Activation Of Integrin Α5β1 Research Articles

Eliminating the invading extracellular and intracellular FnBp+ bacteria from respiratory epithelial cells by autophagy mediated through FnBp-Fn-Integrin α5β1 axis.

We previously found that the respiratory epithelial cells could eliminate the invaded group A streptococcus (GAS) through autophagy induced by binding a fibronectin (Fn) binding protein (FnBp) expressed on the surface of GAS to plasma protein Fn and its receptor integrin α5β1 of epithelial cells. Is autophagy initiated by FnBp+ bacteria via FnBp-Fn-Integrin α5β1 axis a common event in respiratory epithelial cells? We chose Staphylococcus aureus (S. aureus/S. a) and Listeria monocytogenes (L. monocytogenes/L. m) as representatives of extracellular and intracellular FnBp+ bacteria, respectively. The FnBp of them was purified and the protein function was confirmed by western blot, viable bacteria count, confocal and pull-down. The key molecule downstream of the action axis was detected by IP, mass spectrometry and bio-informatics analysis. We found that different FnBp from both S. aureus and L. monocytogenes could initiate autophagy through FnBp-Fn-integrin α5β1 axis and this could be considered a universal event, by which host tries to remove invading bacteria from epithelial cells. Importantly, we firstly reported that S100A8, as a key molecule downstream of integrin β1 chain, is highly expressed upon activation of integrin α5β1, which in turn up-regulates autophagy. Various FnBp from FnBp+ bacteria have the ability to initiate autophagy via FnBp-Fn-Integrin α5β1 axis to promote the removal of invading bacteria from epithelial cells in the presence of fewer invaders. S100A8 is a key molecule downstream of Integrin α5β1 in this autophagy pathway.

Regulation of integrin α5β1-mediated Staphylococcus aureus cellular invasion by the septin cytoskeleton

Staphylococcus aureus, a Gram-positive bacterial pathogen, is an urgent health threat causing a wide range of clinical infections. Originally viewed as a strict extracellular pathogen, accumulating evidence has revealed S. aureus to be a facultative intracellular pathogen subverting host cell signalling to support invasion. The majority of clinical isolates produce fibronectin-binding proteins A and B (FnBPA and FnBPB) to interact with host integrin α5β1, a key component of focal adhesions. S. aureus binding of integrin α5β1 promotes its clustering on the host cell surface, triggering activation of focal adhesion kinase (FAK) and cytoskeleton rearrangements to promote bacterial invasion into non-phagocytic cells. Here, we discover that septins, a component of the cytoskeleton that assembles on membranes, are recruited as collar-like structures with actin to S. aureus invasion sites engaging integrin α5β1. To investigate septin recruitment to the plasma membrane in a bacteria-free system, we used FnBPA-coated latex beads and showed that septins are recruited upon activation of integrin α5β1. SEPT2 depletion reduced S. aureus invasion, but increased surface expression of integrin α5 and adhesion of S. aureus to host cells. Consistent with this, SEPT2 depletion increased cellular protein levels of integrin α5 and β1 subunits, as well as FAK. Collectively, these results provide insights into regulation of integrin α5β1 and invasion of S. aureus by the septin cytoskeleton.

Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1.

Reticular adhesions (RAs) consist of integrin αvβ5 and harbor flat clathrin lattices (FCLs), long-lasting structures with similar molecular composition as clathrin-mediated endocytosis (CME) carriers. Why FCLs and RAs colocalize is not known. Here, we show that RAs are assembled at FCLs in a process controlled by fibronectin (FN) and its receptor, integrin α5β1. We observed that cells on FN-rich matrices displayed fewer FCLs and RAs. CME machinery inhibition abolished RAs and live-cell imaging showed that RA establishment requires FCL coassembly. The inhibitory activity of FN was mediated by the activation of integrin α5β1 at Tensin1-positive fibrillar adhesions. Conventionally, endocytosis disassembles cellular adhesions by internalizing their components. Our results present a novel paradigm in the relationship between these two processes by showing that endocytic proteins can actively function in the assembly of cell adhesions. Furthermore, we show this novel adhesion assembly mechanism is coupled to cell migration via unique crosstalk between cell-matrix adhesions.

Coupling of Integrin α5 to Annexin A2 by Flow Drives Endothelial Activation.

Atherosclerosis preferentially occurs at specific sites of the vasculature where endothelial cells (ECs) are exposed to disturbed blood flow. Translocation of integrin α5 to lipid rafts promotes integrin activation and ligation, which is critical for oscillatory shear stress (OSS)-induced EC activation. However, the underlying mechanism of OSS promoted integrin α5 lipid raft translocation has remained largely unknown. The objective of this study was to specify the mechanotransduction mechanism of OSS-induced integrin α5 translocation and subsequent EC activation. Mass spectrometry studies identified endothelial ANXA2 (annexin A2) as a potential carrier allowing integrin α5β1 to traffic in response to OSS. Interference by siRNA of AnxA2 in ECs greatly decreased OSS-induced integrin α5β1 translocation to lipid rafts, EC activation, and monocyte adhesion. Pharmacological and genetic inhibition of PTP1B (protein tyrosine phosphatase 1B) blunted OSS-induced integrin α5β1 activation, which is dependent on Piezo1-mediated calcium influx in ECs. Furthermore, ANXA2 was identified as a direct substrate of activated PTP1B by mass spectrometry. Using bioluminescence resonance energy transfer assay, PTP1B-dephosphorylated ANXA2 at Y24 was found to lead to conformational freedom of the C-terminal core domain from the N-terminal domain of ANXA2. Immunoprecipitation assays showed that this unmasked ANXA2-C-terminal core domain specifically binds to an integrin α5 nonconserved cytoplasmic domain but not β1. Importantly, ectopic lentiviral overexpression of an ANXA2Y24F mutant increased and shRNA against Ptp1B decreased integrin α5β1 ligation, inflammatory signaling, and progression of plaques at atheroprone sites in apolipoprotein E (ApoE)-/- mice. However, the antiatherosclerotic effect of Ptp1B shRNA was abolished in AnxA2-/-ApoE-/- mice. Our data elucidate a novel endothelial mechanotransduction molecular mechanism linking atheroprone flow and activation of integrin α5β1, thereby identifying a class of potential therapeutic targets for atherosclerosis. Graphic Abstract: An graphic abstract is available for this article.

Synthetic α5β1 integrin ligand PHSRN is proangiogenic and neuroprotective in cerebral ischemic stroke

Ischemic stroke is the leading cause of disability and death worldwide. An effective therapeutic approach is urgently needed. Stroke-induced angiogenesis and neurogenesis are essential mechanisms in the long-term repair. Extracellular matrix proteins are also involved in tissue self-repair. Recently, a PHSRN (Pro-His-Ser-Arg-Asn) peptide from the fibronectin synergistic motif that can promote wound healing in epithelia and induce endothelial proliferation and cancer cell migration was identified. The therapeutic potential of this peptide in stroke is unknown. Here, we examined the potential of PHSRN in stroke therapy using an ischemic rat model of middle cerebral artery occlusion (MCAO). PHSRN reduced the infarct volume in MCAO rats, improved neurological function, and alleviated motor function impairment. PHSRN targeted the damaged brain region and distributed to endothelial cells after intraperitoneal injection. PHSRN significantly promoted angiogenesis and vascular endothelial growth factor secretion through activation of integrin α5β1 and its downstream intracellular signals, e.g., focal adhesion kinase, Ras, cRaf, and extracellular-signal-regulated kinase. PHSRN treatment also stimulated neurogenesis in MCAO rats, and maintained neuronal survival and neuronal morphologic complexity via induction of VEGF secretion. Together, these results provide insights into the role of integrin α5β1 following ischemia and support the feasibility of using PHSRN peptide in stroke therapy.

Estradiol Facilitates Functional Integration of iPSC-Derived Dopaminergic Neurons into Striatal Neuronal Circuits via Activation of Integrin α5β1.

SummaryFor cell transplantation therapy for Parkinson's disease (PD) to be realized, the grafted neurons should be integrated into the host neuronal circuit to restore the lost neuronal function. Here, using wheat-germ agglutinin-based transsynaptic tracing, we show that integrin α5 is selectively expressed in striatal neurons that are innervated by midbrain dopaminergic (DA) neurons. In addition, we found that integrin α5β1 was activated by the administration of estradiol-2-benzoate (E2B) in striatal neurons of adult female rats. Importantly, we observed that the systemic administration of E2B into hemi-parkinsonian rat models facilitates the functional integration of grafted DA neurons derived from human induced pluripotent stem cells into the host striatal neuronal circuit via the activation of integrin α5β1. Finally, methamphetamine-induced abnormal rotation was recovered earlier in E2B-administered rats than in rats that received other regimens. Our results suggest that the simultaneous administration of E2B with stem cell-derived DA progenitors can enhance the efficacy of cell transplantation therapy for PD.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.

Epidermal growth factor receptor transactivation and fibronectin matrix assembly by the G‐protein coupled receptor, GPER, requires a transmembrane signaling complex consisting of PTPN12, integrin α5β1, and MMP‐3

G‐protein estrogen receptor (GPER) positively associates with breast tumor progression, including tumor size and extramammary metastases. Via activation of integrin α5β1, GPER coordinates two cellular activities that facilitate breast tumor cell survival, fibronectin matrix assembly and the release of membrane‐tethered proHB‐EGF. RNAi knockdown of either MMP‐3 or PTPN12 inhibits both of these cellular processes regulated by effectors on opposing sides of the plasma membrane. Moreover, PTPN12 knockdown also negatively influences the ability of GPER to promote the centripetal movement of integrin α5β1 from focal to fibrillar adhesions, the site of fibronectin fibril formation, and results in the accumulation of the Shc adaptor protein on the cytoplasmic tail of integrin α5β1. In support of the concept that PTPN12 is necessary for activated integrin α5β1, SKBR3 cells stably expressing PTPN12 shRNA showed reduced formation of anchorage independent colonies. Efforts to further understand the influence of PTPN12 knockdown on “inside‐out” integrin signaling events required for EGFR transactivation are underway with particular interest in understanding its impact on MMP‐3 activation and HB‐EGF release. A better understand of the molecular mechanism of GPER induced EGFR transactivation may lead to the development of future therapeutic strategies that target this receptor and prevent metastatic disease.

Abstract B42: Modulation of integrin-mediated cell adhesion by ADAM15 disintegrin-like domain

Abstract Integrin-mediated cell adhesion on extracellular matrix works essentially on numerous physiological processes such as cell adhesion, migration and angiogenesis. As integrin ανβ3 and integrin α5β1 are the representative receptors for cell adhesion and due to their important function in cancer biology, the antagonists targeting the integrins has been sought for long. Lately, the interactions between ADAM (A Disintegrin And Metalloprotenase) 15, the only member in ADAM family with Arg-Gly-Asp (RGD) motif, and the ανβ3 and α5β1 intergins was observed. For that, we experimented on the cellular function of recombinant ADAM15-derived disintegrin-like domain containing Asp-Typ-Lys-Arg-Gly-Asp (rNWKRGD) in integrin-mediated cell adhesion. The binding affinity of HUVEC, COS-1, MCF-7 and MDAH 2274 cells to various extracellular matrix proteins were increased by the disintegrin-like domain in a dose-dependent manner, and the presence of inhibitory integrin α5β1 antibody thoroughly abrogated the rNWKRGD-stimulated binding. By mutagenic analysis, it was founded that RGD motif is essential for the binding. In comparison, saxatilin, RGD disintegrin from snake venom, inhibited binding of cells to ECM proteins. Through flow cytometric analysis, we confirmed that β1 integrin on the cell surface was activated by rNWKGRD. All these results indicated that the activation of integrin α5β1 mediates the rNWKRGD-stimulated cell adhesion. Therefore rNWKRGD could function as the potential activator of cell migration and proliferation. Although disintegrin is known as inhibitor of cell migration and proliferation, it is possible that ADAM15 disintegrin domain could have a regulatory role in integrin function such as angiogenesis and invasion of cancer cells. (This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (No. 2009-0081759), KIST grant, and the Brain Korea 21 (BK21).) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B42.