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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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Effect of siRNA-insensitive MMP-2 overexpression and rhMMP-2 supplementation on pM-inhibited IL-6/Stat3 signaling activation. A, After 48 hours of treatment with mock, pSV, pM, pM-FL-A141G, pSV+pM-FL-A141G, pM+pM-FL-A141G, rhMMP-2, pSV+rhMMP-2, and pM+rhMMP-2 as described in Materials and Methods, whole cell lysates were subjected to Western blotting. Blots were representative of at least three independent repetitions and GAPDH probing confirmed equal loading. B, Effect of MMP-2 overexpression by siRNA-insensitive pM-FL-A141G treatment or rhMMP-2 supplementation on pM-inhibited MMP-2/α5β1 integrin binding. Whole cell lysates were (200 µg each) were immunoprecipitated with anti-α5β1 integrin antibody (10 µL each sample) using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to Western blotting. The mock sample immunoprecipitated with non-specific IgG was loaded as the negative control. Input shows whole cell lysate (50 µg) of mock sample served as the positive control. Blots were probed with anti-MMP-2 antibody and representative blots from three independent repetitions were shown. Adjacent bar diagram shows the densitometric analyses of the relative band intensity represented as mean±SE values obtained from three repetitions. The significant differences among various treatment groups were indicated by *at p<0.05. IP blots were stripped and subsequently re-probed with anti-α5, anti-β1 and anti-Myc antibodies. C, EMSA was performed using Panomics EMSA kit following the manufacturer’s instructions as described in Materials and Methods. The mock sample was pre-incubated with Stat3α antibody for 30 minutes at room temperature and loaded for supershift. Representative blots from three experimental replicates were shown. D, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif). In short, cells were fixed in 37% formaldehyde and chromatin was sheared by sonication and immunoprecipitated using anti-Stat3 antibody and normal sheep IgG (Nsp-IgG) antibodies by incubation at 4°C for 2–4 hours on a rotor. The immunoprecipitates was collected by using magnetic protein G beads (Millipore) and the chromatin was reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences. Pre-immunoprecipitated samples were used as input controls for PCR amplification of GAPDH and normal sheep IgG (Nsp-IgG) incubation was used for the negative control. E, BrDU assay was performed following manufacturer’s instructions to obtain mean±SE values of BrDU incorporation percentage from three repetitions and significance was represented by * at p<0.05 and ** p<0.01.
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Figure 6: Effect of siRNA-insensitive MMP-2 overexpression and rhMMP-2 supplementation on pM-inhibited IL-6/Stat3 signaling activation. A, After 48 hours of treatment with mock, pSV, pM, pM-FL-A141G, pSV+pM-FL-A141G, pM+pM-FL-A141G, rhMMP-2, pSV+rhMMP-2, and pM+rhMMP-2 as described in Materials and Methods, whole cell lysates were subjected to Western blotting. Blots were representative of at least three independent repetitions and GAPDH probing confirmed equal loading. B, Effect of MMP-2 overexpression by siRNA-insensitive pM-FL-A141G treatment or rhMMP-2 supplementation on pM-inhibited MMP-2/α5β1 integrin binding. Whole cell lysates were (200 µg each) were immunoprecipitated with anti-α5β1 integrin antibody (10 µL each sample) using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to Western blotting. The mock sample immunoprecipitated with non-specific IgG was loaded as the negative control. Input shows whole cell lysate (50 µg) of mock sample served as the positive control. Blots were probed with anti-MMP-2 antibody and representative blots from three independent repetitions were shown. Adjacent bar diagram shows the densitometric analyses of the relative band intensity represented as mean±SE values obtained from three repetitions. The significant differences among various treatment groups were indicated by *at p<0.05. IP blots were stripped and subsequently re-probed with anti-α5, anti-β1 and anti-Myc antibodies. C, EMSA was performed using Panomics EMSA kit following the manufacturer’s instructions as described in Materials and Methods. The mock sample was pre-incubated with Stat3α antibody for 30 minutes at room temperature and loaded for supershift. Representative blots from three experimental replicates were shown. D, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif). In short, cells were fixed in 37% formaldehyde and chromatin was sheared by sonication and immunoprecipitated using anti-Stat3 antibody and normal sheep IgG (Nsp-IgG) antibodies by incubation at 4°C for 2–4 hours on a rotor. The immunoprecipitates was collected by using magnetic protein G beads (Millipore) and the chromatin was reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences. Pre-immunoprecipitated samples were used as input controls for PCR amplification of GAPDH and normal sheep IgG (Nsp-IgG) incubation was used for the negative control. E, BrDU assay was performed following manufacturer’s instructions to obtain mean±SE values of BrDU incorporation percentage from three repetitions and significance was represented by * at p<0.05 and ** p<0.01.

Mentions: To establish specificity of pM-inhibited IL-6/Stat3 activation and to avoid any off-target consequences, we treated mock-, pSV- and pM-treated cells with siRNA-insensitive MMP-2 overexpression plasmid (pM-FL-A141G) or rhMMP-2 as described in Materials and Methods. The expression levels of α5, β1, phospho-Stat3, CyclinD1 and c-Myc were elevated in both pM-FL-A141G and rhMMP-2 treated cells, implicating a possible role of MMP-2 in the regulation of the α5β1-mediated Stat3 activation and cell proliferation (Figure 6A). As shown in Fig. 6B, the loss-and-gain of function approach by pM and pM-FL-A141G or rhMMP-2 treatments showed a significantly enhanced MMP-2/α5β1 association by complex formation in rhMMP-2- or pM-FL-A141G-treated cells and decreased association in pM-treated cells. Immunoprecipitation with non-specific (Nsp-IgG) did not show precipitation and re-probing the IP blots with anti-α5, anti-β1 and anti-Myc antibodies further confirmed the band specificity. Densitometric analysis of relative binding confirmed that the pM+rhMMP-2 or pM+pM-FL-A141G combination treatment noticeably counteracted the rhMMP-2- or pM-FL-A141G-elevated MMP-2/α5β1 binding, which further evidences direct substantial MMP-2/α5β1 integrin interaction in both 4910 and 5310 cells. In correlation with MMP-2 overexpression-induced MMP-2/α5β1 binding, EMSA displayed obvious elevation in Stat3 DNA-binding activity in rhMMP-2 treatment which is reduced in pM+rhMMP-2-treated cells confirming the role of MMP-2 in maintaining constitutive Stat3 activity (Figure 6C). A noticeable elevation in Stat3 occupancy on to CylcinD1 and c-Myc promoters was observed in pM-FL-A141G- or rhMMP-2-treated 4910 and 5310 cells. In corroboration with the reversal of MMP-2/α5β1 complex formation in pM+rhMMP-2 and pM+pM-FL-A141G treated cells, a clear reversal in Stat3 promoter recruitment was observed suggesting a role of MMP-2 in α5β1-mediated Stat3 activation and promoter binding (Figure 6D). Additionally, the pM-induced loss of cell proliferation was reversed in pM+rhMMP-2 or pM+pM-FL-A141G treatments whereas individual rhMMP-2 or pM-FL-A141G treatments alone enhanced the proliferation rate in both cell lines indicating the role of MMP-2 in maintenance of tumor cell proliferation (Figure 6E).


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 siRNA-insensitive MMP-2 overexpression and rhMMP-2 supplementation on pM-inhibited IL-6/Stat3 signaling activation. A, After 48 hours of treatment with mock, pSV, pM, pM-FL-A141G, pSV+pM-FL-A141G, pM+pM-FL-A141G, rhMMP-2, pSV+rhMMP-2, and pM+rhMMP-2 as described in Materials and Methods, whole cell lysates were subjected to Western blotting. Blots were representative of at least three independent repetitions and GAPDH probing confirmed equal loading. B, Effect of MMP-2 overexpression by siRNA-insensitive pM-FL-A141G treatment or rhMMP-2 supplementation on pM-inhibited MMP-2/α5β1 integrin binding. Whole cell lysates were (200 µg each) were immunoprecipitated with anti-α5β1 integrin antibody (10 µL each sample) using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to Western blotting. The mock sample immunoprecipitated with non-specific IgG was loaded as the negative control. Input shows whole cell lysate (50 µg) of mock sample served as the positive control. Blots were probed with anti-MMP-2 antibody and representative blots from three independent repetitions were shown. Adjacent bar diagram shows the densitometric analyses of the relative band intensity represented as mean±SE values obtained from three repetitions. The significant differences among various treatment groups were indicated by *at p<0.05. IP blots were stripped and subsequently re-probed with anti-α5, anti-β1 and anti-Myc antibodies. C, EMSA was performed using Panomics EMSA kit following the manufacturer’s instructions as described in Materials and Methods. The mock sample was pre-incubated with Stat3α antibody for 30 minutes at room temperature and loaded for supershift. Representative blots from three experimental replicates were shown. D, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif). In short, cells were fixed in 37% formaldehyde and chromatin was sheared by sonication and immunoprecipitated using anti-Stat3 antibody and normal sheep IgG (Nsp-IgG) antibodies by incubation at 4°C for 2–4 hours on a rotor. The immunoprecipitates was collected by using magnetic protein G beads (Millipore) and the chromatin was reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences. Pre-immunoprecipitated samples were used as input controls for PCR amplification of GAPDH and normal sheep IgG (Nsp-IgG) incubation was used for the negative control. E, BrDU assay was performed following manufacturer’s instructions to obtain mean±SE values of BrDU incorporation percentage from three repetitions and significance was represented by * at p<0.05 and ** p<0.01.
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Related In: Results  -  Collection

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

Figure 6: Effect of siRNA-insensitive MMP-2 overexpression and rhMMP-2 supplementation on pM-inhibited IL-6/Stat3 signaling activation. A, After 48 hours of treatment with mock, pSV, pM, pM-FL-A141G, pSV+pM-FL-A141G, pM+pM-FL-A141G, rhMMP-2, pSV+rhMMP-2, and pM+rhMMP-2 as described in Materials and Methods, whole cell lysates were subjected to Western blotting. Blots were representative of at least three independent repetitions and GAPDH probing confirmed equal loading. B, Effect of MMP-2 overexpression by siRNA-insensitive pM-FL-A141G treatment or rhMMP-2 supplementation on pM-inhibited MMP-2/α5β1 integrin binding. Whole cell lysates were (200 µg each) were immunoprecipitated with anti-α5β1 integrin antibody (10 µL each sample) using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to Western blotting. The mock sample immunoprecipitated with non-specific IgG was loaded as the negative control. Input shows whole cell lysate (50 µg) of mock sample served as the positive control. Blots were probed with anti-MMP-2 antibody and representative blots from three independent repetitions were shown. Adjacent bar diagram shows the densitometric analyses of the relative band intensity represented as mean±SE values obtained from three repetitions. The significant differences among various treatment groups were indicated by *at p<0.05. IP blots were stripped and subsequently re-probed with anti-α5, anti-β1 and anti-Myc antibodies. C, EMSA was performed using Panomics EMSA kit following the manufacturer’s instructions as described in Materials and Methods. The mock sample was pre-incubated with Stat3α antibody for 30 minutes at room temperature and loaded for supershift. Representative blots from three experimental replicates were shown. D, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif). In short, cells were fixed in 37% formaldehyde and chromatin was sheared by sonication and immunoprecipitated using anti-Stat3 antibody and normal sheep IgG (Nsp-IgG) antibodies by incubation at 4°C for 2–4 hours on a rotor. The immunoprecipitates was collected by using magnetic protein G beads (Millipore) and the chromatin was reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences. Pre-immunoprecipitated samples were used as input controls for PCR amplification of GAPDH and normal sheep IgG (Nsp-IgG) incubation was used for the negative control. E, BrDU assay was performed following manufacturer’s instructions to obtain mean±SE values of BrDU incorporation percentage from three repetitions and significance was represented by * at p<0.05 and ** p<0.01.
Mentions: To establish specificity of pM-inhibited IL-6/Stat3 activation and to avoid any off-target consequences, we treated mock-, pSV- and pM-treated cells with siRNA-insensitive MMP-2 overexpression plasmid (pM-FL-A141G) or rhMMP-2 as described in Materials and Methods. The expression levels of α5, β1, phospho-Stat3, CyclinD1 and c-Myc were elevated in both pM-FL-A141G and rhMMP-2 treated cells, implicating a possible role of MMP-2 in the regulation of the α5β1-mediated Stat3 activation and cell proliferation (Figure 6A). As shown in Fig. 6B, the loss-and-gain of function approach by pM and pM-FL-A141G or rhMMP-2 treatments showed a significantly enhanced MMP-2/α5β1 association by complex formation in rhMMP-2- or pM-FL-A141G-treated cells and decreased association in pM-treated cells. Immunoprecipitation with non-specific (Nsp-IgG) did not show precipitation and re-probing the IP blots with anti-α5, anti-β1 and anti-Myc antibodies further confirmed the band specificity. Densitometric analysis of relative binding confirmed that the pM+rhMMP-2 or pM+pM-FL-A141G combination treatment noticeably counteracted the rhMMP-2- or pM-FL-A141G-elevated MMP-2/α5β1 binding, which further evidences direct substantial MMP-2/α5β1 integrin interaction in both 4910 and 5310 cells. In correlation with MMP-2 overexpression-induced MMP-2/α5β1 binding, EMSA displayed obvious elevation in Stat3 DNA-binding activity in rhMMP-2 treatment which is reduced in pM+rhMMP-2-treated cells confirming the role of MMP-2 in maintaining constitutive Stat3 activity (Figure 6C). A noticeable elevation in Stat3 occupancy on to CylcinD1 and c-Myc promoters was observed in pM-FL-A141G- or rhMMP-2-treated 4910 and 5310 cells. In corroboration with the reversal of MMP-2/α5β1 complex formation in pM+rhMMP-2 and pM+pM-FL-A141G treated cells, a clear reversal in Stat3 promoter recruitment was observed suggesting a role of MMP-2 in α5β1-mediated Stat3 activation and promoter binding (Figure 6D). Additionally, the pM-induced loss of cell proliferation was reversed in pM+rhMMP-2 or pM+pM-FL-A141G treatments whereas individual rhMMP-2 or pM-FL-A141G treatments alone enhanced the proliferation rate in both cell lines indicating the role of MMP-2 in maintenance of tumor cell proliferation (Figure 6E).

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