphorylation at Y397 were enhanced on PDA substrate, but the F-actin cytoskeleton was not altered, suggesting MSC migration on PDA was regulated by integrin initiated FAK signalling. This study strengthens the biofunctionality of PDA coating for regulating stem cells and offering a way of facilitating tissue integration of devices.Graphic abstractPolydopamine-coated substrate induces increased fibronectin deposition of mesenchymal stem cells, and promotes cell migration via integrin-initiated FAK signaling, compared to non-coated polystyrene-based standard tissue culture surface. In this way, multifunctional PDA coating could support in vivo tissue integration on implant surface and promote in vitro organoid formation.

Anatomy of the Extracellular Clasps That Regulate Platelet αIIbβ3 and αvβ3 Function

The integrins αIIbβ3 and αvβ3 on circulating platelets are maintained in inactive states by an intra-molecular clasp involving the cytoplasmic, transmembrane, and extracellular stalk domains of their α and β subunits. Following platelet stimulation, αIIbβ3 and αvβ3 rapidly undergo global rearrangements in which their clasps are disrupted and their headpieces open to expose a ligand binding site. Protein-protein interfaces, such as those of the αIIbβ3 and αvβ3 clasps, are usually large complementary surfaces in which specific amino acids, termed energetic “hot spots”, contribute disproportionately to the binding free energy. Previously, we scanned the β3 EGF-3, EGF-4, and βTD domains computationally with alanine residues and identified a limited number of interface residues that stabilized the extracellular clasps of αIIbβ3 and αvβ3. Here, we have used computational alanine scanning to identify corresponding “hot spots” in the Calf-1 and Calf-2 domains of the αIIb and αv components of the αIIbβ3 and αvβ3 clasps. The identified alanine substitutions were predicted to destabilize the respective stalk heterodimers. We identified 10 αIIb substitutions and 11 αv substitutions that would destabilize the stalk interfaces by a change in binding free energy (ΔΔG) of >0.3 kcal/mol. To confirm that a ΔΔG as small as 0.3 kcal/mol is sufficient to destabilize the β3 integrin stalk interfaces, we introduced 8 of the 10 αIIb substitutions into full-length αIIb, co-expressed the mutated αIIb with wild type β3 in CHO cells, and measured spontaneous binding of Alexa Fluor 488-labeled fibrinogen to the cells by flow cytometry. αIIbR751Aβ3 did not express, implying that R751 is important for correct αIIb folding and/or αIIbβ3 assembly. Each of the other mutations expressed and caused constitutive αIIbβ3 activation. Importantly, αIIbβ3 containing alanine substitutions whose effect on ΔΔG was <0.3 kcal/mol was inactive. Nonetheless, while the alanine scanning algorithm correctly identified destabilizing alanine substitutions, it did not correctly rank their functional effects. Hot spots could occur because they bury specific side chains in the protein-protein interface. While we found a modest correlation between ΔΔG and the buried surface area of specific αIIb side chains (R2=0.7), there was no correlation with αv (R2=0.1), a result consistent with an analysis of a compiled database of 2325 similar alanine mutants (Bogan and Thorn, J Mol Biol 280:1-9,1998). This suggests that other mechanisms, such as inter-subunit hydrogen bonds, are involved. Previously, in studies of the growth hormone-growth hormone receptor complex, Clackson and Wells (Science 267:383-86,1995) found that functionally important residues in the interface of the complex were in direct contact. To determine if this is the case for αIIbβ3 and αvβ3, we mapped the mutation-sensitive positions onto the αIIbβ3 and αvβ3 crystal structures to localize the αIIb, αv, and β3 hot spots in models of the αIIbβ3 and αvβ3 stalks. The energetic hot spots we identified in αIIb and αv were located in a strip along one face of the Calf-1 and Calf-2 domains. Similarly, the β3 hot spots were located in a strip along on one face of the EGF-3, EGF-4, and βTD domains. However, the hot spots were not contiguous and there was only partial overlap of the β3 side chains that interacted with αIIb versus αv. On the other hand, in models of the assembled stalk heterodimers, there was remarkable complementarity of the computationally identified hot spots in the αIIb and αv stalks with those in β3, indicating that only a limited number of specific inter-subunit interactions actually make significant contributions to stalk assembly. These results confirm the utility of computational alanine scanning for identifying and localizing functionally important interaction sites in αIIbβ3 and αvβ3. They also demonstrate that although the same surface of β3 interacts with the αIIb and αv stalks, unique, as well as overlapping, side chain interactions are responsible for maintaining the inactive state of the respective integrins. They also suggest that stabilizing the extracellular stalk heterodimers may be a way to attenuate β3 integrin activation. DisclosuresNo relevant conflicts of interest to declare.

Structure of an Extended β Integrin

Integrins are a family of 24 α/β heterodimeric adhesion receptors that transmit bidirectional signals across the cell membrane. Each integrin subunit contains a large extracellular domain composed of headpiece and leg piece, a transmembrane domain and usually a short cytoplasmic tail. The α headpiece consists of the common β-propeller and thigh domains, and an extra αI domain inserted into the β-propeller domain in a subset of α subunits such as αL. The α leg piece includes calf-1 and -2 domains. The β headpiece contains βI, which is a structural homolog of αI domain, hybrid, PSI, and I-EGF1 domains. The β leg piece contains I-EGF2-4 and β-tail domains. Structural studies in the last 15 years, largely based on the two β3 integrins, αIIbβ3 and αVβ3, greatly advanced our understanding of integrin conformational regulation upon activation. The platelet specific αIIbβ3 plays an essential role in hemostasis and thrombosis, while the αVβ3 is important for the growth and survival of many cell types. In the resting state, integrin adopts a bent conformation with the headpiece folded onto the leg piece. Crystallographic and EM studies of αIIbβ3 and αVβ3 revealed two major conformational rearrangements, the swing-out motion of the hybrid domain, resulting in headpiece opening, and the switchblade-like movement of the headpiece from the leg domains, leading to integrin extension and leg separation. Despite the visualization of β3 extension by EM at very low resolution, a high-resolution extended β3 structure has been missing.A Leu33Pro polymorphism at the β3 PSI domain defines the human platelet alloantigen 1 (HPA-1) system. The mismatched HPA-1a/b (Leu33/Pro33) is a major cause of two severe bleeding disorders, fetal/neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP) due to the generation of anti-HPA-1 alloantibodies. In addition, it has been controversial whether the Pro33 variant can enhance β3 activity, being a risk factor for thrombosis. It is also unknown whether the Leu33Pro substitution affects the conformation of PSI domain, which undergoes outward movement along with the headpiece opening and extension.It has been difficult to obtain the crystal structure of a full-length extended integrin largely due to the conformational heterogeneity and flexibility, especially in the lower leg domains. In this study, we engineered a chimeric β3 fragment, denoted as cβ3-E2, including the domains of PSI, hybrid, I-EGF1, I-EGF2, and the βI replaced with the αL αI domain, which enables the expression of β3 in the absence of α subunit. We determined the crystal structures of cβ3-E2 in both Leu33 and Pro33 forms. Strikingly, two conformations of cβ3-E2, an intermediate state and a fully extended state are simultaneously present in one crystal. The β3 extension occurs at the I-EGF1 and I-EGF2 interface, denoted as β-knee. Our structure revealed the structural changes of the flexible loop, C473-C486 or C1-C2 loop of I-EGF2 in the β-knee, which has been shown to modulate integrin activation by mutagenesis studies. No significant structural differences were observed between the PSI domains having Leu33 and Pro33. We next compared the ligand binding of β3-Leu33 and β3-Pro33 induced by Mn2+ or talin head domain. No significant differences were observed either in αIIbβ3 or αVβ3, indicating that the Leu33Pro substitution may not affect β3 integrin activation. However, the PSI C26-C38 loop harboring the Leu33Pro polymorphism moves closer to the I-EGF1 domain in the extended conformation. In addition, we found that a completely conserved arginine (Arg93 in β3) at the PSI/hybrid domain interface is important for maintaining the resting integrin state since the alanine or glutamine substitution renders β1, β2 and β3 integrin hyperactive. Interestingly, a naturally occurring β3-Arg93Gln substitution has been shown to disrupt the HPA-1a epitope. Finally, based on our cβ3-E2 structure, we built the extended full-length β3 structures with either closed or open headpiece, which can perfectly fit into the EM structures of extended αIIbβ3 with either closed or open headpiece, respectively. In summary, our study provides structural details of an extended β3 integrin, showing the conformational changes at the PSI, I-EGF1 and I-EGF2 domains, demonstrating their importance in regulating integrin activation and alloimmune response. DisclosuresNo relevant conflicts of interest to declare.

β2 integrin activation and signal transduction in leukocyte recruitment.

Leukocyte recruitment is a critical step in the pathogenesis of inflammatory and immunological responses. Cell adhesion molecules (CAMs) are involved in controlling cell movements and the recruitment process, and the integrin family of CAMs plays a key role. During cell movement, integrin function is dynamically and precisely regulated. However, this balance might be broken under pathological conditions. Thus, the functional regulation and molecular mechanisms of integrins related to diseases are often a focus of research. Integrin β2 is one of the most commonly expressed integrins in leukocytes that mediate leukocyte adhesion and migration, and it plays an important role in immune responses and inflammation. In this review, we focus on specific functions of integrin β2 in leukocyte recruitment, the conformational changes and signal transduction of integrin β2 activation, the similarities between murine and human factors, and how new insights into these processes can inform future therapies for inflammation and immune diseases.

MYH9-dependent polarization of ATG9B promotes colorectal cancer metastasis by accelerating focal adhesion assembly

Tumour metastasis is a major reason accounting for the poor prognosis of colorectal cancer (CRC), and the discovery of targets in the primary tumours that can predict the risk of CRC metastasis is now urgently needed. In this study, we identified autophagy-related protein 9B (ATG9B) as a key potential target gene for CRC metastasis. High expression of ATG9B in tumour significantly increased the risk of metastasis and poor prognosis of CRC. Mechanistically, we further find that ATG9B promoted CRC invasion mainly through autophagy-independent manner. MYH9 is the pivotal interacting protein for ATG9B functioning, which directly binds to cytoplasmic peptide segments aa368–411 of ATG9B by its head domain. Furthermore, the combination of ATG9B and MYH9 enhance the stability of each other by decreasing their binding to E3 ubiquitin ligase STUB1, therefore preventing them from ubiquitin-mediated degradation, which further amplified the effect of ATG9B and MYH9 in CRC cells. During CRC cell invasion, ATG9B is transported to the cell edge with the assistance of MYH9 and accelerates focal adhesion (FA) assembly through mediating the interaction of endocytosed integrin β1 and Talin-1, which facilitated to integrin β1 activation. Clinically, upregulated expression of ATG9B in human CRC tissue is always accompanied with highly elevated expression of MYH9 and associated with advanced CRC stage and poor prognosis. Taken together, this study highlighted the important role of ATG9B in CRC metastasis by promoting focal adhesion assembly, and ATG9B together with MYH9 can provide a pair of potential therapeutic targets for preventing CRC progression.

Suppression of the fibrotic encapsulation of silicone implants by inhibiting the mechanical activation of pro-fibrotic TGF-β.

The fibrotic encapsulation of implants involves the mechanical activation of myofibroblasts and of pro-fibrotic transforming growth factor beta 1 (TGF-β1). Here, we show that both softening of the implant surfaces and inhibition of the activation of TGF-β1 reduce the fibrotic encapsulation of subcutaneous silicone implants in mice. Conventionally stiff silicones (elastic modulus, ~2 MPa) coated with a soft silicone layer (elastic modulus, ~2 kPa) reduced collagen deposition as well as myofibroblast activation without affecting the numbers of macrophages and their polarization states. Instead, fibroblasts around stiff implants exhibited enhanced intracellular stress, increased the recruitment of αv and β1 integrins, and activated TGF-β1 signalling. In vitro, the recruitment of αv integrin to focal adhesions and the activation of β1 integrin and of TGF-β were higher in myofibroblasts grown on latency-associated peptide (LAP)-coated stiff silicones than on soft silicones. Antagonizing αv integrin binding to LAP through the small-molecule inhibitor CWHM-12 suppressed active TGF-β signalling, myofibroblast activation and the fibrotic encapsulation of stiff subcutaneous implants in mice.

The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active β1-integrin in adult tendon cells

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal–regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin–containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp–mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.

The Antibody Receptor Fc Gamma Receptor IIIb Induces Calcium Entry via Transient Receptor Potential Melastatin 2 in Human Neutrophils.

Human neutrophils express two unique antibody receptors for IgG, the FcγRIIa and the FcγRIIIb. FcγRIIa contains an immunoreceptor tyrosine-based activation motif (ITAM) sequence within its cytoplasmic tail, which is important for initiating signaling. In contrast, FcγRIIIb is a glycosylphosphatidylinositol (GPI)-linked receptor with no cytoplasmic tail. Although, the initial signaling mechanism for FcγRIIIb remains unknown, it is clear that both receptors are capable of initiating distinct neutrophil cellular functions. For example, FcγRIIa is known to induce an increase in L-selectin expression and efficient phagocytosis, while FcγRIIIb does not promote these responses. In contrast, FcγRIIIb has been reported to induce actin polymerization, activation of β1 integrins, and formation of neutrophils extracellular traps (NET) much more efficiently than FcγRIIa. Another function where these receptors seem to act differently is the increase of cytoplasmic calcium concentration. It has been known for a long time that FcγRIIa induces production of inositol triphosphate (IP3) to release calcium from intracellular stores, while FcγRIIIb does not use this phospholipid. Thus, the mechanism for FcγRIIIb-mediated calcium rise remains unknown. Transient Receptor Potential Melastatin 2 (TRPM2) is a calcium permeable channel expressed in many cell types including vascular smooth cells, endothelial cells and leukocytes. TRPM2 can be activated by protein kinase C (PKC) and by oxidative stress. Because we previously found that FcγRIIIb stimulation leading to NET formation involves PKC activation and reactive oxygen species (ROS) production, in this report we explored whether TRPM2 is activated via FcγRIIIb and mediates calcium rise in human neutrophils. Calcium rise was monitored after Fcγ receptors were stimulated by specific monoclonal antibodies in Fura-2-loaded neutrophils. The bacterial peptide fMLF and FcγRIIa induced a calcium rise coming initially from internal pools. In contrast, FcγRIIIb caused a calcium rise by inducing calcium entry from the extracellular medium. In addition, in the presence of 2-aminoethoxydiphenyl borate (2-APB) or of clotrimazole, two inhibitors of TRPM2, FcγRIIIb-induced calcium rise was blocked. fMLF- or FcγRIIa-induced calcium rise was not affected by these inhibitors. These data suggest for the first time that FcγRIIIb aggregation activates TRPM2, to induce an increase in cytoplasmic calcium concentration through calcium internalization in human neutrophils.

Mitofusin-2 regulates leukocyte adhesion through the maturation of beta2 integrin activation in differentiation

Abstract Neutrophils are critical in inflammation and innate immunity. Neutrophil adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β2-integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in mitofusin-2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP receptors and β2 integrins as well as the inhibited β2 integrin activation, as assessed by conformational-specific antibodies. Mitofusin-2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by identified markers CD35 and CD87. Reversed to results in overall HL60 cells, mitofusin-2 knockdown in β2-integrin-activation-matured cells (CD87high population) enhanced integrin activation. Our study illustrates the function of mitofusin-2 in leukocyte adhesion and may provide new insights into the development and treatment of mitofusin-2-deficiency-related disease.

Mitofusin‐2 regulates leukocyte adhesion through the maturation of β2 integrin activation in differentiation

Neutrophils are critical in inflammation and innate immunity. Neutrophil adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β2-integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in mitofusin-2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP receptors and β2 integrins as well as the inhibited β2 integrin activation, as assessed by conformational-specific antibodies. Mitofusin-2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by identified markers CD35 and CD87. Reversed to results in overall HL60 cells, mitofusin-2 knockdown in β2-integrin-activation-matured cells (CD87high population) enhanced integrin activation. Our study illustrates the function of mitofusin-2 in leukocyte adhesion and may provide new insights into the development and treatment of mitofusin-2-deficiency-related disease.

KRIT1 stabilizes endothelial adherens junctions independent of Rap1 via regulation of β1‐integrin

The junctional scaffold KRIT1 interacts with adherens junctions (AJ) proteins to stabilize endothelial cell-cell contacts. Loss of KRIT1 function leads to Cerebral Cavernous Malformations (CCM), a disease characterized by leaky vascular lesions as the result of perturbed endothelial proliferation and inflammatory signaling. As a true Rap1 effector, KRIT1 localizes to and associates with AJ after binding active Rap1. Inhibition of this interaction by mutation of the Rap1-binding site (KRIT1-R452E) prevents KRIT1 from localizing to AJ and stabilizing barrier function. However, it remains unclear whether KRIT1 can stabilize endothelial junctions independently of Rap1. In order to study the mechanisms of KRIT1-mediated junction stabilization, we targeted mCherry-tagged KRIT1-R452E to the plasma membrane using a C-terminal lipid modification (MT-R452E). Immunofluorescence studies indicated that this construct localized to cell-cell contacts similar to WT-KRIT1 in endothelial cells depleted of endogenous KRIT1 (KRIT1 shRNA). While MT-R452E partially rescued AJ staining in these cells, it was unable to rescue barrier function, suggesting that both KRIT1 and Rap1 may be necessary at AJ to stabilize the endothelial barrier. As Rap1 binding may also regulate a putative intramolecular interaction between KRIT1's first NPxY motif and its C-terminal PTB domain, we sought to evaluate whether stabilization of endothelial junctions was dependent on KRIT1 conformation. The NPxY motif of MT-R452E was mutated (N192A-Y195A) to yield a constitutively open, membrane-targeted construct (MT-R452E-ΔNPxY). Immunofluorescence confirmed that this construct localized to cell-cell contacts, and appeared to rescue AJ staining after loss of endogenous KRIT1. Interestingly, open conformation R452E lacking any targeting sequence (R452E-ΔNPxY) also rescued AJ staining despite remaining primarily cytosolic. Further, both open conformation constructs rescued barrier function independent of Rap1 binding, indicating that KRIT1's junction stabilizing effects may be exerted without binding of Rap1 or localization to cell-cell contacts. As this NPxY (192-195) motif mediates binding to ICAP1a, which has been shown to regulate β1-integrin activity, we next examined whether junctional stability was associated with changes in β1-integrin activation. Using an activation-specific antibody against β1-integrin, we observed an increase in large fibrillar adhesions in cells depleted of KRIT1 (KRIT1 shRNA), or rescued with either R452E or MT-R452E, constructs which notably failed to rescue loss of barrier function. Conversely, expression of WT, MT-R452E-ΔNPXY, and R452E-ΔNPXY constructs, which do rescue barrier function, was associated with smaller, peripheral staining of focal contacts. These results suggest that KRIT1 may stabilize endothelial cell junctions independent of Rap1 by regulating β1-integrin-containing adhesive complexes. We expect that these novel findings will further our understanding of junctional physiology while facilitating the development of novel CCM therapeutics.

Involvement of Mechanosensitive Channel Piezo1 in Renal Fibrosis

Renal fibrosis is the final common pathway of numerous progressive kidney diseases, characterized by deposition of extracellular matrix in the tubulointerstitial areas, leading to scar formation and increased tissue stiffness of kidney. The underlying mechanisms of renal fibrosis are not fully identified. Piezo1 is known as a mechanosensitive nonselective cation channel, playing an important role in many physiological and pathological processes. The purpose of the current study is to investigate the role of Piezo1 in renal fibrosis and potential molecular mechanism. In human fibrotic kidneys, Piezo1 protein expression was markedly upregulated. The abundance of Piezo1 protein in kidneys of mice was instantly increased after 30 minutes of unilateral ureteral obstruction (UUO), and persistently increased at the 3rd and the 7th days after UUO. Inhibition of Piezo1 with GsMTx4 dramatically ameliorated UUO-induced renal fibrosis. Mechanical stretch induced Piezo1 activation and epithelial-mesenchymal transition (EMT) in human proximal tubular HK2 cells, as seen in increased abundance of a fibrotic marker fibronectin and decreased expression of an epithelial marker E-cadherin, which were greatly reversed by inhibition or silence of Piezo1. Similarly, in HK2 cells, TGF-β1-induced EMT was significantly inhibited by GsMTx4, whereas a specific Piezo1 agonist Yoda1 markedly induced EMT. In addition, the increased expression of Piezo1 protein was found when HK2 cells were cultured in stiff polyacrylamide hydrogels, mimicking the natural accumulation of ECM. Stimulation of Piezo1 by Yoda1 caused calcium influx in association with activation of calpain2 and integrinβ1. Also, Yoda1 promoted an interaction between ECM and integrinβ1 in HK2 cells. In conclusion, activation of Piezo1 is involved in the development of renal fibrosis and EMT in HK2 cells, likely through calcium-calpain2-integrinβ1 pathway.