YouDong Pan

Cation-π interaction regulates ligand-binding affinity and signaling of integrin α4β7

Integrin α4β7 mediates rolling and firm adhesion of leucocytes, two of the critical steps in leukocyte migration and tissue specific homing. Affinity of α4β7 for ligand is dynamically regulated by three interlinked metal ion-binding sites in β7-subunit I domain. In this study, we found that Phe185 (F185), a highly conserved aromatic residue in β7-subunit, links the specificity-determining loop and the synergistic metal ion-binding site (SyMBS) through cation-π interaction. Mutations of F185 that disrupted the SyMBS cation-F185 interaction led to deficient firm cell adhesion mediated by high affinity α4β7, and only slightly affected rolling adhesion mediated by low affinity α4β7. Disruption of SyMBS cation-F185 interaction induced partial extension of integrin ectodomain and separation of cytoplasmic tails, and impaired α4β7-mediated bidirectional signaling. In addition, loss of SyMBS cation-F185 interaction increased paxillin expression and promoted paxillin-integrin binding, leading to deficient cell spreading. Furthermore, integrin α4β7-mediated cell migration was decreased by the abolishment of SyMBS cation-F185 interaction. Thus, these findings reveal a cation-π interaction playing vital roles in the regulation of integrin affinity, signaling, and biological functions.

Distinct Chemokine Signaling Regulates Integrin Ligand Specificity to Dictate Tissue-Specific Lymphocyte Homing

Immune surveillance and host defense depend on the precisely regulated trafficking of lymphocytes. Integrin α4β7 mediates lymphocyte homing to the gut through its interaction with mucosal vascular address in cell adhesion molecule-1 (MAdCAM-1). α4β7 also binds vascular cell adhesion molecule-1 (VCAM-1), which is expressed in other tissues. To maintain the tissue specificity of lymphocyte homing, α4β7 must distinguish one ligand from the other. Here, we demonstrate that α4β7 is activated by different chemokines in a ligand-specific manner. CCL25 stimulation promotes α4β7-mediated lymphocyte adhesion to MAdCAM-1 but suppresses adhesion to VCAM-1, whereas CXCL10 stimulation has the opposite effect. Using separate pathways, CCL25 and CXCL10 stimulate differential phosphorylation states of the β7 tail and distinct talin and kindlin-3 binding patterns, resulting in different binding affinities of MAdCAM-1 and VCAM-1 to α4β7. Thus, our findings provide a mechanism for lymphocyte traffic control through the unique ligand-specific regulation of integrin adhesion by different chemokines.

The CC′ and DE Loops in Ig Domains 1 and 2 of MAdCAM-1 Play Different Roles in MAdCAM-1 Binding to Low- and High-affinity Integrin α4β7

Lymphocyte homing is regulated by the dynamic interaction between integrins and their ligands. Integrin α4β7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Although previous studies have revealed some mechanisms of α4β7-MAdCAM-1 binding, little is known about the different molecular bases of the low- and high-affinity α4β7-MAdCAM-1 interactions, which mediate rolling and firm adhesion of lymphocytes, respectively. Here, we found that two loops in immunoglobulin domains 1 and 2 (D1 and D2) of MAdCAM-1 played different roles in MAdCAM-1 binding to low-affinity (inactive) and high-affinity (activated) α4β7. The Asp-42 in the CC′ loop of D1 was indispensable for MAdCAM-1 binding to both low-affinity and high-affinity α4β7. The other CC′ loop residues except for Arg-39 and Ser-44 were essential for MAdCAM-1 binding to both inactive α4β7 and α4β7 activated by SDF-1α or talin, but not required for MAdCAM-1 binding to Mn2+-activated α4β7. Single amino acid substitution of the DE loop residues mildly decreased MAdCAM-1 binding to both inactive and activated α4β7. Notably, removal of the DE loop greatly impaired MAdCAM-1 binding to inactive and SDF-1α- or talin-activated α4β7, but only decreased 60% of MAdCAM-1 binding to Mn2+-activated α4β7. Moreover, DE loop residues were important for stabilizing the low-affinity α4β7-MAdCAM-1 interaction. Thus, our findings demonstrate the distinct roles of the CC′ and DE loops in the recognition of MAdCAM-1 by low- and high-affinity α4β7 and suggest that the inactive α4β7 and α4β7 activated by different stimuli have distinct conformations with different structural requirements for MAdCAM-1 binding.

Lithocholic acid‐induced placental tumor necrosis factor‐α upregulation and syncytiotrophoblast cell apoptosis in intrahepatic cholestasis of pregnancy

To investigate tumor necrosis factor (TNF)‐α expression and its relationship with serum bile acids in placental trophoblasts from patients with intrahepatic cholestasis of pregnancy (ICP).

Static Compression Induces Ecm Remodeling and Integrin α2β1 Expression and Signaling in a Rat Tail Caudal Intervertebral Disc Degeneration Model

Study Design. A three-level rat tail caudal intervertebral disc (IVD) degeneration (IVDD) model was established to study effects of static compression on extracellular matrix (ECM) remodeling and integrin signaling in IVDs during IVDD. Objective. The aim of this study was to investigate the effect of compression force on ECM remodeling and integrin signaling in IVDs during IVDD. Summary of Background Data. Integrins sense mechanical environment alteration via binding to ECM ligands and trigger intracellular signaling for pathological ECM remodeling during IVDD. However, the role of compression force in ECM remodeling and integrin signaling during IVDD remains elusive. Methods. Compared with the classical one-level rat tail IVDD model that exerts axial stress on the 8th to 9th caudal vertebral bodies, a three-level model was established by using an Ilizarov-type apparatus to exert stress on the 7th to 10th caudal vertebral bodies in rat tails for four weeks. To exclude side effects from surgical stab injury on manipulated discs, intact coccygeal (Co) disc Co8–9 was analyzed. Results. In three-level IVDD model, significant degeneration of the Co8–9 disc was observed. Quantitative real-time polymerase chain reaction (qRT-PCR) showed elevated mRNA expression of collagen types I, III, and V; matrix metalloproteinases (MMPs) 2, 3, 9, 13, 14; and decreased mRNA expression of collagen type II in Co8–9 disc. Compression loading altered the expression of integrin &agr;2&bgr;1 (upregulated) and &agr;10&bgr;1 (downregulated) in NP cells, and activated integrin downstream signaling. By contrast, one-level model showed more severe disc degeneration and ECM remodeling. Integrin &agr;1, &agr;2, &agr;11, and &bgr;1 were upregulated, whereas &agr;10 was downregulated. Similar activation of integrin signaling was observed. Conclusion. Static compression altered collagen and MMP expression, and promoted &bgr;1 integrin expression and signaling in IVD. Compared with one-level rat tail IVDD model, three-level model showed milder effects on disc degeneration, ECM remodeling, and integrin expression, suggesting one-level model might involve other causes that induce IVDD via mechanisms independent of compression force. Level of Evidence: N/A

Study on the regulation of cell adhesion molecule expression and function in placenta from women with intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a common complication of pregnancy manifested as skin pruritus of cholestasis. ICP occurs mainly in the second or third trimester of pregnancy and may cause fetal distress, unexpected intrauterine fetal death and does serious harm to maternal and fetal health. The pathogenesis of ICP is still unclear. In ICP placentas, placental syncytiotrophoblasts are the most direct contact between maternal high bile acid environment and fetus. Our previous study found that in ICP placental syncytiotrophoblasts, both mRNA expression level and protein expression level of vascular cell adhesion molecule-1 (VCAM-1), were significantly elevated. Since VCAM-1 is important in inflammatory injury of lymphocytes, we speculate that ICP pathogenesis may be associated with VCAM-1 up-regulation which may lead to inflammatory injury and cause intrauterine fetal distress, intrauterine fetal death and other adverse outcomes. Elucidation of this mechanism should help reveal the ICP pathogenesis and facilitate the clinical treatment of intrauterine fetal death.

Disruption of disulfide restriction at integrin knees induces activation and ligand-independent signaling of α4β7

Summary Control of integrin activation and signaling plays crucial roles in cell adhesion, spreading and migration. Here, we report that selective breakage of two conserved disulfide bonds located at the knees of integrin &agr;4C589–C594 and &bgr;7C494–C526 activated &agr;4&bgr;7. This activated integrin had a unique structure that was different from the typical extended conformation of active integrin. In addition, these activated &agr;4&bgr;7 integrins spontaneously clustered on the cell membrane and triggered integrin downstream signaling independent of ligand binding. Although these disulfide bonds were not broken during &agr;4&bgr;7 activation by inside-out signaling or Mn2+, they could be specifically reduced by 0.1 mM dithiothreitol, a reducing strength that could be produced in vivo under certain conditions. Our findings reveal a novel mechanism of integrin activation under specific reducing conditions by which integrin can signal and promote cell spreading in the absence of ligand.

The Unique Disulfide Bond-stabilized W1 β4-β1 Loop in the α4 β-Propeller Domain Regulates Integrin α4β7 Affinity and Signaling

Background: Integrin α4β7 is unique for mediating rolling and firm adhesion of lymphocytes pre- and post-activation. Results: Disrupting the disulfide bond-stabilized W1 β4-β1 loop in the α4 β-propeller domain impaired α4β7-mediated, low-affinity ligand binding and bidirectional signaling. Conclusion: The W1 β4-β1 loop regulates integrin ligand binding and signaling. Significance: Our findings reveal a particular molecular basis for α4β7-mediated rolling cell adhesion. Integrin α4β7 mediates rolling and firm adhesion of lymphocytes pre- and post-activation, which is distinct from most integrins only mediating firm cell adhesion upon activation. This two-phase cell adhesion suggests a unique molecular basis for the dynamic interaction of α4β7 with its ligand, mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Here we report that a disulfide bond-stabilized W1 β4-β1 loop in α4 β-propeller domain plays critical roles in regulating integrin α4β7 affinity and signaling. Either breaking the disulfide bond or deleting the disulfide bond-occluded segment in the W1 β4-β1 loop inhibited rolling cell adhesion supported by the low-affinity interaction between MAdCAM-1 and inactive α4β7 but negligibly affected firm cell adhesion supported by the high-affinity interaction between MAdCAM-1 and Mn2+-activated α4β7. Additionally, disrupting the disulfide bond or deleting the disulfide bond-occluded segment not only blocked the conformational change and activation of α4β7 triggered by talin or phorbol-12-myristate-13-acetate via inside-out signaling but also disrupted integrin-mediated outside-in signaling and impaired phosphorylation of focal adhesion kinase and paxillin. Thus, these findings reveal a particular molecular basis for α4β7-mediated rolling cell adhesion and a novel regulatory element of integrin affinity and signaling.