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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 IL-6 overexpression on pM-inhibited constitutive Stat3 phosphorylation, DNA-binding activity and proliferation in glioma xenograft cells. A, Western blotting was performed using 4910 and 5310 whole cell lysates at 48 hours post-transfection. GAPDH probing was used to check equal loading. B, Nuclear extracts were prepared using Biovision cytoplasmic/nuclear extraction kit and subjected to EMSA using Panomics EMSA kit as per the manufacturer’s instructions. Representative blots from three independent repetitions were shown. For supershift analysis, mock sample was loaded after pre-incubation with STAT3α antibody for 30 min at room temperature. C, 4910 and 5310 cells were seeded (1×103 cells/well) in 96-well plates and subjected to different treatments for 48 hours as described in Materials and Methods. BrDU cell proliferation assay was performed % BrDU incorporation obtained from three individual repetitions was represented as mean±SE and significance is shown by * at p<0.05 and ** p<0.01. D, Western blotting was performed using whole cell lysates to check cleaved PARP, cleaved caspase-3, CyclinD1 and c-Myc expression levels. Blots are representative of three experimental replicates and GAPDH probing was used to confirm equal loading.
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Figure 5: Effect of IL-6 overexpression on pM-inhibited constitutive Stat3 phosphorylation, DNA-binding activity and proliferation in glioma xenograft cells. A, Western blotting was performed using 4910 and 5310 whole cell lysates at 48 hours post-transfection. GAPDH probing was used to check equal loading. B, Nuclear extracts were prepared using Biovision cytoplasmic/nuclear extraction kit and subjected to EMSA using Panomics EMSA kit as per the manufacturer’s instructions. Representative blots from three independent repetitions were shown. For supershift analysis, mock sample was loaded after pre-incubation with STAT3α antibody for 30 min at room temperature. C, 4910 and 5310 cells were seeded (1×103 cells/well) in 96-well plates and subjected to different treatments for 48 hours as described in Materials and Methods. BrDU cell proliferation assay was performed % BrDU incorporation obtained from three individual repetitions was represented as mean±SE and significance is shown by * at p<0.05 and ** p<0.01. D, Western blotting was performed using whole cell lysates to check cleaved PARP, cleaved caspase-3, CyclinD1 and c-Myc expression levels. Blots are representative of three experimental replicates and GAPDH probing was used to confirm equal loading.

Mentions: To further evaluate the significance of MMP-2.siRNA-induced IL-6 inhibition on subsequent JAK/Stat3 pathway attenuation, we overexpressed IL-6 and confirmed the enhanced mRNA and protein expression levels of IL-6 in transfected 4910 and 5310 cells after 24 hours (Supplementary Fig. S5A). There was remarkable elevation of gp130, phopsho-JAK1, JAK1, phospho-JAK2, JAK2 and phospho-Stat3 expression levels in cells treated with pIL-6 alone or pSV+pIL-6 compared to either mock- or pSV-treated cells. In contrast the expression levels of IL-6, phospho-JAK2, and phosho-Stat3 in pM+IL-6 treatments were elevated when compared to pM treatment alone and lesser in comparison to either pIL-6 or pSV+pIL-6 treatments, implying a significant reversal of phospho-Stat3 inhibition by IL-6 overexpression in pM-treated cells. The reversal in gp130, phospho-JAK1, JAK1, phospho-JAK2, JAK2 and phospho-Stat3 expression correlated with IL-6 overexpression in pM-treated cells, further implying that the MMP-2 depletion attenuated the IL-6-mediated Stat3 activation (Figure 5A). Corroborating previous reports showing MMP-2 as a potential Stat3 target gene, IL-6 overexpression in turn resulted in remarkable elevation in MMP-2 gelatinolytic activity, which was suppressed in pM+pIL-6 combination treatment (Supplementary Figure S5B). Additionally, EMSA also showed significant reversal of IL-6-induced Stat3-DNA binding activity in pM+pIL-6 treatment suggesting that the loss of MMP-2 activity inhibited the IL-6 mediated Stat3 activation (Figure 5B). Confocal microscopy also revealed elevation in phospho-Stat3 nuclear localization in pIL-6-treated cells compared to mock-treated cells and pM+pIL-6-treated cells showed an expression pattern similar to either mock or pSV treatment confirming the reversal of pM-inhibited phospho-Stat3 nuclear localization in pIL-6-treated cells (Supplementary Figure S5C). The effect of pM and pM+pIL-6 treatments was further checked on cell proliferation using BrDU assay. IL-6 overexpression restored the pM-inhibited proliferation in pM+pIL-6 treatment (Figure 5C). Additionally, we observed a consistent reversal in pM-induced PARP and caspase-3 cleavage, and inhibition in CyclinD1 and c-Myc expression levels in pM+pIL-6-treated cells, which directly indicates that the loss of MMP-2 leads to the suppression of IL-6/Stat3 proliferation signaling (Figure 5D).


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 IL-6 overexpression on pM-inhibited constitutive Stat3 phosphorylation, DNA-binding activity and proliferation in glioma xenograft cells. A, Western blotting was performed using 4910 and 5310 whole cell lysates at 48 hours post-transfection. GAPDH probing was used to check equal loading. B, Nuclear extracts were prepared using Biovision cytoplasmic/nuclear extraction kit and subjected to EMSA using Panomics EMSA kit as per the manufacturer’s instructions. Representative blots from three independent repetitions were shown. For supershift analysis, mock sample was loaded after pre-incubation with STAT3α antibody for 30 min at room temperature. C, 4910 and 5310 cells were seeded (1×103 cells/well) in 96-well plates and subjected to different treatments for 48 hours as described in Materials and Methods. BrDU cell proliferation assay was performed % BrDU incorporation obtained from three individual repetitions was represented as mean±SE and significance is shown by * at p<0.05 and ** p<0.01. D, Western blotting was performed using whole cell lysates to check cleaved PARP, cleaved caspase-3, CyclinD1 and c-Myc expression levels. Blots are representative of three experimental replicates and GAPDH probing was used to confirm equal loading.
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Related In: Results  -  Collection

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

Figure 5: Effect of IL-6 overexpression on pM-inhibited constitutive Stat3 phosphorylation, DNA-binding activity and proliferation in glioma xenograft cells. A, Western blotting was performed using 4910 and 5310 whole cell lysates at 48 hours post-transfection. GAPDH probing was used to check equal loading. B, Nuclear extracts were prepared using Biovision cytoplasmic/nuclear extraction kit and subjected to EMSA using Panomics EMSA kit as per the manufacturer’s instructions. Representative blots from three independent repetitions were shown. For supershift analysis, mock sample was loaded after pre-incubation with STAT3α antibody for 30 min at room temperature. C, 4910 and 5310 cells were seeded (1×103 cells/well) in 96-well plates and subjected to different treatments for 48 hours as described in Materials and Methods. BrDU cell proliferation assay was performed % BrDU incorporation obtained from three individual repetitions was represented as mean±SE and significance is shown by * at p<0.05 and ** p<0.01. D, Western blotting was performed using whole cell lysates to check cleaved PARP, cleaved caspase-3, CyclinD1 and c-Myc expression levels. Blots are representative of three experimental replicates and GAPDH probing was used to confirm equal loading.
Mentions: To further evaluate the significance of MMP-2.siRNA-induced IL-6 inhibition on subsequent JAK/Stat3 pathway attenuation, we overexpressed IL-6 and confirmed the enhanced mRNA and protein expression levels of IL-6 in transfected 4910 and 5310 cells after 24 hours (Supplementary Fig. S5A). There was remarkable elevation of gp130, phopsho-JAK1, JAK1, phospho-JAK2, JAK2 and phospho-Stat3 expression levels in cells treated with pIL-6 alone or pSV+pIL-6 compared to either mock- or pSV-treated cells. In contrast the expression levels of IL-6, phospho-JAK2, and phosho-Stat3 in pM+IL-6 treatments were elevated when compared to pM treatment alone and lesser in comparison to either pIL-6 or pSV+pIL-6 treatments, implying a significant reversal of phospho-Stat3 inhibition by IL-6 overexpression in pM-treated cells. The reversal in gp130, phospho-JAK1, JAK1, phospho-JAK2, JAK2 and phospho-Stat3 expression correlated with IL-6 overexpression in pM-treated cells, further implying that the MMP-2 depletion attenuated the IL-6-mediated Stat3 activation (Figure 5A). Corroborating previous reports showing MMP-2 as a potential Stat3 target gene, IL-6 overexpression in turn resulted in remarkable elevation in MMP-2 gelatinolytic activity, which was suppressed in pM+pIL-6 combination treatment (Supplementary Figure S5B). Additionally, EMSA also showed significant reversal of IL-6-induced Stat3-DNA binding activity in pM+pIL-6 treatment suggesting that the loss of MMP-2 activity inhibited the IL-6 mediated Stat3 activation (Figure 5B). Confocal microscopy also revealed elevation in phospho-Stat3 nuclear localization in pIL-6-treated cells compared to mock-treated cells and pM+pIL-6-treated cells showed an expression pattern similar to either mock or pSV treatment confirming the reversal of pM-inhibited phospho-Stat3 nuclear localization in pIL-6-treated cells (Supplementary Figure S5C). The effect of pM and pM+pIL-6 treatments was further checked on cell proliferation using BrDU assay. IL-6 overexpression restored the pM-inhibited proliferation in pM+pIL-6 treatment (Figure 5C). Additionally, we observed a consistent reversal in pM-induced PARP and caspase-3 cleavage, and inhibition in CyclinD1 and c-Myc expression levels in pM+pIL-6-treated cells, which directly indicates that the loss of MMP-2 leads to the suppression of IL-6/Stat3 proliferation signaling (Figure 5D).

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