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Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma.

Kesanakurti D, Chetty C, Dinh DH, Gujrati M, Rao JS - Oncogene (2012)

Bottom Line: MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters.In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments.Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.

ABSTRACT
Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

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Related in: MedlinePlus

Effect of MMP-2 downregulation on α5 and β1 integrin expression and MMP-2/α5β1 complex formation. A, Whole cell lysates were subjected to western blotting to check the expression levels of α5 and β1 integrins and relative expression levels were quantified using ImageJ (NIH) and plotted as mean±SE values from three replicates and statistical significance was represented by, * at p<0.01. B, Whole cell lysates (200 µg) were immunoprecipitated with antibodies against non-specific IgG, α5 and β1 integrin using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to western blotting. The co-IP blots were stripped and re-probed with respective antibody used for immunoprecipitation. Input samples (whole cell lysates without immunoprecipitation) were subjected to Western blot analysis where GAPDH probing was used to check equal loading. C, 4910 and 5310 cells were seeded in 2-well chamber slides (2×103 per well) and transfected for 48 h as described in Materials and Methods. Cells were fixed, permeabilized and incubated with antibodies specific for α5β1 and MMP-2 (1:100 dilution) for 2 hours at room temperature followed AlexaFluor® secondary antibodies for 1 hour, DAPI stained and mounted. Non-specific IgG staining (Nsp-IgG) served as negative control (Insets). Representative confocal microscopic pictures in randomly selected microscopic fields of three independent experimental replicates were shown. Relative florescence levels representing MMP-2/α5β1 co-localization were estimated by ImageJ 1.42 (NIH) and arbitrary units were plotted in the bar diagram as mean ± SE and significant difference among different treatment groups was denoted by * at p<0.01.
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Figure 2: Effect of MMP-2 downregulation on α5 and β1 integrin expression and MMP-2/α5β1 complex formation. A, Whole cell lysates were subjected to western blotting to check the expression levels of α5 and β1 integrins and relative expression levels were quantified using ImageJ (NIH) and plotted as mean±SE values from three replicates and statistical significance was represented by, * at p<0.01. B, Whole cell lysates (200 µg) were immunoprecipitated with antibodies against non-specific IgG, α5 and β1 integrin using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to western blotting. The co-IP blots were stripped and re-probed with respective antibody used for immunoprecipitation. Input samples (whole cell lysates without immunoprecipitation) were subjected to Western blot analysis where GAPDH probing was used to check equal loading. C, 4910 and 5310 cells were seeded in 2-well chamber slides (2×103 per well) and transfected for 48 h as described in Materials and Methods. Cells were fixed, permeabilized and incubated with antibodies specific for α5β1 and MMP-2 (1:100 dilution) for 2 hours at room temperature followed AlexaFluor® secondary antibodies for 1 hour, DAPI stained and mounted. Non-specific IgG staining (Nsp-IgG) served as negative control (Insets). Representative confocal microscopic pictures in randomly selected microscopic fields of three independent experimental replicates were shown. Relative florescence levels representing MMP-2/α5β1 co-localization were estimated by ImageJ 1.42 (NIH) and arbitrary units were plotted in the bar diagram as mean ± SE and significant difference among different treatment groups was denoted by * at p<0.01.

Mentions: A possible interaction between MMP-2 and α5β1 has been implicated in the modulation of MMP-2 activity and subsequent tumor cell invasion has been reported previously in melanoma and breast cancer cells (15–17). Several reports suggested the positive regulation of survival signaling by α5β1 integrin and targeting by α5β1 antagonists decreased proliferation in tumor cells (37, 38). Based on the previous reports suggesting possible MMP-2/α5β1 integrin interaction and with the prominent proliferation inhibition upon MMP-2 downregulation in our studies we next checked the effect of MMP-2 knockdown on α5 and β1 integrin expression levels. Both α5 and β1 mRNA and protein expression levels were remarkably reduced and densitometric analysis showed up to ~60% reduction in pM-treated 4910 and 5310 cells compared to control counterparts (Supplementary Fig. S1B, Figure 2A). Immunoprecipitation experiments showed immunocomplex formation of MMP-2/α5 and MMP-2/β1 by physical interaction in un-treated 4910 and 5310 cells whereas pM-treated cells show decreased interaction (Figure 2B). Eventually co-IP blots were re-probed with antibody used for immunoprecipitation to check the band specificity, input samples were subjected to Western blotting, and GAPDH levels were estimated to confirm equal loading. Confocal studies signified a strong co-localization of MMP-2 with α5β1 in mock- and pSV-treated 4910 and 5310 cells. On the other hand, pM-transfected cells showed noticeable inhibition in MMP-2 and α5β1 expression, which subsequently led to decreased MMP-2/α5β1 co-localization. Densitometric analysis showed significant reduction (up to ~67%) in MMP-2/α5β1 co-localization upon pM-treatment for 48 hours (Figure 2C). To further verify the MMP-2/α5β1 interaction, we overexpressed siRNA-insensitive Myc-tagged MMP-2 (pM-FL-A141G) in 4910 and 5310 cells. MMP-2 overexpression was confirmed by Western blotting analysis in comparison to mock or pEV controls (Supplementary Fig. S1C). Immunoprecipitation and probing with anti-MMP-2 and anti-Myc antibodies confirmed enhanced MMP-2/α5β1 binding in pM-FL-A141G treated cells compared to mock and pEV control counterparts (Supplementary Fig. S1C). siRNA-insensitive MMP-2 overexpression upon pM-FL-A141G treatment was confirmed by Western blotting (Supplementary Fig. S1D). The prominent decreases in MMP-2 and α5β1 expression levels and interaction in pM treatments and enhancement of MMP-2/α5β1 binding by pM-FL-A141G treatment suggests a role of MMP-2 binding in α5β1 integrin-mediated intracellular signaling. The high expression and co-localization of MMP-2 and α5β1 integrin was also evident in the human glioblastoma tissues implicating the significance of MMP-2/α5β1 binding in glioma malignancy where as normal brain tissues did not show noticeable expression and MMP-2/α5β1 co-localization (Supplementary Figure S2).


Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma.

Kesanakurti D, Chetty C, Dinh DH, Gujrati M, Rao JS - Oncogene (2012)

Effect of MMP-2 downregulation on α5 and β1 integrin expression and MMP-2/α5β1 complex formation. A, Whole cell lysates were subjected to western blotting to check the expression levels of α5 and β1 integrins and relative expression levels were quantified using ImageJ (NIH) and plotted as mean±SE values from three replicates and statistical significance was represented by, * at p<0.01. B, Whole cell lysates (200 µg) were immunoprecipitated with antibodies against non-specific IgG, α5 and β1 integrin using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to western blotting. The co-IP blots were stripped and re-probed with respective antibody used for immunoprecipitation. Input samples (whole cell lysates without immunoprecipitation) were subjected to Western blot analysis where GAPDH probing was used to check equal loading. C, 4910 and 5310 cells were seeded in 2-well chamber slides (2×103 per well) and transfected for 48 h as described in Materials and Methods. Cells were fixed, permeabilized and incubated with antibodies specific for α5β1 and MMP-2 (1:100 dilution) for 2 hours at room temperature followed AlexaFluor® secondary antibodies for 1 hour, DAPI stained and mounted. Non-specific IgG staining (Nsp-IgG) served as negative control (Insets). Representative confocal microscopic pictures in randomly selected microscopic fields of three independent experimental replicates were shown. Relative florescence levels representing MMP-2/α5β1 co-localization were estimated by ImageJ 1.42 (NIH) and arbitrary units were plotted in the bar diagram as mean ± SE and significant difference among different treatment groups was denoted by * at p<0.01.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368064&req=5

Figure 2: Effect of MMP-2 downregulation on α5 and β1 integrin expression and MMP-2/α5β1 complex formation. A, Whole cell lysates were subjected to western blotting to check the expression levels of α5 and β1 integrins and relative expression levels were quantified using ImageJ (NIH) and plotted as mean±SE values from three replicates and statistical significance was represented by, * at p<0.01. B, Whole cell lysates (200 µg) were immunoprecipitated with antibodies against non-specific IgG, α5 and β1 integrin using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to western blotting. The co-IP blots were stripped and re-probed with respective antibody used for immunoprecipitation. Input samples (whole cell lysates without immunoprecipitation) were subjected to Western blot analysis where GAPDH probing was used to check equal loading. C, 4910 and 5310 cells were seeded in 2-well chamber slides (2×103 per well) and transfected for 48 h as described in Materials and Methods. Cells were fixed, permeabilized and incubated with antibodies specific for α5β1 and MMP-2 (1:100 dilution) for 2 hours at room temperature followed AlexaFluor® secondary antibodies for 1 hour, DAPI stained and mounted. Non-specific IgG staining (Nsp-IgG) served as negative control (Insets). Representative confocal microscopic pictures in randomly selected microscopic fields of three independent experimental replicates were shown. Relative florescence levels representing MMP-2/α5β1 co-localization were estimated by ImageJ 1.42 (NIH) and arbitrary units were plotted in the bar diagram as mean ± SE and significant difference among different treatment groups was denoted by * at p<0.01.
Mentions: A possible interaction between MMP-2 and α5β1 has been implicated in the modulation of MMP-2 activity and subsequent tumor cell invasion has been reported previously in melanoma and breast cancer cells (15–17). Several reports suggested the positive regulation of survival signaling by α5β1 integrin and targeting by α5β1 antagonists decreased proliferation in tumor cells (37, 38). Based on the previous reports suggesting possible MMP-2/α5β1 integrin interaction and with the prominent proliferation inhibition upon MMP-2 downregulation in our studies we next checked the effect of MMP-2 knockdown on α5 and β1 integrin expression levels. Both α5 and β1 mRNA and protein expression levels were remarkably reduced and densitometric analysis showed up to ~60% reduction in pM-treated 4910 and 5310 cells compared to control counterparts (Supplementary Fig. S1B, Figure 2A). Immunoprecipitation experiments showed immunocomplex formation of MMP-2/α5 and MMP-2/β1 by physical interaction in un-treated 4910 and 5310 cells whereas pM-treated cells show decreased interaction (Figure 2B). Eventually co-IP blots were re-probed with antibody used for immunoprecipitation to check the band specificity, input samples were subjected to Western blotting, and GAPDH levels were estimated to confirm equal loading. Confocal studies signified a strong co-localization of MMP-2 with α5β1 in mock- and pSV-treated 4910 and 5310 cells. On the other hand, pM-transfected cells showed noticeable inhibition in MMP-2 and α5β1 expression, which subsequently led to decreased MMP-2/α5β1 co-localization. Densitometric analysis showed significant reduction (up to ~67%) in MMP-2/α5β1 co-localization upon pM-treatment for 48 hours (Figure 2C). To further verify the MMP-2/α5β1 interaction, we overexpressed siRNA-insensitive Myc-tagged MMP-2 (pM-FL-A141G) in 4910 and 5310 cells. MMP-2 overexpression was confirmed by Western blotting analysis in comparison to mock or pEV controls (Supplementary Fig. S1C). Immunoprecipitation and probing with anti-MMP-2 and anti-Myc antibodies confirmed enhanced MMP-2/α5β1 binding in pM-FL-A141G treated cells compared to mock and pEV control counterparts (Supplementary Fig. S1C). siRNA-insensitive MMP-2 overexpression upon pM-FL-A141G treatment was confirmed by Western blotting (Supplementary Fig. S1D). The prominent decreases in MMP-2 and α5β1 expression levels and interaction in pM treatments and enhancement of MMP-2/α5β1 binding by pM-FL-A141G treatment suggests a role of MMP-2 binding in α5β1 integrin-mediated intracellular signaling. The high expression and co-localization of MMP-2 and α5β1 integrin was also evident in the human glioblastoma tissues implicating the significance of MMP-2/α5β1 binding in glioma malignancy where as normal brain tissues did not show noticeable expression and MMP-2/α5β1 co-localization (Supplementary Figure S2).

Bottom Line: MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters.In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments.Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.

ABSTRACT
Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

Show MeSH
Related in: MedlinePlus