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OBJECTIVE - To investigate the role of integrin α1β1 in the progression of post-traumatic osteoarthritis (PTOA), and elucidate the contribution of epidermal growth factor receptor (EGFR) signalling to the mechanism by which integrin α1β1 might control PTOA. We hypothesised that integrin α1β1 plays a protective role in the course of PTOA and that the effect of PTOA (e.g., synovitis, loss of cartilage and growth of osteophytes) would be exacerbated in mice lacking integrin α1β1 at every time point post destabilisation of medial meniscus (DMM).
METHODS - DMM or sham surgery was performed on integrin α1-null and wild type (WT) mice and the progression of PTOA analysed at 2, 4, 8 and 12 weeks post-surgery (PS) using micro-computed tomography (microCT), histology, and immunohistochemistry. In addition, the effects of EGFR blockade were examined by treating the mice with the EGFR inhibitor erlotinib.
RESULTS - Integrin α1-null female, but not male, mice showed earlier cartilage degradation post DMM surgery compared to WT controls. Furthermore, erlotinib treatment resulted in significantly less cartilage damage in integrin α1-null but not WT mice. Independent of genotype, erlotinib treatment significantly mitigated the effects of PTOA on many tissues of female mice including meniscal and fabella bone volume, subchondral bone thickness and density and cartilage degradation. In contrast, reduced EGFR signalling had little effect on signs of PTOA in male mice.
CONCLUSION - Integrin α1β1 protects against PTOA-induced cartilage degradation in female mice partially via the reduction of EGFR signalling. Furthermore, reduction of EGFR signalling protects against the development of PTOA in female, but not male mice.
Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-β mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-β receptor (TβRII), which in turn promotes a TβRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TβRII is considered the principal regulatory mechanism of TβRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TβRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TβRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TβRII tail tyrosine residues, resulting in inhibition of TβR-dependent fibrotic signaling. The collagen-binding receptor integrin α1β1 was required for recruitment of TCPTP to the TβRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TβRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1β1 and TβRII that is essential for TβRII-mediated SMAD activation and fibrotic signaling pathways.
BACKGROUND/PURPOSE - The goal of this study was to determine the role of the collagen binding receptor integrin α1β1 in regulating osmotically induced [Ca(2+)]i transients in chondrocytes.
METHOD - The [Ca(2+)]i transient response of chondrocytes to osmotic stress was measured using real-time confocal microscopy. Chondrocytes from wildtype and integrin α1-null mice were imaged ex vivo (in the cartilage of intact murine femora) and in vitro (isolated from the matrix, attached to glass coverslips). Immunocytochemistry was performed to detect the presence of the osmosensor, transient receptor potential vanilloid-4 (TRPV4), and the agonist GSK1016790A (GSK101) was used to test for its functionality on chondrocytes from wildtype and integrin α1-null mice.
RESULTS/INTERPRETATION - Deletion of the integrin α1 subunit inhibited the ability of chondrocytes to respond to a hypo-osmotic stress with [Ca(2+)]i transients ex vivo and in vitro. The percentage of chondrocytes responding ex vivo was smaller than in vitro and of the cells that responded, more single [Ca(2+)]i transients were observed ex vivo compared to in vitro. Immunocytochemistry confirmed the presence of TRPV4 on wildtype and integrin α1-null chondrocytes, however application of GSK101 revealed that TRPV4 could be activated on wildtype but not integrin α1-null chondrocytes. Integrin α1β1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, the mechanism by which integrin α1β1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor TRPV4.
Copyright © 2014 Elsevier Inc. All rights reserved.
Integrin α1β1 binding to collagen IV, which is mediated by the α1-inserted (I) domain, down-regulates collagen synthesis. When unligated, a salt bridge between Arg(287) and Glu(317) is thought to keep this domain in a low affinity conformation. Ligand binding opens the salt bridge leading to a high-affinity conformation. How modulating integrin α1β1 affinity alters collagen homeostasis is unknown. To address this question, we utilized a thermolysin-derived product of the α1α2α1 network of collagen IV (α1α2α1(IV) truncated protomer) that selectively binds integrin α1β1. We show that an E317A substitution enhanced binding to the truncated protomer, consistent with a previous finding that this substitution eliminates the salt bridge. Surprisingly, we show that an R287A substitution did not alter binding, whereas R287E/E317R substitutions enhanced binding to the truncated protomer. NMR spectroscopy and molecular modeling suggested that eliminating the Glu(317) negative charge is sufficient to induce a conformational change toward the open state. Thus, the role played by Glu(317) is largely independent of the salt bridge. We further show that cells expressing E317A or R287E/E317R substitutions have enhanced down-regulation of collagen IV synthesis, which is mediated by the ERK/MAPK pathway. In conclusion, we have demonstrated that modulating the affinity of the extracellular α1 I domain to collagen IV enhances outside-in signaling by potentiating ERK activation and enhancing the down-regulation of collagen synthesis.
Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury.
Integrin alpha1beta1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin alpha1beta1-mediated EGFR activation. Integrin alpha1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin alpha1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin alpha1-null MCs decreases EGFR-mediated ROS production. We further show that integrin alpha1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARgamma or inhibition of ERK increases Cav-1 levels in the integrin alpha1-null MCs. Finally, we show that glomeruli of integrin alpha1-null mice have reduced levels of Cav-1 and activated PPARgamma but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin alpha1beta1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARgamma axis plays a key role in regulating integrin alpha1beta1-dependent Cav-1 expression and consequent EGFR-mediated ROS production.
BACKGROUND - Cells interact with type IV collagen (Col IV) via integrins through the triple-helical and NC1 domains. We examined interactions of human glomerular and proximal tubular epithelial cells with recombinant alpha1 and alpha3 NC1 chains of Col IV, to explore the ability of different cell types to interact with Col IV of different trimer composition.
METHODS - Interactions of TSV-40-immortalized human glomerular epithelial cells (HGECs), HPV-16-immortalized human proximal tubular epithelial (HK-2) cells and primary human mesangial cells (MES) with recombinant alpha1 and alpha3 NC1 chains of Col IV were examined by affinity chromatography and solid-phase binding assays. The expression of integrin-regulated metalloproteinases was examined by zymography.
RESULTS - HGECs bound to both alpha3 and alpha1(IV)NC1, albeit there was preferential binding to alpha3(IV)NC1, through the alpha3beta1 and alpha2beta1 integrin receptors; HK-2 cells and MES bound almost exclusively to alpha1(IV)NC1 via the alpha3beta1/alphavbeta3 and alpha1beta1/alpha2beta1 receptors, respectively. It was demonstrated that the expression of MMP-2 and MMP-9 by HGECs was down-regulated in the presence of alpha3(IV)NC1.
CONCLUSIONS - The observed data indicate that the isoform NC1 chains of Col IV serve for selective, integrin-mediated cell binding which probably triggers different signaling mechanisms, resulting in the activation of specific transcription factors and the modulation of gene expression.
Atherosclerotic plaques express high levels of small proline-rich repeat protein (SPRR3), a previously characterized component of the cornified cell envelope of stratified epithelia, where it is believed to play a role in cellular adaptation to biomechanical stress. We investigated the physiological signals and underlying mechanism(s) that regulate atheroma-enriched SPRR3 expression in vascular smooth muscle cells (VSMCs). We showed that SPRR3 is expressed by VSMCs in both human and mouse atheromas. In cultured arterial VSMCs, mechanical cyclic strain, but neither shear stress nor lipid loading induced SPRR3 expression. Furthermore, this upregulation of SPRR3 expression was dependent on VSMC adherence to type I collagen. To link the mechanoregulation of SPRR3 to specific collagen/integrin interactions, we used blocking antibodies against either integrin alpha1 or alpha2 subunits and VSMCs from mice that lack specific collagen receptors. Our results showed a dependence on the alpha1beta1 integrin for SPRR3 expression induced by cyclic strain. Furthermore, we showed that integrin alpha1 but not alpha2 subunits were expressed on VSMCs within mouse lesions but not in normal arteries. Therefore, we identified the enrichment of the mechanical strain-regulated protein SPRR3 in VSMCs of both human and mouse atherosclerotic lesions whose expression is dependent on the collagen-binding integrin alpha1beta1 on VSMCs. These data suggest that SPRR3 may play a role in VSMC adaptation to local biomechanical stress within the plaque microenvironment.
The collagen-binding integrins alpha1beta1 and alpha2beta1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that alpha1beta1 negatively regulates integrin alpha2beta1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins alpha1beta1 and alpha2beta1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin alpha2beta1, but not alpha1beta1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin alpha2 subunit with that of alpha1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin alpha2 cytoplasmic tail with that of alpha1, decreases cell migration and cord formation, but increases proliferation. When integrin alpha1 and alpha2 subunits are co-expressed in UB cells, the alpha1 subunit negatively regulates integrin alpha2beta1-dependent cord formation, adhesion and migration and this inhibition requires expression of both alpha1 and alpha2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the alpha2 integrin subunit, as well as the alpha1 integrin subunit, regulate integrin alpha2beta1 cell function.
The collagen IV binding receptor integrin alpha1beta1 has been shown to regulate lung cancer due to its proangiogenic properties; however, it is unclear whether this receptor also plays a direct role in promoting primary lung tumors. To investigate this possibility, integrin alpha1-null mice were crossed with KrasLA2 mice that carry an oncogenic mutation of the Kras gene (G12D) and develop spontaneous primary tumors with features of non-small cell lung cancer. We provide evidence that KrasLA2/alpha1-null mice have a decreased incidence of primary lung tumors and longer survival compared with KrasLA2/alpha1 wild-type controls. Tumors from KrasLA2/alpha1-null mice were also smaller, less vascularized, and exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end staining, respectively. Moreover, tumors from the KrasLA2/alpha1-null mice showed diminished extracellular signal-regulated kinase (ERK) but enhanced p38 mitogen-activated protein kinase activation. Primary lung tumor epithelial cells isolated from KrasLA2/alpha1-null mice showed a significant decrease in anchorage-independent colony formation, collagen-mediated cell proliferation, ERK activation, and, most importantly, tumorigenicity when injected into nude mice compared with KrasLA2/alpha1 wild-type tumor cells. These results indicate that loss of the integrin alpha1 subunit decreases the incidence and growth of lung epithelial tumors initiated by oncogenic Kras, suggesting that both Kras and integrin alpha1beta1 cooperate to drive the growth of non-small cell lung cancer in vivo.