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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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MMP-2 downregulation inhibits Stat3 phosphorylation, DNA-binding activity and recruitment on to CyclinD1 and c-Myc promoters. A, At 48 h post-transfection, whole 4910 and 5310 cell lysates were subjected to Western blot analysis. Subsequently, blots were stripped and reprobed with GAPDH to confirm equal loading. B, Immunoflourescence was performed as described in Materials and Methods to analyze the expression levels of phospho-Stat3. DAPI was used for nuclear counterstaining and confocal micrographs representative of numerous randomly selected microscopic fields in at least 3 independent experiments was shown. C, EMSA showing Stat3 DNA-binding activity. Nuclear extracts were prepared using Biovision cytoplasmic/nuclear extraction kit and subjected to EMSA using Panomics EMSA kit following manufacturer’s instructions. The mock sample was pre-incubated with STAT3α antibody for 30 minutes at room temperature and then loaded for supershift and representative blots from three independent repetitions were shown. D, ChIP assay was performed in mock-, pSV- and pM-treated cells using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA was collected using magnetic protein G beads and the chromatin 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 in PCR amplification of GAPDH to monitor equal loading and IP with normal sheep IgG (Nsp-IgG) served as negative control. E, Whole 4910 and 5310 cell lysates were subjected to Western blotting and blots were re-probed with GAPDH to assure equal loading.
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Figure 4: MMP-2 downregulation inhibits Stat3 phosphorylation, DNA-binding activity and recruitment on to CyclinD1 and c-Myc promoters. A, At 48 h post-transfection, whole 4910 and 5310 cell lysates were subjected to Western blot analysis. Subsequently, blots were stripped and reprobed with GAPDH to confirm equal loading. B, Immunoflourescence was performed as described in Materials and Methods to analyze the expression levels of phospho-Stat3. DAPI was used for nuclear counterstaining and confocal micrographs representative of numerous randomly selected microscopic fields in at least 3 independent experiments was shown. C, EMSA showing Stat3 DNA-binding activity. Nuclear extracts were prepared using Biovision cytoplasmic/nuclear extraction kit and subjected to EMSA using Panomics EMSA kit following manufacturer’s instructions. The mock sample was pre-incubated with STAT3α antibody for 30 minutes at room temperature and then loaded for supershift and representative blots from three independent repetitions were shown. D, ChIP assay was performed in mock-, pSV- and pM-treated cells using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA was collected using magnetic protein G beads and the chromatin 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 in PCR amplification of GAPDH to monitor equal loading and IP with normal sheep IgG (Nsp-IgG) served as negative control. E, Whole 4910 and 5310 cell lysates were subjected to Western blotting and blots were re-probed with GAPDH to assure equal loading.

Mentions: Western blotting showed considerable decrease in gp130, phospho-JAK1, JAK1, phospho-JAK2, JAK2 and phospho-Stat3 (Tyr-705 and Ser-727) expression levels with no noticeable change in total Stat3 in pM-treated 4910 and 5310 cells whereas constitutive Stat3 activation were documented in both mock- and pSV-treated controls (Figure 4A). Even though the inhibition in Stat3 phosphorylation was evident at 24 h we observed further decrease at 48 hours post pM-transfection indicating that this effect was time-dependent (data not shown). Fluorescence microscopy showed conspicuous reduction in phospho-Stat3 expression and subsequent nuclear translocation in MMP-2 knockdown cells. In contrast, mock- and pSV-treated cells show high expression of phospho-Stat3 distributed throughout cytoplasm, with prominent nuclear localization (Figure 4B). To further assess the effect of pM on Stat3 DNA-binding activity, EMSA was performed which revealed a high Stat3 activity in both mock- and pSV-treated 4910 and 5310 controls. Conversely, a prominent decrease in DNA-binding activity was evidenced in 48 h pM-transfected cells implying that the loss of MMP-2 leads to suppression of constitutive STAT3 activation (Figure 4C). Further, the recruitment of Stat3 was assessed by ChIP assay which showed a strong occupancy of Stat3 at CyclinD1 and c-Myc promoters in both mock- and pSV-treated 4910 and 5310 cells. In contrast, MMP-2 suppression using pM noticeably inhibited the Stat3 recruitment on to CyclinD1 and c-Myc promoters suggesting the downregulation of these proteins (Figure 4D). Western blotting showed a remarkable downregulation in Stat3 target proteins CyclinD1, c-Myc, Bcl-xL, Bcl2 and Survivin in pM-treated 4910 and 5310 cells in comparison to control counterparts (Figure 4E). Eventually, to rule out the off-target effects of pM on negative regulation of α5β1-mediated IL-6/Stat3 activation and proliferation in 4910 and 5310 cell lines, parallel MMP-2 knockdown experiments were also performed using pM-1 (siRNA in pcDNA3.0 targeting the 1073–1094 position in MMP-2 cDNA), pM-2 (sc-29398) and siRNA-specific scrambled vectors pSV-1 and pSV-2. Comparable to pM-induced MMP-2 knockdown, pM-1 and pM-2 treatments also resulted in a noticeable decrease in proliferation compared to respective pSV-1 and pSV-2 controls (Supplementary Fig. S3A). The conditioned media from pM-1 and pM-2 treated cells showed drastic reduction in the secreted levels of IL-6, IL-8 and IL-10 in corroboration with the downregulation of cellular expression levels of α5 integrin, β1 integrin, IL-6, phospho-Stat3, (Supplementary Fig. S3B, S3C). The effect of MMP-2 downregulation was also evaluated in another two human glioma cell lines, U251 and U87, which confirmed the noticeable decrease in proliferation and suppression of α5, β1, IL-6, expression and JAK1, JAK2 and Stat3 phosphorylation levels (Supplementary Fig. S4).


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)

MMP-2 downregulation inhibits Stat3 phosphorylation, DNA-binding activity and recruitment on to CyclinD1 and c-Myc promoters. A, At 48 h post-transfection, whole 4910 and 5310 cell lysates were subjected to Western blot analysis. Subsequently, blots were stripped and reprobed with GAPDH to confirm equal loading. B, Immunoflourescence was performed as described in Materials and Methods to analyze the expression levels of phospho-Stat3. DAPI was used for nuclear counterstaining and confocal micrographs representative of numerous randomly selected microscopic fields in at least 3 independent experiments was shown. C, EMSA showing Stat3 DNA-binding activity. Nuclear extracts were prepared using Biovision cytoplasmic/nuclear extraction kit and subjected to EMSA using Panomics EMSA kit following manufacturer’s instructions. The mock sample was pre-incubated with STAT3α antibody for 30 minutes at room temperature and then loaded for supershift and representative blots from three independent repetitions were shown. D, ChIP assay was performed in mock-, pSV- and pM-treated cells using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA was collected using magnetic protein G beads and the chromatin 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 in PCR amplification of GAPDH to monitor equal loading and IP with normal sheep IgG (Nsp-IgG) served as negative control. E, Whole 4910 and 5310 cell lysates were subjected to Western blotting and blots were re-probed with GAPDH to assure equal loading.
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Related In: Results  -  Collection

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Figure 4: MMP-2 downregulation inhibits Stat3 phosphorylation, DNA-binding activity and recruitment on to CyclinD1 and c-Myc promoters. A, At 48 h post-transfection, whole 4910 and 5310 cell lysates were subjected to Western blot analysis. Subsequently, blots were stripped and reprobed with GAPDH to confirm equal loading. B, Immunoflourescence was performed as described in Materials and Methods to analyze the expression levels of phospho-Stat3. DAPI was used for nuclear counterstaining and confocal micrographs representative of numerous randomly selected microscopic fields in at least 3 independent experiments was shown. C, EMSA showing Stat3 DNA-binding activity. Nuclear extracts were prepared using Biovision cytoplasmic/nuclear extraction kit and subjected to EMSA using Panomics EMSA kit following manufacturer’s instructions. The mock sample was pre-incubated with STAT3α antibody for 30 minutes at room temperature and then loaded for supershift and representative blots from three independent repetitions were shown. D, ChIP assay was performed in mock-, pSV- and pM-treated cells using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA was collected using magnetic protein G beads and the chromatin 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 in PCR amplification of GAPDH to monitor equal loading and IP with normal sheep IgG (Nsp-IgG) served as negative control. E, Whole 4910 and 5310 cell lysates were subjected to Western blotting and blots were re-probed with GAPDH to assure equal loading.
Mentions: Western blotting showed considerable decrease in gp130, phospho-JAK1, JAK1, phospho-JAK2, JAK2 and phospho-Stat3 (Tyr-705 and Ser-727) expression levels with no noticeable change in total Stat3 in pM-treated 4910 and 5310 cells whereas constitutive Stat3 activation were documented in both mock- and pSV-treated controls (Figure 4A). Even though the inhibition in Stat3 phosphorylation was evident at 24 h we observed further decrease at 48 hours post pM-transfection indicating that this effect was time-dependent (data not shown). Fluorescence microscopy showed conspicuous reduction in phospho-Stat3 expression and subsequent nuclear translocation in MMP-2 knockdown cells. In contrast, mock- and pSV-treated cells show high expression of phospho-Stat3 distributed throughout cytoplasm, with prominent nuclear localization (Figure 4B). To further assess the effect of pM on Stat3 DNA-binding activity, EMSA was performed which revealed a high Stat3 activity in both mock- and pSV-treated 4910 and 5310 controls. Conversely, a prominent decrease in DNA-binding activity was evidenced in 48 h pM-transfected cells implying that the loss of MMP-2 leads to suppression of constitutive STAT3 activation (Figure 4C). Further, the recruitment of Stat3 was assessed by ChIP assay which showed a strong occupancy of Stat3 at CyclinD1 and c-Myc promoters in both mock- and pSV-treated 4910 and 5310 cells. In contrast, MMP-2 suppression using pM noticeably inhibited the Stat3 recruitment on to CyclinD1 and c-Myc promoters suggesting the downregulation of these proteins (Figure 4D). Western blotting showed a remarkable downregulation in Stat3 target proteins CyclinD1, c-Myc, Bcl-xL, Bcl2 and Survivin in pM-treated 4910 and 5310 cells in comparison to control counterparts (Figure 4E). Eventually, to rule out the off-target effects of pM on negative regulation of α5β1-mediated IL-6/Stat3 activation and proliferation in 4910 and 5310 cell lines, parallel MMP-2 knockdown experiments were also performed using pM-1 (siRNA in pcDNA3.0 targeting the 1073–1094 position in MMP-2 cDNA), pM-2 (sc-29398) and siRNA-specific scrambled vectors pSV-1 and pSV-2. Comparable to pM-induced MMP-2 knockdown, pM-1 and pM-2 treatments also resulted in a noticeable decrease in proliferation compared to respective pSV-1 and pSV-2 controls (Supplementary Fig. S3A). The conditioned media from pM-1 and pM-2 treated cells showed drastic reduction in the secreted levels of IL-6, IL-8 and IL-10 in corroboration with the downregulation of cellular expression levels of α5 integrin, β1 integrin, IL-6, phospho-Stat3, (Supplementary Fig. S3B, S3C). The effect of MMP-2 downregulation was also evaluated in another two human glioma cell lines, U251 and U87, which confirmed the noticeable decrease in proliferation and suppression of α5, β1, IL-6, expression and JAK1, JAK2 and Stat3 phosphorylation levels (Supplementary Fig. S4).

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