Beta1 Integrin Subunit Research Articles

Pelvic Chlamydial Infection Predisposes to Ectopic Pregnancy by Upregulating Integrin β1 to Promote Embryo-tubal Attachment

Tubal ectopic pregnancies are a leading cause of global maternal morbidity and mortality. Previous infection with Chlamydia trachomatis is a major risk factor for tubal embryo implantation but the biological mechanism behind this association is unclear. Successful intra-uterine embryo implantation is associated with increased expression of endometrial “receptivity” integrins (cell adhesion molecules). We examined integrin expression in Fallopian tubes of women with previous C. trachomatis infection, in mice experimentally infected with C. trachomatis, in immortalised human oviductal epithelial cells (OE-E6/E7) and in an in vitro model of human embryo attachment (trophoblast spheroid-OE-E6/7 cell co-culture). Previous exposure with C. trachomatis increased Fallopian tube/oviduct integrin-subunit beta-1 (ITGB1) in women and mice compared to controls. C. trachomatis increased OE-E6/E7 cell ITGB1 expression and promoted trophoblast attachment to OE-E6/E7 cells which was negated by anti-ITGB1-antibody. We demonstrate that infection with C. trachomatis increases tubal ITGB1 expression, predisposing to tubal embryo attachment and ectopic pregnancy.

Effect of 2,3',4,4',5-Pentachlorobiphenyl Exposure on Endometrial Receptivity and the Methylation of HOXA10.

Polychlorinated biphenyls (PCBs) are one of the most common endocrine-disrupting chemicals and have obvious toxicity on human reproductive development. The aim of our study was to investigate the toxicity of chronic 2,3',4,4',5-pentachlorobiphenyl (PCB 118) exposure on embryo implantation and endometrial receptivity, with the possible mechanism of DNA methylation involved. Virgin CD-1 female mice (3 weeks old) were housed and orally treated with PCB 118 (0, 1, 10, 100 μg/kg) for a month. After mating with fertile males, the pregnant mice were killed on gestation day 4.5. Compared with the control group, implantation failures were observed in 1 μg/kg PCB 118- and 100 μg/kg PCB 118-treated groups. Abnormal endometrial morphology with open uterine lumens and densely compact stromal cells and poorly developed pinopodes were substantially in response to PCB 118 doses above, as well as the significant downregulation of implantation-associated genes (estrogen receptor 1, homeobox A10 [HOXA10], integrin subunit beta 3) and hypermethylation in the promoter region of HOXA10 further. It was confirmed that chronic exposure to PCB 118 produced an increased number of implantation failures in association with a defective uterine morphology during the implantation period. Alterations in methylation of HOXA10 could explain, at least in part, the mechanism of effects of PCB 118 exposure on the implantation process.

Evaluation of ITGB2 (CD18) and SELL (CD62L) genes expression and methylation of ITGB2 promoter region in patients with systemic sclerosis.

Systemic sclerosis (SSc), an autoimmune disease of connective tissue, is characterized by inflammation, fibrosis, and vessel endothelial damage. Products of Integrin subunit beta 2 (ITGB2) and selectin L (SELL) genes participate in several functional pathways of immune system. The aim of this investigation was to survey the transcript level of ITGB2 and SELL genes as well as methylation status of CpG sites in promoter region of differently expressed gene in PBMCs of SSc patients. PBMCs were isolated from whole blood of 50 SSc patients and 30 healthy controls. Total RNA and DNA contents of PBMCs were extracted. Gene expression was analyzed by real-time PCR using the SYBR Green PCR Master Mix. To investigate the methylation status of CpG sites, DNA samples were treated by bisulfite, amplified through nested PCR, and sequenced through Sanger difficult sequencing method. ITGB2 gene in PBMCs of SSc patients was overexpressed significantly in comparison to healthy controls. However, no altered SELL expression was observed. Three CpG sites of 12, 13 and 14 were significantly hypomethylated in patients group, despite overall methylation status of ITGB2 gene promoter revealed no significant difference between study groups. There was no statistically significant correlation between methylation status of ITGB2 promoter and the gene expression in patients. Regarding to lack of correlation of increased expression of ITGB2 with its promoter hypomethylation in SSc patients, our study suggests that upregulation of ITGB2 in PBMCs from SSc patients is probably due to another mechanism other than methylation alteration.

A novel circular RNA, hsa_circ_0046701, promotes carcinogenesis by increasing the expression of miR-142-3p target ITGB8 in glioma

In the present study, we identified a novel circular RNA (circRNA), hsa_circ_0046701, in glioma cells. We measured the expression of hsa_circ_0046701 using qRT-PCR in glioma tissues and cell lines, and explored its functions using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and Transwell assays. Luciferase reporter assays were performed to validate the correlation between microRNA miR-142-3p and hsa_circ_0046701 or integrin subunit beta 8 (ITGB8). The results showed that hsa_circ_0046701 was significantly upregulated in glioma tissues and cell lines, and knockdown of hsa_circ_0046701 inhibited cell proliferation and invasion. Luciferase reporter assays indicated that hsa_circ_0046701 functions as a sponge for miR-142-3p and regulates the expression of ITGB8. Subsequently, functional assays revealed that silencing of hsa_circ_0046701 could upregulate miR-142-3p, resulting in downregulation of ITGB8. The results demonstrated that the hsa_circ_0046701/miR-142-3p/ITGB8 axis might play critical regulatory roles in the pathogenesis and development of glioma.

Simulated Microgravity Condition Alters the Gene Expression of some ECM and Adhesion Molecules in Adipose Derived Stem Cells.

Adipose- derived stem cells (ADSCs) are widely used for tissue engineering and regenerative medicine. The beneficial effects of ADSCs on wound healing have already been reported. Remodeling of extracellular matrix (ECM) is the most important physiological event during wound healing. ECM is sensitive to mechanical stresses and the expression of its components can be therefore influenced. The aim of this study was to investigate the effect of simulated microgravity on gene expression of some ECM and adhesion molecules in human ADSCs. After isolation and characterization of ADSCs, cells were exposed to simulated microgravity for 1, 3 and 7 days. Real-time PCR, fluorescence immunocytochemistry, and MTT assay were performed to evaluate the alterations of integrin subunit beta 1 (ITGB1), collagen type 3 (ColIII), matrix metalloproteinase-1 (MMP1), CD44, fibrillin (FBN1), vimentin (VIM) genes, and ColIII protein levels as well as cells viability. Microgravity simulation increased the expression of ITGB1, ColIII, MMP1, and CD44 and declined the expression of FBN1 and VIM genes. ColIII protein levels also increased. There were no significant changes in the viability of cells cultured in microgravity. Since the high expression of ECM components is known as one of the fibroblast markers, our data suggest that pretreatment of ADSCs by simulated microgravity may increase their differentiation capacity towards fibroblastic cells. Microgravity had not adversely affected the viability of ADSCs, and it is likely to be used alone or in combination with biochemical inducers for cell manipulation.

Effect of Simulated Microgravity Conditions on Differentiation of Adipose Derived Stem Cells towards Fibroblasts Using Connective Tissue Growth Factor.

Background: Mesenchymal stem cells (MSCs) are multipotent cells able to differentiating into a variety of mesenchymal tissues including osteoblasts, adipocytes and several other tissues. Objectives: Differentiation of MSCs into fibroblast cells in vitro is an attractive strategy to achieve fibroblast cell and use them for purposes such as regeneration medicine. The goal of this study was investigate the simulated microgravity effect on differentiation of Adipose Derived Stem Cells (ADSCs) to fibroblasts. Materials and Methods: To fibroblast differentiation 100 ng.mL-1 of connective tissue growth factor (CTGF), and for simulation microgravity, 2D clinostat was used. After isolation the human ADSCs from adipose, cells were passaged, and at passages 3 they were used for characterization and subsequent steps. After 7 days of CTGF and simulated microgravity treatment, proliferation, and differentiation were analyzed collectively by MTT assay, quantitative PCR analyses, and Immunocytochemistry staining. Results: MTT assay revealed that CTGF stimulate the proliferation but simulated microgravity didn't have statistically significant effect on cell proliferation. In RNA level the expression of these genes are investigated: collagen type I (COLI), elastin (ELA), collagen type III (ColIII), Matrix Metalloproteinases I(MMP1), Fibronectin 1 (FN1), CD44, Fibroblast Specific protein (FSP-1), Integrin Subunit Beta 1 (ITGB1), Vimentin (VIM) and Fibrillin (FBN). We found that expression of ELN, FN1, FSP1, COL1A1, ITGB1, MMP1 and COL3A1 in both condition, and VIM and FBN1 just in differentiation medium in normal gravity increased. In protein level the expression of COL III and ELN in simulated microgravity increased. Conclusions: These findings collectively demonstrate that the simulated microgravity condition alters the marker fibroblast gene expression in fibroblast differentiation process.

Galectin-3 Mediates Tumor Cell–Stroma Interactions by Activating Pancreatic Stellate Cells to Produce Cytokines via Integrin Signaling

Pancreatic ductal adenocarcinoma (PDAC) is characterized by activated pancreatic stellate cells (PSCs), abundance of extracellular matrix (ECM), and production of cytokines and chemokines. Galectin 3 (GAL3), a β-galactoside-specific lectin, contributes to PDAC development but its effects on the stroma and cytokine production are unclear. The effect of recombinant human GAL3 (rGAL3) on activation of PSCs, production of cytokines, and ECM proteins was determined by proliferation, invasion, cytokine array, and quantitative polymerase chain reaction. We assessed co-cultures of PDAC cells with GAL3 genetic alterations with PSCs. Production of interleukin 8 (IL8) and activities of nuclear factor (NF)-κB were determined by enzyme-linked immunosorbent assay and luciferase reporter analyses. We studied the effects of inhibitors of NF-κB and integrin-linked kinase (ILK) on pathways activated by rGAL3. In analyses of the Gene Expression Omnibus database and our dataset, we observed higher levels of GAL3, IL8, and other cytokines in PDAC than in nontumor tissues. Production of IL8, granulocyte-macrophage colony-stimulating factor, chemokine ligand 1, and C-C motif chemokine ligand 2 increased in PSCs exposed to rGAL3 compared with controls. Culture of PSCs with PDAC cells that express different levels of GAL3 resulted in proliferation and invasion of PSCs that increased with level of GAL3. GAL3 stimulated transcription of IL8 through integrin subunit beta 1 (ITGB1) on PSCs, which activates NF-κB through ILK. Inhibitors of ILK or NF-κB or a neutralizing antibody against ITGB1 blocked transcription and production of IL8 from PSCs induced by rGAL3. The GAL3 inhibitor significantly reduced growth and metastases of orthotopic tumors that formed from PDAC and PSC cells co-implanted in mice. GAL3 activates PSC cells to produce inflammatory cytokines via ITGB1signaling to ILK and activation of NF-κB. Inhibition of this pathway reduced growth and metastases of pancreatic orthotopic tumors in mice.

WNK lysine deficient protein kinase 1 regulates human endometrial stromal cell decidualization, proliferation, and migration in part through mitogen-activated protein kinase 7.

The differentiation of endometrial stromal cells into decidual cells, termed decidualization, is an integral step in the establishment of pregnancy. The mitogen-activated protein kinase homolog, WNK lysine deficient protein kinase 1 (WNK1), is activated downstream of epidermal growth factor receptor during decidualization. Primary human endometrial stromal cells (HESCs) were subjected to small interfering RNA knockdown of WNK1 followed by in vitro decidualization. This abrogated expression of the decidual marker genes, insulin like growth factor binding protein 1 (IGFBP1) and prolactin (PRL), and prevented adoption of decidual cell morphology. Analysis of the WNK1-dependent transcriptome by RNA-Seq demonstrated that WNK1 regulates the expression of 1858 genes during decidualization. Gene ontology and upstream regulator pathway analysis showed that WNK1 regulates cell migration, differentiation, and proliferation. WNK1 was required for many of the gene expression changes that drive decidualization, including the induction of the inflammatory cytokines, C-C motif chemokine ligand 8 (CCL8), interleukin 1 beta (IL1B), and interleukin 15 (IL15), and the repression of transforming growth factor-beta (TGF-beta) pathway genes, including early growth response 2 (EGR2), SMAD family member 3 (SMAD3), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 4 (ITGA4), and integrin subunit beta 3 (ITGB3). In addition to abrogating decidualization, WNK1 knockdown decreased the migration and proliferation of HESCs. Furthermore, mitogen-activated protein kinase 7 (MAPK7), a known downstream target of WNK1, was activated during decidualization in a WNK1-dependent manner. Small interfering RNA knockdown of MAPK7 demonstrated that MAPK7 regulates a subset of WNK1-regulated genes and controls the migration and proliferation of HESCs. These results indicate that WNK1 and MAPK7 promote migration and proliferation during decidualization and regulate the expression of inflammatory cytokines and TGF-beta pathway genes in HESCs.

Knockdown of Snail inhibits epithelial-mesenchymal transition of human laryngeal squamous cell carcinoma Hep-2 cells through the vitamin D receptor signaling pathway.

It has been well documented that Snail plays a decisive role in various tumors. However, the direct effect of Snail on laryngeal squamous cell carcinoma (LSCC) has not been elaborated. In this study, we firstly detected the expression of Snail in 14 samples of patients with LSCC and found that its content was high in cancer tissues compared with adjacent tissues. Then we established LSCC Hep-2 cells with Snail silencing and validated the knockdown efficiency by Western blotting and real-time PCR. Results showed that silencing of Snail significantly inhibited the ability of adhesion, migration, and invasion of Hep-2 cells. Further study revealed that knockdown of Snail suppressed the epithelial-mesenchymal transition (EMT) process of Hep-2 cells, as evidenced by downregulation of matrix metallopeptidase (MMP)-2, MMP-9, integrin subunit beta 1 (ITGβ1), β-catenin, vimentin, N-cadherin, and fibronectin and upregulation of vitamin D receptor (VDR) and E-cadherin. Additionally, transfection with the small interfering RNA of VDR reversed the effect induced by Snail silencing in Hep-2 cells. Taken together, these results demonstrate that knockdown of Snail can inhibit the EMT process of LSCC cells through the VDR signaling pathway in vitro.

Abstract 4847: NME1 mediates a switch in beta integrin subunits that correlates with prolonged patient survival

Abstract Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility, invasion, and metastasis, but the molecular mechanisms underlying this activity are not completely understood. Herein we report a novel mechanism through which NME1 modulates focal adhesion dynamics via regulation of integrins β1 and β3. Stable expression of NME1 significantly altered focal adhesion turnover at the cell periphery. Interestingly, over-expression of NME1 resulted in a switch from predominantly fast recycling α4β1 integrins to slower recycling αvβ3 integrins. Contrary to its regulation of other cell surface receptors, the inhibition of integrin β1 and induction of integrin β3 by NME1 was found to occur at the transcriptional level rather than through dynamin mediated endocytosis. Induction of integrin β3 required both the 3-5’ exonuclease and nucleoside diphosphate kinase (NDPK) activities of NME1, which are also required for its metastasis suppressor activities in vivo. Further suggesting that the induction of integrin β3 is involved in the metastasis suppression function of NME1, knockdown of integrin β3 significantly increased the invasion capability of cells expressing NME1 in vitro compared to control cells. Analysis of metastatic melanoma patients in the TCGA showed individuals with a 1.5x or greater increase in integrin β3 mRNA had a significantly longer overall survival times. Additionally, an inverse correlation was observed between NME1 and integrin β1 mRNA in an independent microarray of primary melanomas. The inverse correlation of NME1 and integrin β1 RNA was also a strong predictor of prolonged distant disease free and overall survival in patients with the basal-like subtype of breast carcinoma. Together, these data strongly suggest NME1 prevents metastasis of human melanoma and some types of breast cancers by altering beta integrin expression to reduce recycling of focal adhesions and, ultimately suppress of cell motility. Citation Format: M. Kathryn Leonard, Grace Snow, Marián Novak, Joseph R. McCorkle, Xiuwei H. Yang, Alexey Belkin, David M. Kaetzel. NME1 mediates a switch in beta integrin subunits that correlates with prolonged patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4847. doi:10.1158/1538-7445.AM2017-4847

Abstract 1535: Angiogenesis-related gene expression profile of a novel antiangiogenic agent F16 in human vascular endothelial cells

Abstract Angiogenesis is a key process in tumor propagation, progression, and metastasis. This process is tightly modulated by a wide range of growth factors, receptor tyrosine kinases, cytokines, matrix metalloproteinases, and transcription factors. Distinctively, vascular endothelial growth factor (VEGF) and their receptors have been identified as prominent stimulators of tumor angiogenesis. Therefore, inhibiting VEGF-induced tumor angiogenesis has been explored extensively for cancer therapy. In this context, F16, a novel small molecule possessing a unique molecular configuration to block VEGFR2, has been subjected to intensive preclinical investigations. The F16 molecule exhibits in vitro antiangiogenic activity via inhibiting endothelial cell proliferation, migration, and tube formation. In addition, F16 was found to significantly inhibit the in vivo angiogenesis in chick chorioallantoic membranes as well as in athymic nude mice with xenograft tumors. As a consequence of the antiangiogenic effects, F16 was able to effectively control the tumor growth in mice with xenograft tumors. In the present study, we aimed to comprehensively interrogate the impact of F16 treatment on the expression of a panel of angiogenesis-related genes to gain insight into the underlying molecular mode of action. We analyzed the expression of 84 genes that are known to be associated with angiogenesis in human umbilical vein endothelial cells (HUVECs). Our pilot study identified upregulation of some key genes in response to F16 treatment which include tissue inhibitor of metalloproteinase 1 (TIMP1), angiopoietin-like 4 (ANGPTL 4), chondromodulin 1 (LECT 1), and placenta growth factor (PIGF). On the other hand, several genes involved in promoting angiogenesis were downregulated, which include matrix metallopeptidase 9 (MMP9), integrin subunit beta 3 (ITGβ 3), insulin growth factor 1 (IGF 1), transforming growth factor alpha (TGF α), interleukin 6 (IL 6), leptin (LEP), thrombospondin -1, -2 (THBS -1, -2), tyrosine kinase (TEK), VEGF B and C. The differential genes expression related to pro- and anti-angiogenic growth factors coincide very well with our previous observation of F16 inhibiting endothelial cell proliferation, migration and tube formation. Based on the Gene Expression Profile (GEP) observed in our experiments, we speculate that F16 can induce TIMP1 which in turn could suppress the MMP9 signaling leading to the inhibition of endothelial cell migration. This disruption of TIMP1/MMP9 pathway offers an interesting foundation for functional studies that can be performed to confirm the role of F16 as a potential MMP9 inhibitor, which may provide extended benefits during the use of this drug for treating highly metastatic cancers. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Khalid Alhazzani, Ali Alaseem, Thiagarajan Venkatesan, Appu Rathinavelu. Angiogenesis-related gene expression profile of a novel antiangiogenic agent F16 in human vascular endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1535. doi:10.1158/1538-7445.AM2017-1535

Polymorphisms of Platelet Glycoprotein Receptors and Cell Adhesion Molecules in Fetuses with Fetal Growth Restriction and Their Mothers As Detected with Pyrosequencing.

Vascular thrombotic tendency may lead to fetal growth restriction (FGR). Altered platelet function and genetic heterogeneity may play a role in this procedure. We investigated whether maternal or fetal genotypic frequencies of genes polymorphisms for certain platelet receptor and cell adhesion molecules are altered in FGR. We compared the maternal and fetal genotypic frequencies of single nucleotide polymorphisms (SNPs) in four genes coding for platelet receptors and cell adhesion molecules [integrin alpha subunit 2 (ITGA2)C807T, integrin subunit beta 3(ITGB3) T1565C, platelet cell adhesion protein 1 (PECAM1) CTG-GTG and selectin P(SELP)A/C]. A total of 32 fetuses with fetal growth restriction and their mothers were matched with 18 normal controls. Using maternal venous blood and umbilical cord blood samples, nucleotide sequences were determined from pyrograms. Genotypic frequencies were calculated and analyzed using appropriate tests and logistic regression. There was no statistical difference in the proportion of heterozygotes or homozygotes for any of the genotypic frequencies between FGR and control groups in mothers or fetuses. Our study demonstrated no association of maternal or fetal ITGA2 C807T SNP, ITGB3 T1565C SNP, PECAM1 CTG - GTG and SELP A/C polymorphisms with FGR.

P.2.012 - Oxytocin promotes neurotrophic growth, increases integrin subunit beta 3 (ITGB3) and ameliorates depressive- and anxiety-like behaviour in rats

P.2.012 - Oxytocin promotes neurotrophic growth, increases integrin subunit beta 3 (ITGB3) and ameliorates depressive- and anxiety-like behaviour in rats

Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli.

Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs) could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA) polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffness <10 MPa. Like chondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, β1, and β3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1) in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin β1 plays a pivotal role in this process.

Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera

ABSTRACTPurpose: The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research.Methods: Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods.Results: Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing.Conclusion: The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.

Abstract P117: A Conserved Trans-membrane Domain Lysine in the Integrin Beta1 Subunit Regulates Renal Collecting Duct Cell Function

Integrins are heterodimeric trans-membrane receptor proteins that mediate the interaction of cells with extracellular matrix proteins (ECM). They mediate various growth factor dependent cell-signaling pathways during the development of fibrosis that is characteristic of all forms of chronic kidney diseases. Although integrin β1 is the most abundant integrin subunit in kidney and can form complexes with 12 different α subunits, integrin β3 is the best studied integrin β subunit and serves as the canonical model for integrin function based on the high sequence homology between the trans-membrane and cytosolic domains of integrin β subunits. A conserved lysine residue towards the C-terminus of integrin β3 subunit is reported to be important for regulating the activation of integrin αIIbβ3 complexes; however the functional importance of this lysine is unknown in β1 integrins. We investigated the role of this lysine residue in integrin β1-dependent kidney collecting duct cell function. We expressed the mutant protein where the lysine is mutated to glutamic acid in collecting duct cells null for integrin β1. Collecting duct cells expressing mutant protein had decreased the adhesion of cells to collagen IV mediated by integrin α1β1 by 80% (0.95 vs 0.18). This mutation also decreased the ability of IMCD cells adhesion to collagen I mediated by integrin α2β1 by 82% (0.78 vs 0.15). In contrast to earlier reports in integrin β3, this mutation did not significantly alter the amount of active integrin β1 on the cell surface as estimated by FACS analysis; however we did observe a decrease in conformation specific antibody binding on cells adhered to collagen (0.70 vs 0.30). We also investigated the role of this lysine residue in complex formation of purified integrin β1 with integrin α1 and α2 TM/CT domains in phospholipid bicelles using fluorescence anisotropy. The dissociation constant for binding was estimated to be &gt;3.2 mol and mutation of lysine residue did not significantly alter their binding ability. This contrasted with integrin αIIb β3 where we found fourfold decrease in binding ability (Kd 0.09 ± 0.03 mol and 0.33 ± 0.05 mol). Our data clearly suggest that conserved transmembrane lysine in both integrin β3 and integrin β1 regulate cell functions by distinct mechanisms.

Elucidation of the Roles of Tumor Integrin β1 in the Extravasation Stage of the Metastasis Cascade.

Tumor integrin β1 (ITGB1) contributes to primary tumor growth and metastasis, but its specific roles in extravasation have not yet been clearly elucidated. In this study, we engineered a three-dimensional microfluidic model of the human microvasculature to recapitulate the environment wherein extravasation takes place and assess the consequences of β1 depletion in cancer cells. Combined with confocal imaging, these tools allowed us to decipher the detailed morphology of transmigrating tumor cells and associated endothelial cells in vitro at high spatio-temporal resolution not easily achieved in conventional transmigration assays. Dynamic imaging revealed that β1-depleted cells lacked the ability to sustain protrusions into the subendothelial matrix in contrast with control cells. Specifically, adhesion via α3β1 and α6β1 to subendothelial laminin was a critical prerequisite for successful transmigration. β1 was required to invade past the endothelial basement membrane, whereas its attenuation in a syngeneic tumor model resulted in reduced metastatic colonization of the lung, an effect not observed upon depletion of other integrin alpha and beta subunits. Collectively, our findings in this novel model of the extravasation microenvironment revealed a critical requirement for β1 in several steps of extravasation, providing new insights into the mechanisms underlying metastasis. Cancer Res; 76(9); 2513-24. ©2016 AACR.

Abstract 306: Role of tumor beta-1 integrin in the tumor cell extravasation cascade

Abstract It has been shown that the tumor beta-1 integrin subunit plays an important role in primary tumor growth and metastasis. However, the specific role of beta-1 in the tumor cell extravasation cascade has not yet been clearly elucidated. This is partly due to the lack of extravasation models that possess high throughput and spatio-temporal resolution of each step in the cascade, which consists of tumor-endothelium arrest, transendothelial migration, and migration into the sub-endothelial matrix. To address this question, we employ multiple in vitro microfluidic platforms to recapitulate the extravasation microenvironment, allowing us to decipher in high spatio-temporal resolution, the specific extravasation defects associated with shRNA mediated beta-1 integrin knockdown in MDA-MB-231, MA2 and SUM 159 cell lines. These models include perfusable HUVEC microvascular networks embedded in a fibrin-collagen hydrogel and an upright HUVEC monolayer on a collagen gel that allows for detailed in-plane observation of 3D extravasation events. We first show that beta-1 knockdown drastically decreases extravasation efficiency in both assays, and further validated the defect in an in vivo mouse lung metastasis model. To explain the overall decreased ability to extravasate, we employed our assays to visualize and quantify each step in the extravasation cascade. First, we show that the retention rate of beta-1 knockdowns is significantly decreased under flow in microvascular networks. This suggests that beta-1 mediates adhesion to the endothelium, which was confirmed by a reduced adhesion rate under shear flow on a planar monolayer. Next, using the upright monolayer assay, we observed via time-lapse confocal microscopy that while beta-1 knockdowns assume a rounded morphology and extends nearly no protrusions past the endothelial barrier, their ability to open the endothelium was not significantly affected. However, beta-1 knockdowns were unable to fully invade past the endothelium and remained intercalated between endothelial cells. Further analysis revealed that extravasated beta-1 knockdown cells remained closely associated with the endothelium, while control cells migrated farther out into the sub-endothelial matrix. Immunostaining of the basement membrane protein collagen IV showed that transmigrated beta-1 knockdown cells were found trapped between the endothelial and col IV layers, suggesting that the close association with the endothelium is partly due to the inability to breach the basement membrane. Taken together, our results indicate that beta-1 is required for tumor cell extravasation by mediating tumor-endothelial adhesion and invasion into the subendothelial matrix, post-endothelial breaching. Citation Format: Michelle B. Chen, John M. Lamar, Roger D. Kamm, Richard O. Hynes. Role of tumor beta-1 integrin in the tumor cell extravasation cascade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 306. doi:10.1158/1538-7445.AM2015-306

Abstract 3257: NME1 mediates a switch in expression of beta integrin subunits that correlates with prolonged patient survival

Abstract Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the molecular mechanisms underlying this activity are not completely understood. Herein we report a novel mechanism through which NME1 modulates focal adhesion dynamics via regulation of integrin β1. Stable expression of NME1 significantly altered focal adhesion turnover at the cell periphery. Interestingly, over-expression of NME1 resulted in a switch in from predominantly fast recycling α4β1 integrins to slower recycling αvβ3 integrins. Contrary to its regulation of other cell surface receptors, the inhibition of integrin β1 by NME1 was found to occur at the transcriptional level rather than through dynamin-mediated endocytosis. This effect of NME1 required both its 3-5′ exonuclease and nucleoside diphosphate kinase (NDPK) activities, which are also required for its metastasis suppressor activities in vivo. Furthermore, an inverse correlation was observed between NME1 and integrin β1 mRNA in a large cohort of primary melanoma biopsies. The inverse correlation of NME1 and integrin β1 RNA was also a strong predictor of prolonged distant disease-free and overall survival in patients with the basal-like subtype of breast carcinoma. Together, these data strongly suggest NME1 suppresses metastasis of human melanoma and some types of breast cancers by inhibiting integrin β1 expression, which reduces recycling of focal adhesions and suppresses cell motility. Citation Format: M. Kathryn Leonard, Marian Novak, Xiuwei Yang, Alexey Belkin, David Kaetzel. NME1 mediates a switch in expression of beta integrin subunits that correlates with prolonged patient survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3257. doi:10.1158/1538-7445.AM2015-3257

The role of integrins in glioma biology and anti-glioma therapies.

Integrins are a group of molecules expressed by various cells including glioma cells and endothelial cells within the tumor. There are 18 known alpha and beta integrin subunits which form a heterodimer. Integrins regulate different cellular processes such as proliferation, adhesion, motility and survival as shown in numerous preclinical models. Furthermore, integrins control the activity of the transforming growth factor (TGF)-beta pathway and are involved in the process of angiogenesis which is indispensable for continued tumor growth. Because of the high expression levels of some integrins on glioma cells and their numerous functions, inhibition of integrin signaling has been considered a promising strategy for the treatment of glioma patients. Besides blocking antibodies which are currently under clinical investigation in other cancer entities, the integrin inhibitor cilengitide has been tested within several trials in glioblastoma patients over the last years. Cilengitide is a cyclic RDG peptide which targets integrins alphvbeta3 and alphavbeta5. Based on the results of smaller, initial trials suggesting an activity of cilengitide against glioblastoma, 2 larger trials were subsequently performed. However, both trials, which combined temozolomide-based chemoradiation with cilengitide failed to demonstrate an improved outcome with the addition of cilengitide. Ongoing translational analyses suggest that integrin levels in the tumor tissue are neither prognostic nor predict response to cilengitide. While the clinical development of cilengitide has been stopped, integrin inhibition with more effective agents may still be a promising approach in clinical neurooncology.