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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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Abrogation of IL-6/Stat3 activation by α5β1 integrin function blocking glioma xenograft cells. A, Effect of Fibronectin adhesion induced α5β1 signaling activation on pM-inhibited Stat3 activation. The 4910 and 5310 cells were transfected with mock, pSV and pM for 24 hours as described in Materials and Methods. Cells were detached from culture plates by trypsinization and re-plated on FN-coated plates and cultured for another 24 hours. Whole cell lysates were subjected western blotting and representative blots showing expression levels of phospho-Stat3, CyclinD1 and c-Myc were obtained from three individual repetitions. B, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences where pre-immunoprecipitated input samples were used for GAPDH PCR amplification and normal sheep IgG (Nsp-IgG) immunoprecipitates was loaded as negative control. C, Cells were treated with mock, pSV, pM, rhMMP-2 and α5β1 blocking antibody for 48 hours as described in Materials and Methods and whole cell lysates were subjected to western blotting to determine the expression levels of IL-6, phospho-Stat3, CyclinD1 and c-Myc. Blots were stripped and re-probed with GAPDH to confirm equal loading.
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Figure 7: Abrogation of IL-6/Stat3 activation by α5β1 integrin function blocking glioma xenograft cells. A, Effect of Fibronectin adhesion induced α5β1 signaling activation on pM-inhibited Stat3 activation. The 4910 and 5310 cells were transfected with mock, pSV and pM for 24 hours as described in Materials and Methods. Cells were detached from culture plates by trypsinization and re-plated on FN-coated plates and cultured for another 24 hours. Whole cell lysates were subjected western blotting and representative blots showing expression levels of phospho-Stat3, CyclinD1 and c-Myc were obtained from three individual repetitions. B, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences where pre-immunoprecipitated input samples were used for GAPDH PCR amplification and normal sheep IgG (Nsp-IgG) immunoprecipitates was loaded as negative control. C, Cells were treated with mock, pSV, pM, rhMMP-2 and α5β1 blocking antibody for 48 hours as described in Materials and Methods and whole cell lysates were subjected to western blotting to determine the expression levels of IL-6, phospho-Stat3, CyclinD1 and c-Myc. Blots were stripped and re-probed with GAPDH to confirm equal loading.

Mentions: To further assess the pM-inhibited α5β1 signaling, we elevated the β1 signaling by FN adhesion in mock-, pSV- and pM-treated cells. Western blotting indicated the α5β1-mediated elevation in phospho-Stat3, CyclinD1 and c-Myc expression levels were decreased in pM-treated cells evidencing the role of MMP-2 in the regulation of α5β1-mediated Stat3 activation (Figure 7A). The essential functional role of MMP-2/α5β1 interaction on rhMMP-2-induced and pM-inhibited activation of IL-6/Stat3 was verified by treating the cells with α5β1 blocking antibody in combination with pM and rhMMP-2 treatments. The effect of α5β1 blocking antibody on pM-inhibited IL-6/Stat3 activation and subsequent cell survival was checked to investigate the role of MMP-2/α5β1 coupling in these tumor cell lines. Blocking of α5β1 integrin further reduced the pM-inhibited recruitment of Stat3 on both CyclinD1 and c-Myc promoters suggesting the functional significance of MMP-2 and α5β1 integrin co-operativity (Figure 7B). Supplementation of α5β1 blocking antibody to rhMMP-2-treated 4910 and 5310 cells substantially decreased the rhMMP-2-induced expression levels of IL-6, phospho-Stat3, CyclinD1 and c-Myc. On the other hand, augmentation of α5β1 blocking antibody to pM-treated cells further decreased the pM-inhibited expression levels of IL-6, phopsho-Stat3, CyclinD1 and c-Myc expression levels implying the significance of MMP-2 binding in α5β1-mediated Stat3 activation and cell survival. The expression levels of Stat3 target proteins CyclinD1 and c-Myc were severely reduced and almost undetectable in pM+anti-α5β1 integrin treatments in both the cell lines when compared to either pM- or anti-α5β1 integrin-treatments indicating that the loss of MMP-2 augments the inhibitory effects of α5β1 blocking antibody (Figure 7C). The combination of pM and α5β1 substantially inhibited the rhMMP-2-induced Stat3 activation and expression of CyclinD1 and c-Myc proteins when compared to either pM or anti-α5β1 treatments alone, which indicates that the disruption of MMP-2 and α5β1 association inhibits IL-6/Stat3 survival signaling leads to decrease in proliferation in 4910 and 5310 glioma xenograft cells.


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)

Abrogation of IL-6/Stat3 activation by α5β1 integrin function blocking glioma xenograft cells. A, Effect of Fibronectin adhesion induced α5β1 signaling activation on pM-inhibited Stat3 activation. The 4910 and 5310 cells were transfected with mock, pSV and pM for 24 hours as described in Materials and Methods. Cells were detached from culture plates by trypsinization and re-plated on FN-coated plates and cultured for another 24 hours. Whole cell lysates were subjected western blotting and representative blots showing expression levels of phospho-Stat3, CyclinD1 and c-Myc were obtained from three individual repetitions. B, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences where pre-immunoprecipitated input samples were used for GAPDH PCR amplification and normal sheep IgG (Nsp-IgG) immunoprecipitates was loaded as negative control. C, Cells were treated with mock, pSV, pM, rhMMP-2 and α5β1 blocking antibody for 48 hours as described in Materials and Methods and whole cell lysates were subjected to western blotting to determine the expression levels of IL-6, phospho-Stat3, CyclinD1 and c-Myc. Blots were stripped and re-probed with GAPDH to confirm equal loading.
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Related In: Results  -  Collection

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

Figure 7: Abrogation of IL-6/Stat3 activation by α5β1 integrin function blocking glioma xenograft cells. A, Effect of Fibronectin adhesion induced α5β1 signaling activation on pM-inhibited Stat3 activation. The 4910 and 5310 cells were transfected with mock, pSV and pM for 24 hours as described in Materials and Methods. Cells were detached from culture plates by trypsinization and re-plated on FN-coated plates and cultured for another 24 hours. Whole cell lysates were subjected western blotting and representative blots showing expression levels of phospho-Stat3, CyclinD1 and c-Myc were obtained from three individual repetitions. B, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences where pre-immunoprecipitated input samples were used for GAPDH PCR amplification and normal sheep IgG (Nsp-IgG) immunoprecipitates was loaded as negative control. C, Cells were treated with mock, pSV, pM, rhMMP-2 and α5β1 blocking antibody for 48 hours as described in Materials and Methods and whole cell lysates were subjected to western blotting to determine the expression levels of IL-6, phospho-Stat3, CyclinD1 and c-Myc. Blots were stripped and re-probed with GAPDH to confirm equal loading.
Mentions: To further assess the pM-inhibited α5β1 signaling, we elevated the β1 signaling by FN adhesion in mock-, pSV- and pM-treated cells. Western blotting indicated the α5β1-mediated elevation in phospho-Stat3, CyclinD1 and c-Myc expression levels were decreased in pM-treated cells evidencing the role of MMP-2 in the regulation of α5β1-mediated Stat3 activation (Figure 7A). The essential functional role of MMP-2/α5β1 interaction on rhMMP-2-induced and pM-inhibited activation of IL-6/Stat3 was verified by treating the cells with α5β1 blocking antibody in combination with pM and rhMMP-2 treatments. The effect of α5β1 blocking antibody on pM-inhibited IL-6/Stat3 activation and subsequent cell survival was checked to investigate the role of MMP-2/α5β1 coupling in these tumor cell lines. Blocking of α5β1 integrin further reduced the pM-inhibited recruitment of Stat3 on both CyclinD1 and c-Myc promoters suggesting the functional significance of MMP-2 and α5β1 integrin co-operativity (Figure 7B). Supplementation of α5β1 blocking antibody to rhMMP-2-treated 4910 and 5310 cells substantially decreased the rhMMP-2-induced expression levels of IL-6, phospho-Stat3, CyclinD1 and c-Myc. On the other hand, augmentation of α5β1 blocking antibody to pM-treated cells further decreased the pM-inhibited expression levels of IL-6, phopsho-Stat3, CyclinD1 and c-Myc expression levels implying the significance of MMP-2 binding in α5β1-mediated Stat3 activation and cell survival. The expression levels of Stat3 target proteins CyclinD1 and c-Myc were severely reduced and almost undetectable in pM+anti-α5β1 integrin treatments in both the cell lines when compared to either pM- or anti-α5β1 integrin-treatments indicating that the loss of MMP-2 augments the inhibitory effects of α5β1 blocking antibody (Figure 7C). The combination of pM and α5β1 substantially inhibited the rhMMP-2-induced Stat3 activation and expression of CyclinD1 and c-Myc proteins when compared to either pM or anti-α5β1 treatments alone, which indicates that the disruption of MMP-2 and α5β1 association inhibits IL-6/Stat3 survival signaling leads to decrease in proliferation in 4910 and 5310 glioma xenograft cells.

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