Integrin mRNA Levels Research Articles

2-hydroxy-4-methoxy benzaldehyde from Hemidesmus indicus root extract suppresses toll-like receptor2-mediated migration and invasive mechanisms in rheumatoid arthritis

IntroductionElevated expression of Toll-like receptor2 (TLR2) in synovial fibroblasts, endothelial and other infiltrating cells in rheumatoid arthritis promotes their migration and invasion. Our aim was to isolate a TLR2 inhibitor from Hemidesmus indicus root extract and test its effectiveness in regulating migration and invasion of fibroblast-like synoviocytes, endothelial cells and ex vivo synovial explant outgrowth. MethodFractionation of TLR2 inhibitor by a bioassay, followed by testing the inhibitor in the cultures of isolated fibroblast-like synoviocytes, endothelial cells and ex vivo synovial explant outgrowth in the presence of a TLR2 agonist, Pam3CSK4 was undertaken. ResultsTLR2 inhibition using 2-hydroxy-4-methoxy benzaldehyde significantly lowered Pam3CSK4-induced cell migration, invasion, MMP-1, MMP-3, Pro-MMP-2 and Pro-MMP-9 expression in fibroblast-like synovial cells isolated from the discarded biopsy specimens of rheumatoid arthritis patients as well as in ex vivo synovial explant culture. TLR2 inhibition lowered active Rac1 protein and β1 integrin mRNA levels in synovial fibroblasts and suppressed filopodia and lamellipodia formation in response to Pam3CSK4. In endothelial cells, 2-hydroxy-4-methoxy benzaldehyde inhibited Pam3CSK4-stimulated vascular endothelial growth factor (VEGF) and ICAM-1 expression and thereby lowered tube formation and endothelial migration. TLR2 inhibition using 2-hydroxy-4-methoxy benzaldehyde also attenuated the Pam3CSK4-evoked outgrowth formation in ex vivo synovial explants. 2-hydroxy-4-methoxy benzaldehyde targeted TLR2-MyD88 interactions and prevented dimerization. Conclusions2-hydroxy-4-methoxy benzaldehyde is the active constituent in the roots of H indicus that appears to act through TLR2 inhibition, followed by modulation of the agonist-induced cell migration, invasion and angiogenesis.

Semaphorin 7A knockdown improves injury and prevents endothelial-to-mesenchymal transition in ox-LDL-induced HUVECs by regulating β1 integrin expression.

Atherosclerosis is the most common cause of cardiovascular disease and is accompanied by high mortality rates and a poor prognosis. Semaphorin 7A (Sema7A) and its receptor β1 integrin have been reported to participate in the development of atherosclerosis. However, the role of Sema7A and β1 integrin in endothelial cell injury and endothelial-to-mesenchymal transition (EMT) in atherosclerosis remains undetermined, to the best of our knowledge. The mRNA and protein expression levels of Sema7A and β1 integrin in HUVECs were analyzed using reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses, respectively. HUVECs were induced with 50 µg/ml oxidized low-density lipoprotein (ox-LDL) to establish an atherosclerosis cell model. Cell viability was measured using Cell Counting Kit-8 assay and the production of IL-1β, IL-6 and C-C motif chemokine ligand 2 was determined using ELISA. The expression levels of cell adhesion factors, intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 were analyzed using RT-qPCR and western blot analyses. Cell apoptosis was detected using flow cytometry and western blotting. The levels of EMT-related markers were evaluated using RT-qPCR, western blotting and immunofluorescence staining. The results of the present study revealed that the expression levels of Sema7A and β1 integrin were significantly upregulated in ox-LDL-treated HUVECs. Treatment with ox-LDL significantly decreased cell viability, and increased the levels of inflammatory and adhesion factors, the cell apoptotic rate and the expression levels of EMT-related proteins. Knockdown of Sema7A reversed the ox-LDL-induced inflammatory responses and EMT, while the overexpression of β1 integrin reversed the Sema7A-mediated inhibitory effects on ox-LDL-treated HUVECs. In conclusion, the findings of the present study indicated that Sema7A and β1 integrin may play significant roles in atherosclerosis by mediating endothelial cell injury and EMT progression.

Abstract LB256: Dynamic kindlin-2 complexes containing a laminin-binding integrin are responsive to hypoxia

Abstract The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) invasion, metastasis and decreased cancer-specific survival. Kindlins are integrin adaptors and mechanosensory focal adhesion (FA) proteins that activate and cluster integrins in response to structural ECM alterations in the tumor microenvironment. Hypoxia is a well-known inducer of tumor migration and invasion, results in ECM remodeling, and is physiologically relevant for PCa. The study's objective was to determine if integrin-kindlin-2 adhesion complexes were responsive to hypoxia and the dynamic distribution of integrin-kindlin-2 complexes using immunofluorescence microscopy (IFM). The methods included a combination of biochemical analysis of kindlin-2 adhesion complexes, qRT-PCR, immunoblotting, and immunocytochemistry. DU145 cells were analyzed in HEPES-containing media with 10% FBS under hypoxic conditions (1% O2) continuously exposed for 4, 8, 12, and 16 hours. The results showed that hypoxia increased the mRNA expression of CAIX, a well-known HIF-1α target gene, within 4 hours, resulting in 7-fold maximum expression. Similarly, VEGF-A mRNA was responsive to hypoxia as expected. In contrast, HIF-1α, α6 integrin, and kindlin-2 mRNA levels remained unchanged as compared to normoxia. Strikingly, we found that kindlin-2-containing adhesion complexes increased under hypoxia conditions as compared to normoxia and detected by immunoprecipitation, the complexes contained α6β1 integrin. It is important to note that the constitutive levels of either α6 integrin or kindlin-2 were not altered by hypoxia. Using IFM under normoxic conditions, we confirmed the location of kindlin-2 within phosphorylated (Y31) paxillin (pPXN)-containing FAs and within extended plasma membrane domains exclusive of pPXN. However, under hypoxic conditions, an increased reorganization of pPXN-containing kindlin-2 complexes occurred within 4 hours, was continuously changing up to 16 hours, with increasing fibrillar forms of FAs. In addition, kindlin-2 was observed as increased in plasma membrane protrusions devoid pPXN. The dynamic nature of the hypoxia-driven FAs was observed by a time course analysis and indicated the apparent assembly and disassembly of the structures during 16-hours of PCa cells exposed to hypoxia. We conclude that kindlin-2-integrin complexes are responsive to hypoxia and contain the unexpected α6β1 integrin in the membrane exclusive of FAs. (Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226). Citation Format: Daniel Hernandez-Cortes, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Dynamic kindlin-2 complexes containing a laminin-binding integrin are responsive to hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB256.

Simvastatin ameliorates altered mechanotransduction in uterine leiomyoma cells

Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress β1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies. This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 μM, or control) for 48 hours. Protein and mRNA levels of β1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction. We found that simvastatin significantly reduced the protein expression of β1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in β1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1. The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.

Pectasol-C Modified Citrus Pectin targets Galectin-3-induced STAT3 activation and synergize paclitaxel cytotoxic effect on ovarian cancer spheroids.

Here we sought to determine the relationship between STAT3 activity and Galectin‐3 (Gal‐3) and to investigate the cytotoxic effect of PectaSol‐C Modified Citrus Pectin (Pect‐MCP) as a specific competitive inhibitor of Galectin‐3 (Gal‐3) in combination with Paclitaxel (PTX) to kill the ovarian cancer cell SKOV‐3 multicellular tumor spheroid (MCTS). To this order, SKOV‐3 cells in 2D and 3D cultures were treated with exogenous Gal‐3 for the assessment of STAT3 activity. Two‐way ANOVA main effect and IC50 of each drug Paclitaxel (PTX) and Pect‐MCP or in combination were obtained from MTT assay results. The phosphorylated STAT3 levels, migration, invasion, integrin mRNA and p‐AKTser473 levels were assessed in the absence or presence of each drug alone or in combination. Gal‐3 expression levels were assessed in human serous ovarian cancer (SOC) specimens and its correlation with different integrin mRNA levels was further assessed. Our results showed that Gal‐3 expression level was significantly increased in MCTS compared to monolayer SKOV‐3 cells which triggered STAT3 phosphorylation. Moreover, Pect‐MCP synergized with PTX to kill SKOV3 MCTS through abrogation of STAT3 activity and reduced expression of its downstream target HIF‐1α, reduced integrin mRNA levels, and subsequently decreased AKT activity. There were higher expression levels of Gal‐3 in human high‐grade SOC specimens compared to the normal ovary and borderline SOC which positively and significantly correlated with α5, β2 and β6 integrin mRNA levels. Together, these results revealed for the first time that Pect‐MCP could be considered as a potential drug to enhance the PTX effect on ovarian cancer cells MCTS through inhibition of STAT3 activity.

Evaluation of hepatic integrin \u03b1v\u03b23 expression in non-alcoholic steatohepatitis (NASH) model mouse by 18F-FPP-RGD2 PET

BackgroundActivated hepatic stellate cells (HSCs), which express integrin αvβ3, are a major fibrogenic factor in NASH pathophysiology. 18F-labeled cyclic arginine-glycine-aspartic acid penta-peptide (18F-FPP-RGD2) has been used as a PET probe for tumors expressing integrin αvβ3. The aim of this study was to assess the potential of PET with 18F-FPP-RGD2 to detect hepatic integrin αvβ3 expression in non-alcoholic steatohepatitis (NASH) model mice.ResultsThirty-two male C57BL/6 mice aged 6 weeks were fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) for 3 and 8 weeks. 18F-FPP-RGD2 PET imaging of the liver was performed at 3 and 8 weeks after CDAHFD feeding. After PET scanning, levels of hepatic integrin αvβ, 3α-smooth muscle actin (α-SMA), and collagen type 1 alpha 1(col1a1) were measured. Histopathological analysis of hepatic steatosis, inflammation, and fibrosis, as well as blood biochemistry analysis, was also performed. CDAHFD for 3 and 8 weeks produced a moderate-to-severe steatosis and inflammation of the liver in mice. NAFLD activity score (NAS) in mice fed the CDAHFD for 3 and 8 weeks were more than 4 indicating NASH or borderline NASH pathology. Fibrosis was observed only in mice fed the CDAHFD for 8 weeks. PET imaging showed that the hepatic standardized uptake value, SUV80–90 min, was increased with prolonged CDAHFD feeding compared with the respective controls (CDAHFD 3 weeks 0.32 ± 0.06 vs 0.48 ± 0.05, p < 0.01; CDAHFD 8 weeks 0.35 ± 0.04 vs 0.75 ± 0.07, p < 0.01, respectively). Prolonged CDAHFD feeding increased hepatic mRNA and protein levels of integrin αv and β3 at 3 and 8 weeks. Hepatic 18F-FPP-RGD2 uptake and amount of integrin αv and β3 protein were well correlated (r = 0.593, p < 0.05 and r = 0.835, p < 0.001, respectively). Hepatic 18F-FPP-RGD2 uptake also showed a positive correlation with Sirius red-positive area.ConclusionsThe hepatic uptake of 18F-FPP-RGD2 correlated well with integrin αv and β3 expression and histological fibrosis in a mouse model of NASH, suggesting the predictability of fibrosis in NASH pathology.

Therapeutic effects of tyroservatide on metastasis of lung cancer and its mechanism affecting integrin-focal adhesion kinase signal transduction.

Tyroservatide (YSV) can inhibit the growth and metastasis of mouse lung cancer significantly. This study investigated the therapeutic effects of tripeptide YSV on metastasis of human lung cancer cells and explored its possible mechanism that affects integrin–focal adhesion kinase (FAK) signal transduction in tumor cells. YSV significantly inhibited the adhesion and the invasion of highly metastatic human lung cancer cell lines 95D, A549, and NCI-H1299. In addition, YSV significantly inhibited phosphorylation of FAK Tyr397 and FAK Tyr576/577 in the 95D, A549, and NCI-H1299 human lung cancer cells in vitro. And the mRNA level and protein expression of FAK in these human lung cancer cells decreased at the same time. YSV also significantly inhibited mRNA and protein levels of integrin β1 and integrin β3 in the 95D, A549, and NCI-H1299 human lung cancer cells. Our research showed that YSV inhibited adhesion and invasion of human lung cancer cells and exhibited therapeutic effects on metastasis of lung cancer.

PHLPP negatively regulates cell motility through inhibition of Akt activity and integrin expression in pancreatic cancer cells.

Pancreatic adenocarcinoma is currently the fourth leading cause for cancer-related mortality. Malignant progression of pancreatic cancer depends not only on rapid proliferation of tumor cells but also on increased cell motility. In this study, we showed that increased PHLPP expression significantly reduced the rate of migration in pancreatic ductal adenocarcinoma (PDAC) cells whereas knockdown of PHLPP had the opposite effect. In addition, cell motility at the individual cell level was negatively regulated by PHLPP as determined using time-lapse imaging. Interestingly, the expression of β1 and β4 integrin proteins were decreased in PHLPP overexpressing cells and increased in PHLPP knockdown cells whereas the mRNA levels of integrin were not altered by changes in PHLPP expression. In determining the molecular mechanism underlying PHLPP-mediated regulation of integrin expression, we found that inhibition of lysosome activity rescued integrin expression in PHLPP overexpressing cells, thus suggesting that PHLPP negatively controls cell motility by inhibiting Akt activity to promote lysosome-dependent degradation of integrins. Functionally, the increased cell migration observed in PHLPP knockdown cells was effectively blocked by the neutralizing antibodies against β1 or β4 integrin. Taken together, our study identified a tumor suppressor role of PHLPP in suppressing cell motility by negatively regulating integrin expression in pancreatic cancer cells.

Alpha7 Beta1 Integrin mRNA and Protein Responses to Repeated Bouts of Eccentric Contractions in Human Skeletal Muscle

High force, eccentric contractions disrupt skeletal muscle and have a negative impact on muscle function. Muscle exposed to repeated bouts of eccentric contractions demonstrates a protective adaptation insomuch that the negative responses in muscle are attenuated following the second bout of eccentric contractions. Acute alterations in the muscle cytoskeleton following eccentric contractions may contribute to this protective phenomenon in skeletal muscle known as the repeated bout effect. The alpha7 beta1 integrin has been associated with resistance to muscle damage in animal models. PURPOSE: To determine the gene and protein response of the alpha7 beta1 integrin protein following repeated bouts of eccentric contractions (EC). METHODS:Fourteen inactive men (n=7; 23.3 ± 2.1yrs; 178.5 ± 8.3cm and 84.8 ± 23.4kg) and women (n=7; 25.6 ± 2.5yrs; 166.0 ± 8.3cm and 76.6 ± 32.7kg) participated in this study. Subjects performed 2 bouts of EC separated by 28d. Muscle biopsies were taken before (baseline) the first bout (B1), and at 2d and 27d post-B1. At 28d post-B1, subjects completed a second bout of EC (B2) and a muscle biopsy was taken 2d post-B2. The alpha7 beta1 integrin mRNA response was assessed via qRT-PCR and protein levels were evaluated with immunoblotting. RESULTS: Integrin mRNA levels were significantly increased at 2d following B1 (4.5 fold, p<0.05). No further changes in integrin mRNA levels were noted at B2 or 2d post B2. In spite of mRNA responses, protein levels of alpha7 beta1 integrin were unchanged among the various sampling time-points. CONCLUSIONS: These data demonstrate that the alpha7 beta1 integrin gene is sensitive to a single bout of EC in human skeletal muscle. However individual variation in the alpha7 beta1 integrin protein response to repeated bouts of EC does not presently suggest a major role for this integrin protein in the repeated bout effect.

FAK Inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth.

Ovarian cancer ascites fluid contains matrix proteins that can impact tumor growth via integrin receptor binding. In human ovarian tumor tissue arrays, we find that activation of the cytoplasmic focal adhesion (FAK) tyrosine kinase parallels increased tumor stage, β5 integrin, and osteopontin matrix staining. Elevated osteopontin, β5 integrin, and FAK mRNA levels are associated with decreased serous ovarian cancer patient survival. FAK remains active within ovarian cancer cells grown as spheroids, and anchorage-independent growth analyses of seven ovarian carcinoma cell lines identified sensitive (HEY, OVCAR8) and resistant (SKOV3-IP, OVCAR10) cells to 0.1 μmol/L FAK inhibitor (VS-4718, formerly PND-1186) treatment. VS-4718 promoted HEY and OVCAR8 G0-G1 cell-cycle arrest followed by cell death, whereas growth of SKOV3-IP and OVCAR10 cells was resistant to 1.0 μmol/L VS-4718. In HEY cells, genetic or pharmacological FAK inhibition prevented tumor growth in mice with corresponding reductions in β5 integrin and osteopontin expression. β5 knockdown reduced HEY cell growth in soft agar, tumor growth in mice, and both FAK Y397 phosphorylation and osteopontin expression in spheroids. FAK inhibitor-resistant (SKOV3-IP, OVCAR10) cells exhibited anchorage-independent Akt S473 phosphorylation, and expression of membrane-targeted and active Akt in sensitive cells (HEY, OVCAR8) increased growth but did not create a FAK inhibitor-resistant phenotype. These results link osteopontin, β5 integrin, and FAK in promoting ovarian tumor progression. β5 integrin expression may serve as a biomarker for serous ovarian carcinoma cells that possess active FAK signaling.

Integrins αvβ6, α6β4 and α3β1 are down‐regulated in cholangiolocellular carcinoma but not cholangiocarcinoma

The aim of this study was to evaluate integrin expression and immunolocalization of the extracellular matrix proteins in cholangiolocellular carcinoma (CoCC). Tissue specimens of 23 CoCC, 28 cholangiocarcinomas (CCC), 42 hepatocellular carcinomas (HCC) and 11 classical type combined hepatocellular cholangiocarcinomas (CHC) were immunostained for β6, β4 and α3 integrins, fibronectin and laminin. ITGB6, B4 and A3 mRNA levels in six HCC cell lines, five CCC cell lines and two CHC cell lines were quantified by quantitative reverse transcription polymerase chain reaction. Little or no positivity for β6, β4 and α3 integrins was shown in 91%, 91% and 52% of CoCC and 100%, 98% and 81% of HCC, respectively, according to immunostaining, whereas intense positive staining for these integrins was demonstrated in 64%, 96% and 75% of CCC, respectively. There was a close correlation between β4 and α3 integrin expression and intracytoplasmic laminin in CoCC, CCC and HCC, but not between β6 expression and its ligand, fibronectin. Integrin mRNA levels were increased in four of five CCC cell lines, but nearly undetectable in five of six HCC cell lines and one CHC cell line. Tubular differentiation of a CHC cell line cultured in collagen gel matrix induced upregulation of these integrins. Our results first indicated downregulation of αvβ6, α6β4 and α3β1 integrins in CoCC, in contrast to its high expression in CCC, suggesting a diagnostic value of integrins in the differential diagnosis of CoCC and CCC, as well as a useful inducible marker of the intermediate features of CoCC.

Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions, Changes Matrix Architecture, and Induces an Inflammatory Phenotype

Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α11 mRNA and protein expression, and an increase in α2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced.

Dexamethasone increases αvβ3 integrin expression and affinity through a calcineurin/NFAT pathway

The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvβ3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvβ3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvβ3 integrin was the result of an increase in the expression of the β3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p<0.04) in β3 integrin mRNA by day 2 compared to control and remained elevated for 6days of treatment and then an additional 10days once the DEX was removed. The increase in β3 integrin mRNA levels required only 1day of DEX treatment to increase levels for 4days in the absence of DEX. In contrast, DEX did not alter β1 integrin mRNA or protein levels. The DEX-induced upregulation of β3 integrin mRNA was partly due to an increase in its half-life to 60.7h from 22.5h in control cultures (p<0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in β3 integrin mRNA. In summary, the DEX-induced increase in β3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvβ3 integrin and the upregulation of the β3 integrin subunit through the calcineurin/NFAT pathway.

Abstract LB-350: Abelson interactor-1 (Abi1) expression levels as a prognostic and drug resistance marker in Bcr-Abl positive leukemias.

Abstract In hematological malignancies, quiescent leukemic stem cells are responsible for persistence of minimal residual disease and relapse. Emerging evidence points to the involvement of the bone marrow microenvironment in survival and systemic retention of leukemic stem/progenitor cells. Integrins, particularly α4β1, which controls hematopoietic stem cell trafficking, were shown to be required for leukemic stem cells lodging to the bone marrow niche, and were shown to be crucial for the persistence of minimal residual disease. The molecular mechanism governing these processes is not known. We have just recently obtained evidence that newly identified α4 integrin-Abelson interactor 1 (Abi1) signaling cross-talk is involved in acquired drug resistance in Bcr-Abl positive leukemic cells. Our data also indicated that Abi1 and α4 integrin may be useful as prognostic markers of treatment outcome in patients with relapsing Bcr-Abl positive leukemia. Comparison of mRNA levels of Abi1 and α4 integrin in Bcr-Abl positive CD34+ progenitor cells isolated from blood or bone marrow of CML (chronic myelogenous leukemia) patients at diagnosis and at relapse revealed significant decrease in mRNA levels of Abi1 and increase in mRNA levels of α4 integrin in relapsing CD34+ progenitor cells. Subsequent analyses performed on various imatinib mesylate (IM) resistant cell lines including K562, LAMA-84 or Bcr-Abl supplemented Ba/F3 cells, confirmed abnormal expression of Abi1 and α4 integrin, both mRNA and protein levels. IM resistant cells also exhibited acquired anchorage-dependent phenotype and enhanced adhesive properties. Further detailed analyses of α4 integrin-dependent pathways revealed significant de-regulation of integrin α4 downstream signaling being due to the abnormal expression of Abi1, what resulted in elevated levels of phospho-Akt (Ser472), phospho-Erk (Thr202/Tyr204) and abnormal JNK/SAPK and p38MAPK phosphorylation signals. In summary, our results not only indicate that Abi1 and α4 integrin may serve as potentially useful prognostic markers of treatment outcome in patients with relapsing Bcr-Abl positive leukemia, but also suggest that newly identified α4 integrin-Abi1-Bcr-Abl cross-talk may play a significant role in the interactions between leukemic progenitor cells and the bone marrow microenvironment, and alterations in this signaling mediate acquisition of the drug resistant phenotype of leukemic cells and contribute to the mechanisms of acquired drug resistance. Citation Format: Anna D. Chorzalska, Ibrahem Salloum, Hammad Shafqat, Saad Khan, Philip Marjon, Vincent Falanga, Ting Zhao, John Reagan, Eric Winer, Samer Al-Homsi, Nicola Kouttab, Patrycja M. Dubielecka. Abelson interactor-1 (Abi1) expression levels as a prognostic and drug resistance marker in Bcr-Abl positive leukemias. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-350. doi:10.1158/1538-7445.AM2013-LB-350

α6β4 Integrin, a Master Regulator of Expression of Integrins in Human Keratinocytes

Three major laminin and collagen-binding integrins in skin (α6β4, α3β1, and α2β1) are involved in keratinocyte adhesion to the dermis and dissemination of skin cells during wound healing and/or tumorigenesis. Knockdown of α6 integrin in keratinocytes not only results in motility defects but also leads to decreased surface expression of the α2, α3, and β4 integrin subunits. Whereas α2 integrin mRNA levels are decreased in α6 integrin knockdown cells, α3 and β4 integrin mRNAs levels are unaffected. Expression of either α6 or α3 integrin in α6 integrin knockdown cells restores α2 integrin mRNA levels. Moreover, re-expression of α6 integrin increases β4 integrin protein at the cell surface, which results in an increase in α3 integrin expression via activation of initiation factor 4E-binding protein 1. Our data indicate that the α6β4 integrin is a master regulator of transcription and translation of other integrin subunits and underscore its pivotal role in wound healing and cancer.

Comparison of FRPE and Human Embryonic Stem Cell–Derived RPE Behavior on Aged Human Bruch's Membrane

To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruch's membrane (BM) from aged and AMD donors. hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21. hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67-positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2. Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM.

S022023 RGD導入フィブロイン平面上に播種された軟骨細胞への低出力超音波パルス刺激の影響([S02202]細胞および分子のマイクロ・ナノスケール解析(2))

Several reports suggest that low-intensity pulsed ultrasound stimulation (LIPUS) facilitates chondrogenesis. Recently it has been suggested that LIPUS may be transmitted via Integrin. In this study, the Arg-Gly-Asp (RGD) amino acid sequence, which is a ligand of Integrin, was induced to the fibroin substrates by either gene transfer or physical mixing, and the variation of chondrocyte response to LIPUS was evaluated. Chondrocytes were seeded on three kinds of fibroin substrates: 1 wild-type, 2 transgenic and 3 mixed. LIPUS stimulation, with spatial and temporal average intensity of 30 mW/cm2, was applied to the chondrocytes at 12, 36, 60 hours and administered for 20 minutes each time. The results showed that GAG production, number of chondrocytes and mRNA levels of aggrecan, Sox 9 and Integrin ^ 1 was enhanced in the transgenic group by LIPUS administration, but these LIPUS-derived changes were not found in the wild-type and mixed groups. We previously reported that the adhesive force between chondrocytes and RGD transgenic fibroin surfaces was higher than that for mixed fibroin, suggesting that adhesive force is translated via RGD which bonds covalently to the fibroin proteins for the transgenic group. The present results suggest that the early biological adhesion via RGD on the transgenic fibroin is sensitive to LIPUS.

Modulation of Cell Adhesion and Migration by the Histone Methyltransferase Subunit mDpy-30 and Its Interacting Proteins

We have previously shown that a subset of mDpy-30, an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT) complex, also localizes at the trans-Golgi network (TGN), where its recruitment is mediated by the TGN-localized ARF guanine nucleotide exchange factor (ArfGEF) BIG1. Depletion of mDpy-30 inhibits the endosome-to-TGN transport of internalized CIMPR receptors and concurrently promotes their accumulation at the cell protrusion. These observations suggest mDpy-30 may play a novel role at the crossroads of endosomal trafficking, nuclear transcription and adhesion/migration. Here we provide novel mechanistic and functional insight into this association. First, we demonstrate a direct interaction between mDpy-30 and BIG1 and locate the binding region in the N-terminus of BIG1. Second, we provide evidence that the depletion or overexpression of mDpy-30 enhances or inhibits cellular adhesion/migration of glioma cells in vitro, respectively. A similar increase in cell adhesion/migration is observed in cells with reduced levels of BIG1 or other H3K4MT subunits. Third, knockdown of mDpy-30, BIG1, or the RbBP5 H3K4MT subunit increases the targeting of β1 integrin to cell protrusions, and suppression of H3K4MT activity by depleting mDpy-30 or RbBP5 leads to increased protein and mRNA levels of β1 integrin. Moreover, stimulation of cell adhesion/migration via mDpy-30 knockdown is abolished after treating cells with a function-blocking antibody to β1 integrin. Taken together, these data indicate that mDpy-30 and its interacting proteins function as a novel class of cellular adhesion/migration modulators partially by affecting the subcellular distribution of endosomal compartments as well as the expression of key adhesion/migration proteins such as β1 integrin.

Molecular and mechano-biology of collagen gel contraction mediated by human MG-63 cells: involvement of specific intracellular signaling pathways and the cytoskeleton

Culture of human osteoblast-like MG-63 cells within collagen gels results in the generation of intrinsic stress. Release of such collagen gels from attachment results in gel contraction and enhanced MMP-1, MMP-3, and alpha2 integrin mRNA levels. To understand the potential role of microtubules and signaling pathways involved in MG-63 cell-mediated contraction and gene expression, cells were cultured in collagen gels. After 24 h collagen gels were released, then immediately treated with nocodazole or specific protein kinase inhibitors. Contraction was assessed, RNA isolated, and real-time PCR analysis performed. Treatment with high concentrations of a microtubule depolymerization agent, nocodazole, enhanced early contraction and led to elevated mRNA levels for MMP-3, whereas low concentrations inhibited contraction at later time points and did not affect mRNA levels. ROCK inhibitor treatment (Y27632) inhibited collagen gel contraction and led to depressed mRNA levels. The ERK1/2 inhibitor U0126 did not affect contraction, but treatment led to depressed MMP-1, MMP-3, and alpha2 mRNA levels. The p38MAPK inhibitor SB203580 modestly affected contraction, but did not affect mRNA levels. These results suggest the potential role of cytoskeletal integrity and multiple kinase signaling pathways in specific bone-remodeling events.

Cell Motility and Spreading Are Suppressed by HOXA4 in Ovarian Cancer Cells: Possible Involvement of β1 Integrin

HOX genes are transcription factors that control morphogenesis, organogenesis and differentiation. Increasing evidence suggests that HOX genes play a role in ovarian cancer progression; however few studies have defined functional roles and mechanisms of action. We showed previously that HOXA4 expression is increased in invasive, compared to noninvasive, epithelial ovarian tumors. However, HOXA4 suppressed cell migration suggesting that elevated HOXA4 expression in invasive tumors constitutes a homeostatic response. In the present study, we used siRNA and forced-expression in multiple cell lines to define the role of HOXA4 in the regulation of transwell migration/invasion and cellular/colony morphology. Knockdown of endogenous HOXA4 increased migration, but not Matrigel invasion, of OVCAR-8 and OVCAR-3 cells. HOXA4 knockdown also increased cell spreading on plastic or fibronectin, reduced cell-cell adhesion, and increased filopodia in two- and three-dimensional cultures. These changes were not associated with significant changes in alphaV or beta3 integrin and E- or N-cadherin. However, down-regulation of HOXA4 significantly reduced beta1 integrin protein levels within cell colonies and cell aggregates, but not of single, nonadherent cells. It had no effect on beta1 integrin, alpha5 integrin, or fibronectin mRNA levels. Conversely, overexpression of HOXA4 in CaOV-3 cells suppressed transwell migration and increased beta1 integrin protein levels. Our results confirm that HOXA4 inhibits cell motility, show that it suppresses cell spreading and filopodia formation while enhancing cell-cell adhesion, and suggest a role for beta1 integrin in mediating these changes. These observations support the hypothesis that overexpression of HOXA4 in invasive ovarian tumors is a homeostatic, invasion-suppressive response.