Integrin Β1 mRNA Research Articles

P1.13-05 Integrin β3 Inhibition Enhances the Antitumor Activity of Alk Inhibitor in Alkrearranged NSCLC

Anaplastic lymphoma kinase (ALK)-positive cancers are sensitive to small molecule ALK kinase inhibitors, but most cases experience failure following treatment. Hence, additional drug targets and combination therapeutic treatments are needed. We investigated gene expression that is regulated by the expression of ALK and explored its roles in cancer progression and therapeutic implication. We screened ALK-rearranged non-small cell lung cancer (NSCLC) cases using immunohistochemistry and fluorescence in situ hybridization, and then conducted multiplex gene expression analysis. We also performed a clinicopathological analysis to validate the findings. Additional cellular experiments, including inhibition and migration assays, and in vivo lung cancer model studies were performed. Among patients with ALK-rearranged NSCLC, integrin β3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin β3 expression were positively correlated, and we discovered that high integrin β3 mRNA expression was associated with metastasis and more advanced tumor stages (P < 0.005; P < 0.05). Furthermore, we found that inhibition of both ALK and integrin β3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05). We discovered a positive correlation between ALK and integrin β3 expression levels in ALK rearranged NSCLC. Our findings suggest that high integrin β3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This demonstrates the prognostic value of integrin β3 and provides a rationale for combination treatment with ALK and integrin β3 inhibitors in patients with ALK-rearranged NSCLC.

Integrin β3 Inhibition Enhances the Antitumor Activity of ALK Inhibitor in ALK-Rearranged NSCLC.

Purpose: Anaplastic lymphoma kinase (ALK)-positive cancers are sensitive to small-molecule ALK kinase inhibitors, but most cases experience failure following treatment. Hence, additional drug targets and combination therapeutic treatments are needed. We investigated gene expression that is regulated by the expression of ALK and explored its roles in cancer progression and therapeutic implication.Experimental Design: We screened ALK-rearranged non-small cell lung cancer (NSCLC) cases using immunohistochemistry and fluorescence in situ hybridization and then conducted multiplex gene expression analysis. We also performed a clinicopathologic analysis to validate the findings. Additional cellular experiments, including inhibition and migration assays, and in vivo lung cancer model studies were performed.Results: Among patients with ALK-rearranged NSCLC, integrin β3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin β3 expression were positively correlated, and we discovered that high integrin β3 mRNA expression was associated with metastasis and more advanced tumor stages (P < 0.005; P < 0.05). Furthermore, we found that inhibition of both ALK and integrin β3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05).Conclusions: We discovered a positive correlation between ALK and integrin β3 expression levels in ALK-rearranged NSCLC. Our findings suggest that high integrin β3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This finding demonstrates the prognostic value of integrin β3 and provides a rationale for combination treatment with ALK and integrin β3 inhibitors in patients with ALK-rearranged NSCLC. Clin Cancer Res; 24(17); 4162-74. ©2018 AACR.

TGF-β1 modulates podocyte migration by regulating the expression of integrin-β1 and -β3 through different signaling pathways

Podocyte migration may lead foot process effacement and proteinuria. Transforming growth factor-β1 (TGF-β1) and integrins are involved in the adhesion and migration of cells. However, the crosstalk of TGF-β1 and integrins is unclear. Here, we examined how TGF-β1 regulates the expression of integrin-β1 and -β3 to modulate podocyte adhesion and migration. Podocytes were exposed to TGF-β1 and/or the inhibitors of Smad2/3, ERK and p38, then the expression of integrin-β1 and -β3 was assessed by Real-time PCR and western blot analyses. Podocyte adhesion and migration were measured under TGF-β1 treatment and/or anti-integrin-β3 antibody by cell adhesion assay and wound healing assay. TGF-β1 had no effect on integrin-β1 mRNA expression. In the analysis of protein expression, TGF-β1 decreased the mature form of integrin-β1, but increased both the precursor form and core peptide of integrin-β1. The inhibitors of ERK and p38, but not Smad2/3, abrogated TGF-β1-induced changes in integrin-β1 protein expression. TGF-β1 increased integrin-β3 mRNA and protein levels. The inhibitors of Smad2/3, ERK and p38 attenuated the TGF-β1-induced increase in integrin-β3 mRNA and protein levels. Podocyte adhesion and migration were enhanced under the stimulation of TGF-β1. The blockade of interactions between integrin-αvβ3 and the extracellular matrix by the anti-integrin-β3 antibody abrogated the TGF-β1-induced enhancement in podocyte adhesion and migration. Our results demonstrate that TGF-β1up-regulates integrin-β3 expression and down-regulates integrin-β1 expression through different pathways. The up-regulation of integrin-β3 expression enhances podocyte migration. This study provides a novel mechanism for TGF-β1 signaling in regulating podocyte migration.

Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection.

In tuberculosis (TB), the innate inflammatory immune response drives tissue destruction, morbidity, and mortality. Monocytes secrete matrix metalloproteinases (MMPs), which have key roles in local tissue destruction and cavitation. We hypothesized that integrin signaling might regulate monocyte MMP secretion in pulmonary TB during cell adhesion to the extracellular matrix (ECM). Adhesion to type I collagen and fibronectin by Mycobacterium tuberculosis–stimulated monocytes increased MMP-1 gene expression by 2.6-fold and 4.3-fold respectively, and secretion by 60% (from 1208.1 ± 186 to 1934.4 ± 135 pg/ml; p < 0.0001) and 63% (1970.3 ± 95 pg/ml; p < 0.001). MMP-10 secretion increased by 90% with binding to type I collagen and 55% with fibronectin, whereas MMP-7 increased 57% with collagen. The ECM did not affect the secretion of tissue inhibitors of metalloproteinases-1 or -2. Integrin αVβ3 surface expression was specifically upregulated in stimulated monocytes and was further increased after adhesion to type I collagen. Binding of either β3 or αV integrin subunits increased MMP-1/10 secretion in M. tuberculosis–stimulated monocytes. In a cohort of TB patients, significantly increased integrin β3 mRNA accumulation in induced sputum was detected, to our knowledge, for the first time, compared with control subjects (p < 0.05). Integrin αVβ3 colocalized with areas of increased and functionally active MMP-1 on infected monocytes, and αVβ3 blockade markedly decreased type I collagen breakdown, and impaired both monocyte adhesion and leukocyte migration in a transwell system (p < 0.0001). In summary, our data demonstrate that M. tuberculosis stimulation upregulates integrin αVβ3 expression on monocytes, which upregulates secretion of MMP-1 and -10 on adhesion to the ECM. This leads to increased monocyte recruitment and collagenase activity, which will drive inflammatory tissue damage.

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

Silencing of β1 integrin regulates airway remodeling by regulating the transcription of SOCE‑associated genes in asthmatic mice.

The incidence of asthma is increasing globally; however, current treatments are only able to cure a certain proportion of patients. There is an urgent need to develop novel therapies. β1 integrin serves a role in the pathophysiology of asthma through the development of airway remodeling. The aim of the present study was to investigate silencing of the β1 integrin gene in pre-clinical models of allergic asthma. BALB/c mice were sensitized with ovalbumin through intraperitoneal injection and repeated aerosolized ovalbumin. A short hairpin RNA of the β1 integrin gene was designed and transfected into mouse models of asthma in vivo, in order to evaluate whether silencing of the β1 integrin gene affects airway smooth muscle cell proliferation and inflammation by regulating the mRNA expression of store-operated Ca2+ entry (SOCE)-associated genes. Silencing the β1 integrin gene may downregulate β1 integrin mRNA while not statistically decreasing α-smooth muscle actin gene expression and airway smooth muscle thickness. β1 integrin silencing was able to downregulate the transcription of SOCE-associated genes to normal levels, including calcium release-activated calcium modulator 1 and short transient receptor potential channel member 1, but not stromal interaction molecule 1, in asthma. Silencing of the β1 integrin gene additionally maintained nuclear factor of activated T-cells cytoplasmic 1 gene expression, and inflammatory cytokines interleukin-4 and interferon-γ at normal levels. The results of the present study provide evidence to suggest that silencing of the β1 integrin gene may be of therapeutic benefit for patients with asthma.

Role of β1-Integrin in Colorectal Cancer: Case-Control Study

Objectives In the metastatic process, interactions between circulating tumor cells (CTCs) and the extracellular matrix or surrounding cells are required. β1-integrin may mediate these interactions. The aim of this study was to investigate whether β1-integrin is associated with the detection of CTCs in colorectal cancer. Methods We enrolled 30 patients with colorectal cancer (experimental group) and 30 patients with benign diseases (control group). Blood samples were obtained from each group, carcinoembryonic antigen (CEA) mRNA for CTCs marker and β1-integrin mRNA levels were estimated by using reverse transcription-polymerase chain reaction, and the results were compared between the two groups. Results CEA mRNA was detected more frequently in colorectal cancer patients than in control patients (P=0.008). CEA mRNA was significantly reduced after surgery in the colorectal cancer patients (P=0.032). β1-integrin mRNA was detected more in colorectal cancer patients than in the patients with benign diseases (P<0.001). In colorectal cancer patients, expression of β1-integrin mRNA was detected more for advanced-stage cancer than for early-stage cancer (P=0.033) and was significantly decreased after surgery (P<0.001). In addition, expression of β1-integrin mRNA was significantly associated with that of CEA mRNA in colorectal cancer patients (P=0.001). Conclusion In conclusion, β1-integrin is a potential prognostic factor following surgical resection in colorectal cancer patients. β1-integrin may be a candidate for use as a marker for early detection of micrometastatic tumor cells and for monitoring the therapeutic response in colorectal cancer patients.

Ubiquitous Release of Exosomal Tumor Suppressor miR-6126 from Ovarian Cancer Cells.

Cancer cells actively promote their tumorigenic behavior by reprogramming gene expression. Loading intraluminal vesicles with specific miRNAs and releasing them into the tumor microenvironment as exosomes is one mechanism of reprogramming whose regulation remains to be elucidated. Here, we report that miR-6126 is ubiquitously released in high abundance from both chemosensitive and chemoresistant ovarian cancer cells via exosomes. Overexpression of miR-6126 was confirmed in healthy ovarian tissue compared with ovarian cancer patient samples and correlated with better overall survival in patients with high-grade serous ovarian cancer. miR-6126 acted as a tumor suppressor by directly targeting integrin-β1, a key regulator of cancer cell metastasis. miR-6126 mimic treatment of cancer cells resulted in increased miR-6126 and decreased integrin-β1 mRNA levels in the exosome. Functional analysis showed that treatment of endothelial cells with miR-6126 mimic significantly reduced tube formation as well as invasion and migration capacities of ovarian cancer cells in vitro Administration of miR-6126 mimic in an orthotopic mouse model of ovarian cancer elicited a relative reduction in tumor growth, proliferating cells, and microvessel density. miR-6126 inhibition promoted oncogenic behavior by leading ovarian cancer cells to release more exosomes. Our findings provide new insights into the role of exosomal miRNA-mediated tumor progression and suggest a new therapeutic approach to disrupt oncogenic phenotypes in tumors. Cancer Res; 76(24); 7194-207. ©2016 AACR.

Endometrial Receptivity Markers in Mice Stimulated With Raloxifene Versus Clomiphene Citrate and Natural Cycles.

Ovulation induction therapy with clomiphene citrate can suppress endometrial receptivity. Raloxifene may be an alternative therapeutic for women with ovulatory disorders. This study aimed to compare the expression of endometrial receptivity markers, including homeobox gene 10 (HOXA10), integrin β3, and leukemia inhibitory factor (LIF), as well as pinopode production during the implantation window in mice stimulated with raloxifene and clomiphene citrate and natural cycles. Thirty-six 8-week-old female Kunming mice were randomly divided into 3 groups (n = 12) and administered daily raloxifene (22 mg/kg), clomiphene citrate (18 mg/kg), and normal saline (1 mL), respectively, by gavage. Two days later, mice were injected with 5 IU human chorionic gonadotropin and mated. Successfully mated female animals were identified with vaginal plugs designated gestation day 1. At day 4.5, pregnant donor mice were euthanized, and uterus samples were collected for immunohistochemistry, quantitative polymerase chain reaction, Western blot, and scanning electron microscopy analyses. Homeobox gene 10, integrin β3, and LIF messenger RNA (mRNA) and protein levels were significantly higher in the raloxifene-treated animals compared with the clomiphene citrate group (all P < .05) but not significantly different from saline group values, except for LIF and integrin β3 mRNA levels (P < .05). Pinopodes were abundant and well developed in the raloxifene and saline groups; however, in the clomiphene citrate-treated mice, fewer and poorly developed pinopodes were obtained. In mice, raloxifene had no effect on HOXA10, integrin β3, and LIF expression as well as pinopode production, suggesting it has no adverse effects on endometrial receptivity. Raloxifene may provide a viable alternative oral ovulation induction agent to clomiphene citrate.

Chronic TGFβ stimulation promotes the metastatic potential of lung cancer cells by Snail protein stabilization through integrin β3-Akt-GSK3β signaling.

Chronic exposure to TGFβ, a frequent occurrence for tumor cells in the tumor microenvironment, confers more aggressive phenotypes on cancer cells by promoting their invasion and migration while at the same time increasing their resistance to the growth-inhibitory effect of TGFβ. In this study, a transdifferentiated (TD) A549 cell model, established by chronically exposing A549 cells to TGFβ, showed highly invasive phenotypes in conjunction with attenuation of Smad-dependent signaling. We show that Snail protein, the mRNA expression of which strongly correlates with a poor prognosis in lung cancer patients, was highly stable in TD cells after TGFβ stimulation. The increased protein stability of Snail in TD cells correlated with elevated inhibitory phosphorylation of GSK3β, resulting from the high Akt activity. Notably, integrin β3, whose expression was markedly increased upon sustained exposure to TGFβ, was responsible for the high Akt activity as well as the increased Snail protein stability in TD cells. Consistently, clinical database analysis on lung cancer patients revealed a negative correlation between overall survival and integrin β3 mRNA levels. Therefore, we suggest that the integrin β3-Akt-GSK3β signaling axis plays an important role in non-canonical TGFβ signaling, determining the invasive properties of tumor cells chronically exposed to TGFβ.

Activin B induces human endometrial cancer cell adhesion, migration and invasion by up-regulating integrin β3 via SMAD2/3 signaling.

Endometrial cancer is the fourth most common female cancer and the most common gynecological malignancy. Although it comprises only ~10% of all endometrial cancers, the serous histological subtype accounts for ~40% of deaths due to its aggressive behavior and propensity to metastasize. Histopathological studies suggest that elevated expression of activin/inhibin βB subunit is associated with reduced survival in non-endometrioid endometrial cancers (type II, mostly serous). However, little is known about the specific roles and mechanisms of activin B (βB dimer) in serous endometrial cancer growth and progression. In the present study, we examined the biological functions of activin B in type II endometrial cancer cell lines, HEC-1B and KLE. Our results demonstrate that treatment with activin B increases cell migration, invasion and adhesion to vitronectin, but does not affect cell viability. Moreover, we show that activin B treatment increases integrin β3 mRNA and protein levels via SMAD2/3-SMAD4 signaling. Importantly, siRNA knockdown studies revealed that integrin β3 is required for basal and activin B-induced cell migration, invasion and adhesion. Our results suggest that activin B-SMAD2/3-integrin β3 signaling could contribute to poor patient survival by promoting the invasion and/or metastasis of type II endometrial cancers.

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

Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity.

Toll-like receptor 2 (TLR2) induces migration and invasive mechanisms in rheumatoid arthritis.

IntroductionThis study investigates the role of Toll-like receptor 2 (TLR2) in the regulation of migratory and invasive mechanisms in rheumatoid arthritis (RA).MethodsInvasion, migration, matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) expression, β-integrin binding, cytoskeletal rearrangement and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation in response to a TLR2-ligand, Pam3CSK4 (1 μg/ml), in ex vivo RA synovial tissue explants, primary RA synovial fibroblasts (RASFC) and microvascular endothelial cells (HMVEC) were assessed by Transwell Matrigel™ invasion chambers, enzyme-linked immunosorbent assay (ELISA), multiplex adhesion binding assay, reverse transcription polymerase chain reaction (RT-PCR), F-actin immunofluorescent staining, matrigel synovial outgrowths, Rac1 pull-down assays/Western blot and zymography. β1-integrin expression in RA/control synovial tissue was assessed by immunohistology. The effect of Pam3CSK4 on cell migration, invasion, MMP-3 and Rac1 activation was examined in the presence or absence of anti-β1-integrin (10 μg/ml) or anti-IgG control (10 μg/ml). The effect of an anti-TLR-2 mAb (OPN301)(1 μg/ml) or immunoglobulin G (IgG) control (1 μg/ml) on RASFC migration and RA synovial tissue MMP activity was assessed by wound assays, ELISA and zymography.ResultsPam3CSK4 significantly induced cell migration, invasion, MMP-1, MMP-3, MMP-2 and MMP-9 expression and induced the MMP-1/TIMP-3 and MMP-3/TIMP-3 ratio in RASFC and explants (p <0.05). β1-integrin expression was significantly higher in RA synovial tissue compared to controls (p <0.05). Pam3CSK4 specifically induced β1-integrin binding in RASFC (p <0.05), with no effect observed for β2-4, β6, αvβ5 or α5β1. Pam3CSK4 increased β1-integrin mRNA expression, Rac1 activation, RASFC outgrowths and altered cytoskeletal dynamic through induction of filopodia formation. Pam3CSK4-regulated cell migration and invasion processes, but not MMP-3, were inhibited in the presence of anti-β1-integrin (p <0.05), with no effect observed for anti-IgG control. Furthermore, anti-β1-integrin inhibited Pam3CSK4-induced Rac1 activation. Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 secretion from RA synovial explant cultures (p <0.05). Incubation of RASFC with OPN301 RA ex vivo conditioned media inhibited migration and invasion compared to IgG control.ConclusionsTLR2 activation induces migrational and invasive mechanisms, which are critically involved in the pathogenesis of RA, suggesting TLR2 as a potential therapeutic target for the treatment of RA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0664-8) contains supplementary material, which is available to authorized users.

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.

Mechanism of embryonic stem cells microenvironment enhancing stemness and inhibiting apoptosis of human limbal stem cells

Background The fate of adult stem cells is associated with its surrounding microenviroment.Our previous work found that embryonic stem cells (ESCs) micro-environment enhance the stemness of human limbal stem cells (LSCs), but its mechanism has not been elucidated. Objective This study was to explore the molecular mechanism of ESC micro-environment enhancing the stemness and inhibiting the apoptosis of LSCs. Methods Human LSCs were cultured by explant culture method with CnT-20 medium and CnT-20+ 20%ES culture supernatant (ESC-CM), respectively.Colony formation assay was used to analyze the proliferation ability of cells.Telomerase reverse transcriptase (TERT) siRNA(19-25nt siRNA) or siRNA(sc-37007) was transfected into the cells of ESC-CM group.Apoptosis and mitochondrial membrane potential were assayed by flow cytometry, and the expressions of telomerase and reactive oxygen species (ROS) in TERT siRNA- or siRNA-F-transfected cells by immunofluorescence and flow cytomery.RT-PCR, immunofluorescence staining and Western blot were employed to determine the expressions of p63, ATP-binding cassette transporer G2 (ABCG2), integrin β1 mRNA and proteins and cytokeratin 3(CK3) in the cells.The levels of focal adhesion kinase (FAK), Akt, glycogen synthase kinase 3β (GSK3β) and p21 protein and phosphorylation proteins in the cells were detected by Western blot. Results The LSCs presented an increased proliferative capacity and passaged to the eighth generation with the colony-forming efficiency (CFE) of (7.6±0.6)% in ESC-CM group, but the cells to the sixth generation with the CFE of (5.6±0.6)%, showing a significant difference between them(t=4.454, P=0.011). The apoptotic rates of the cells from 2 through 6 generations were lower in the ESC-CM group than those in the CnT-20 group (all at P 0.05), but significantly declined expressions of CK3 mRNA and protein were found in the ESC-CM group compared with the CnT-20 group (all at P<0.01). However, the expressions of p63, ABCG2, integrin β1 mRNA and proteins and TERT protein in the second generation of the cells were significantly higher in the ESC-CM group compared with the CnT-20 group (all at P<0.01). The telomerase activity was (4.83±0.67) % in the siRNA-TERT transfected group, which was significantly lower than (46.71±1.22) % of the siRNA-F transfected group (t=52.116, P=0.000). The expression of pFAK, pAkt, pGSK3β proteins were weakened, but the expression of p21 was increased in the ESC-CM group after addition of FAK inhibitor, GSK3β inhibitor and TERT-siRNA transfected group.Mitochondrial membrane potential in the second generation of cells was elevated in the ESC-CM group in comparison with the CnT-20 group and the siRNA-TERT transfected group (all at P<0.01), and the rates of ROS positively reaction was lower in the ESC-CM group and the siRNA-F transfected group than those of the CnT-20 group and siRNA-TERT transfected group (all at P<0.01). Conclusions ESC-CM culture system can effectively keep the stemness of LSCs and inhibit apoptosis.ESC-CM culture system plays functions probably via telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways. Key words: Embryonic stem cells; Niche; Telomerase; Limbal stem cells; Signaling pathway; Apoptosis; Colony-forming units

胶原相关整合素β1在形觉剥夺性近视诱导及恢复期中调节巩膜重塑作用的研究

目的：探讨豚鼠形觉剥夺性近视(form-deprivation myopia，FDM)模型中巩膜胶原相关整合素β1在巩膜重塑中的作用。方法：2~3周龄三色豚鼠128只，分为实验组(n = 64)和正常对照组(n = 64)。实验组采用头套法遮盖右眼进行形觉剥夺，左眼则为自身对照，正常对照组双眼不予任何处理。实验组依据形觉剥夺l周、2周、4周不同时间建立不同近视诱导和进展过程中的动物模型。并通过对形觉剥夺4周动物去除遮盖物，依据去遮盖时间l天、3天、5天、7天及14天建立不同时间点FDM恢复期的动物模型。在各个时间点对各组动物分别进行检测：带状光检影测量屈光度值；游标卡尺测量眼轴长度；苏木素–伊红(HE)染色观察后极部巩膜组织形态学变化；实时荧光定量聚合酶链反应(QRT-PCR)、免疫组织化学方法检测后极部巩膜胶原相关整合素β1 mRNA及蛋白表达情况。结果：形觉剥夺2周、4周后，实验组右眼分别诱导出(−3.00 ± 0.50) D及(−5.50 ± 1.08) D的相对近视，眼轴分别拉长(0.22 ± 0.06) mm及(0.49 ± 0.11) mm，与自身对照组及正常对照组相比，组间差异均有统计学意义(P均 0.05)。去遮盖后，豚鼠右眼重新正视化，与自身对照、正常对照眼相比，去遮盖7天时屈光度差值差异无显著性(P > 0.05)；右眼眼轴长度缩短，14天时眼轴长度差值差异无显著性(P > 0.05)。巩膜形态学观察见形觉剥夺性近视眼豚鼠后极部巩膜胶原纤维排列紊乱，巩膜变薄；QRT-PCR及免疫组织化学染色显示，实验组右眼形觉剥夺l周后眼球后部巩膜整合素β1 mRNA及蛋白表达明显减少，差异有统计学意义(P 0.05)。自身对照组的上述所有检测指标与正常对照组相比，均无明显差异(P > 0.05)。结论：单眼遮盖可以导致FDM的发生；2~3周龄豚鼠去除形觉剥夺后可以重新进行正视化，伴随眼轴长度缩短；去遮盖7天内为眼生物学参数恢复的主要时期；豚鼠形觉剥夺时，后极部巩膜整合素β1表达减少，去遮盖后表达上调，提示整合素β1可能参与了形觉剥夺性近视的发生，其影响巩膜重塑的机制有待进一步研究。 Objective: To investigate the role of collagen-binding integrin β1 in sclera remodeling process of the guinea pig form-deprivation myopia (FDM). Methods: 128 young guinea pigs, aged 2 to 3 weeks, were randomized into experimental group (n = 64) and normal control group (n = 64). FDM models were monocular deprived by placement of a facemask over the right eye in the experimental group, and the fellow eyes served as the contralateral control eyes. The guinea pigs in the normal control group were raised with both eyes untreated. The FDM eyes were monocular deprived for 1, 2 and 4 weeks, and recovered 1, 3, 5, 7, 14 days after occluding for 4 weeks. Optical measurements and axial length were accomplished at all of the time points. The histological analysis was applied to assess the posterior sclera changes, and immunohistochemical staining and quantitative real-time PCR (QRT-PCR) were applied to investigate the expression of integrin β1 protein and mRNA. Results: After 2 and 4 weeks, compared with the fellow eyes and the normal control group, the refraction diopter of the experimental group was (−3.00 ± 0.50) D and (−5.50 ± 1.08) D, and the axial length elongated (0.22 ± 0.06) mm and (0.49 ± 0.11) mm, showing significant differences respectively (P 0.05). After re-exposure, the right eyes of guinea pigs in the experimental group experienced re-normalization and the refraction was rapidly recovered in the initial 7 days (P > 0.05); the axial length gradually shortened, and it was statistically non-significant after 14-day recovery (P > 0.05). The morphological observation of sclera found that the posterior sclera collagen fiber arrangement of guinea pigs with FDM was disordered and the sclera became thinner. Scleral integrin β1 mRNA and protein expression in FDM eyes were significantly lower than those in the controls after monocular deprived 1 week (P 0.05). The difference of all the above statistics between contralateral control eyes and the normal control group was not remarkable (P > 0.05). Conclusion: Form-deprivation myopia was able to be induced by monocular covering. Guinea pigs aged 2 to 3 weeks with form-deprivation can experience re-normalization after recovery, along with the shortening of the axial length. The main recovery stage of ocular biometric parameters was in the initial 7 days. The expression of integrin β1 in FDM sclera was found to be lower, but could be restored during the recovery period. This suggests that collagen- binding integrin β1 may be involved in the pathogenesis of myopia, and further study needs to be done on the mechanism of the scleral remodeling in myopia.

Cyclic stretch-induced TGF-β1 and fibronectin expression is mediated by β1-integrin through c-Src- and STAT3-dependent pathways in renal epithelial cells.

Extracellular matrix (ECM) proteins, including fibronectin, may contribute to the early development and progression of renal interstitial fibrosis associated with chronic renal disease. Recent studies showed that β1-integrin is associated with the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). However, the molecular events responsible for β1-integrin-mediated signaling, following UUO, have yet to be determined. In this study, we investigated the mechanism by which mechanical stretch, an in vitro model for chronic obstructive nephropathy, regulates fibronectin and transforming growth factor-β1 (TGF-β1) expression in cultured human proximal tubular epithelium (HK-2) cells. Mechanical stretch upregulated fibronectin and TGF-β1 expression and activated signal transducer and transcription factor 3 (STAT3) in a time-dependent manner. Stretch-induced fibronectin and TGF-β1 were suppressed by a STAT3 inhibitor, S3I-201, and by small interfering RNA (siRNA) targeting human STAT3 (STAT3 siRNA). Similarly, fibronectin and TGF-β1 expression and STAT3 activation induced by mechanical stretch were suppressed by the Src family kinase inhibitor PP2 and by transfection of HK-2 cells with a dominant-negative mutant of c-Src (DN-Src), whereas PP3, an inactive analog of PP2, had no significant effect. Furthermore, mechanical stretch resulted in increased β1-integrin mRNA and protein levels in HK-2 cells. Furthermore, neutralizing antibody against β1-integrin and silencing of β1-integrin expression with siRNAs resulted in decreased c-Src and STAT3 activation and TGF-β1 and fibronectin expression evoked by mechanical stretch. This work demonstrates, for the first time, a role for β1-integrin in stretch-induced renal fibrosis through the activation of c-Src and STAT3 signaling pathways.

Electroacupuncture inhibits annulus fibrosis cell apoptosis in vivo via TNF-α-TNFR1-caspase-8 and integrin β1/Akt signaling pathways

ObjectiveTo examine whether electroacupuncture (EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis (AF) via tumor necrosis factor-α (NF-α-tumor necrosis factor receptor 1 (TNFR1)-caspase-8 and integrin β1/Akt signaling pathways in a rat model of cervical intervertebral disc degeneration caused by unbalanced dynamic and static forces. MethodsThirty-two Sprague-Dawley rats were included in this study, of which 24 rats underwent surgery to induce cervical intervertebral disc degeneration, while eight rats received EA treatment at Dazhui (GV 14). Immunohistochemical staining was used to detect TNF-α, TNFR1, and caspase-8. Apoptosis of AF cells was examined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The mRNA and protein expression levels of integrin β1 and Akt were evaluated with real-time polymerase chain reaction and western blot analysis, respectively. ResultsTreatment with EA decreased TUNEL-positive AF cells and lowered TNF-α, TNFR1 and caspase-8 positive cells compared with control groups. EA treatment also increased integrin β1 and Akt mRNA and protein levels compared with controls. ConclusionTreatment with EA inhibits AF cell apoptosis through suppression of the TNF-α-TNFR1-caspase-8 signal pathway and increases the expression of integrin β1 and Akt. EA may be a good alternative therapy for treating cervical spondylosis.

COMP-angiopoietin1 potentiates the effects of bone morphogenic protein-2 on ischemic necrosis of the femoral head in rats.

Angiogenesis is considered essential for proper bone regeneration. The purpose of this investigation was to determine if a combined therapy of bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein angiopoietin-1 (COMP-Ang1) can potentiate the therapeutic effect of BMP-2 in a rat model of ischemic necrosis of the femoral head (INFH). INFH was surgically induced in the femoral head of rats, and the animals were divided into the following groups: 1) a sham-operated group (sham group), 2) a bovine serum albumin-injected group (BSA group), 3) a BMP-2-injected group (BMP-2 group), and 4) a COMP-Ang1 and BMP-2-injected group (COMP-Ang1 + BMP-2 group) (n = 20/group). Radiologic, histologic, and histomorphometric assessments were performed to assess femoral head morphology, vascular density, and bone resorption activity. Western blots and immunohistochemical staining were performed to evaluate production of BMP-related signaling proteins in C3H10T1/2 cells and tissues. Real-time RT-PCR was performed to investigate expression of the target integrin gene, and the effect of integrin on C3H10T1/2 cells was determined using a cell adhesion assay. Radiographs obtained six weeks after injection revealed better preservation of the architecture of the femoral head in the COMP-Ang1 + BMP-2 group compared with the BSA and BMP-2 groups. Histological findings indicated increased trabecular bone and vascularity and decreased osteoclast bone resorption activity in the COMP-Ang1 + BMP-2 group compared with those in the BSA and BMP-2 groups. The combination of COMP-Ang1 and BMP-2 increased phosphorylation of Smad1/3/5, p38, and Akt. Increased integrin α3 and β1 mRNA expression in the COMP-Ang1 + BMP-2 group promoted cell adhesion. These results suggest that COMP-Ang1 preserved the necrotic femoral head through the potentiation of BMP-2 signaling pathways and angiogenesis. Combination treatment with COMP-Ang1 and BMP-2 may be a clinically useful therapeutic application in INFH.