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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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Modulation of cytokine release pattern after MMP-2.siRNA (pM) treatment in 4910 and 5310 cells. A, Tumor conditioned media from pSV and pM treatments was collected as described in Materials and Methods and subjected to Western array using Ray Biotech cytokine antibody array #3 following manufacturer’s instructions. The significant change in secreted among different treatments was highlighted with surrounding boxes and represent GM-CSF, IL-6, IL-8, IL-10, TNF-α, Angiogenin, VEGF and PDGF BB. The relative signal intensity was quantified using ImageJ 1.42 software (NIH) and mean±SE were plotted in the adjacent bar diagrams where significant deviation is represented by * p<0.05 and ** p<0.01 respectively. B, Conditioned media from mock, pSV and pM treatments were subjected to Western blot analysis and the representative blots from three independent experiments were presented. C, 4910 and 5310 whole cell lysates were subjected to western blot analyses and densitometric values showing relative expression levels were plotted as mean ± SE from three experimental replicates.
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Figure 3: Modulation of cytokine release pattern after MMP-2.siRNA (pM) treatment in 4910 and 5310 cells. A, Tumor conditioned media from pSV and pM treatments was collected as described in Materials and Methods and subjected to Western array using Ray Biotech cytokine antibody array #3 following manufacturer’s instructions. The significant change in secreted among different treatments was highlighted with surrounding boxes and represent GM-CSF, IL-6, IL-8, IL-10, TNF-α, Angiogenin, VEGF and PDGF BB. The relative signal intensity was quantified using ImageJ 1.42 software (NIH) and mean±SE were plotted in the adjacent bar diagrams where significant deviation is represented by * p<0.05 and ** p<0.01 respectively. B, Conditioned media from mock, pSV and pM treatments were subjected to Western blot analysis and the representative blots from three independent experiments were presented. C, 4910 and 5310 whole cell lysates were subjected to western blot analyses and densitometric values showing relative expression levels were plotted as mean ± SE from three experimental replicates.

Mentions: The tumor microenvironment is characterized by enhanced cytokine levels, which in turn activates signaling pathways facilitating exacerbation of proliferation, differentiation, and invasion of the tumor cells (19). With the prominent reduction of proliferation in MMP-2 knockdown cells, we next sought to evaluate the secretory cytokine profile in the extracellular milieu. As described in Materials and Methods, conditioned media consisting of secretory cytokines and growth factors was collected from pSV- and pM-treated cells and subjected to cytokine array which indicated a prominent modulation in cytokine release pattern with an inhibition in several cytokines including GM-CSF, IL-6, IL-8, IL-10, TNF-α, Angiogenin, VEGF, and PDGF-BB in MMP-2-knockdown cells. Densitometric analysis of cytokine array showed significant (p<0.01) and moderate (p<0.05) differences among pSV and pM treatments. Upon pM-treatment a significant inhibition in GM-CSF, IL-6, IL-8, IL-10, VEGF, and PDGF-BB (at p<0.01) and a moderate decrease in TNF-α (p<0.05) was documented in both 4910 and 5310 cells (Figure 3A). Angiogenin showed a noticeable inhibition of up to ~71% in 4910 and a moderate loss up to ~48% in 5310 cells, respectively. This noticeable reduction in the secretion levels of GM-CSF, IL-6, IL-8, IL-10, TNF-α, Angiogenin, VEGF, PDGF BB was further confirmed by subjecting conditioned media to Western blot analysis in both 4910 and 5310 cell lines (Figure 3B). The significant loss of cytokine secretion is in correlation with the reduction of cellular expression levels of cytokines IL-6, IL-8 and IL-10 in MMP-2 knockdown 4910 and 5310 cells (Figure 3C).


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)

Modulation of cytokine release pattern after MMP-2.siRNA (pM) treatment in 4910 and 5310 cells. A, Tumor conditioned media from pSV and pM treatments was collected as described in Materials and Methods and subjected to Western array using Ray Biotech cytokine antibody array #3 following manufacturer’s instructions. The significant change in secreted among different treatments was highlighted with surrounding boxes and represent GM-CSF, IL-6, IL-8, IL-10, TNF-α, Angiogenin, VEGF and PDGF BB. The relative signal intensity was quantified using ImageJ 1.42 software (NIH) and mean±SE were plotted in the adjacent bar diagrams where significant deviation is represented by * p<0.05 and ** p<0.01 respectively. B, Conditioned media from mock, pSV and pM treatments were subjected to Western blot analysis and the representative blots from three independent experiments were presented. C, 4910 and 5310 whole cell lysates were subjected to western blot analyses and densitometric values showing relative expression levels were plotted as mean ± SE from three experimental replicates.
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Related In: Results  -  Collection

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Figure 3: Modulation of cytokine release pattern after MMP-2.siRNA (pM) treatment in 4910 and 5310 cells. A, Tumor conditioned media from pSV and pM treatments was collected as described in Materials and Methods and subjected to Western array using Ray Biotech cytokine antibody array #3 following manufacturer’s instructions. The significant change in secreted among different treatments was highlighted with surrounding boxes and represent GM-CSF, IL-6, IL-8, IL-10, TNF-α, Angiogenin, VEGF and PDGF BB. The relative signal intensity was quantified using ImageJ 1.42 software (NIH) and mean±SE were plotted in the adjacent bar diagrams where significant deviation is represented by * p<0.05 and ** p<0.01 respectively. B, Conditioned media from mock, pSV and pM treatments were subjected to Western blot analysis and the representative blots from three independent experiments were presented. C, 4910 and 5310 whole cell lysates were subjected to western blot analyses and densitometric values showing relative expression levels were plotted as mean ± SE from three experimental replicates.
Mentions: The tumor microenvironment is characterized by enhanced cytokine levels, which in turn activates signaling pathways facilitating exacerbation of proliferation, differentiation, and invasion of the tumor cells (19). With the prominent reduction of proliferation in MMP-2 knockdown cells, we next sought to evaluate the secretory cytokine profile in the extracellular milieu. As described in Materials and Methods, conditioned media consisting of secretory cytokines and growth factors was collected from pSV- and pM-treated cells and subjected to cytokine array which indicated a prominent modulation in cytokine release pattern with an inhibition in several cytokines including GM-CSF, IL-6, IL-8, IL-10, TNF-α, Angiogenin, VEGF, and PDGF-BB in MMP-2-knockdown cells. Densitometric analysis of cytokine array showed significant (p<0.01) and moderate (p<0.05) differences among pSV and pM treatments. Upon pM-treatment a significant inhibition in GM-CSF, IL-6, IL-8, IL-10, VEGF, and PDGF-BB (at p<0.01) and a moderate decrease in TNF-α (p<0.05) was documented in both 4910 and 5310 cells (Figure 3A). Angiogenin showed a noticeable inhibition of up to ~71% in 4910 and a moderate loss up to ~48% in 5310 cells, respectively. This noticeable reduction in the secretion levels of GM-CSF, IL-6, IL-8, IL-10, TNF-α, Angiogenin, VEGF, PDGF BB was further confirmed by subjecting conditioned media to Western blot analysis in both 4910 and 5310 cell lines (Figure 3B). The significant loss of cytokine secretion is in correlation with the reduction of cellular expression levels of cytokines IL-6, IL-8 and IL-10 in MMP-2 knockdown 4910 and 5310 cells (Figure 3C).

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