Limits...
Matrix metalloproteinase 8 (collagenase 2) induces the expression of interleukins 6 and 8 in breast cancer cells.

Thirkettle S, Decock J, Arnold H, Pennington CJ, Jaworski DM, Edwards DR - J. Biol. Chem. (2013)

Bottom Line: In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line.Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity.This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, United Kingdom.

ABSTRACT
Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.

Show MeSH

Related in: MedlinePlus

MMP-8, IL-6, and IL-8 exist as part of an interconnected regulatory circuit.A, RNA quantification by real-time TaqMan RT-PCR of IL-6 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. B, RNA quantification by real-time TaqMan RT-PCR of IL-8 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. C, RNA quantification by real-time TaqMan RT-PCR of endogenous MMP-8 in naïve, non-transfected MDA-MB-231 cells treated with recombinant human IL-6 and IL-8 for 24 h. *, p < 0.05; **, p < 0.01;***, p < 0.001; ns, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675567&req=5

Figure 5: MMP-8, IL-6, and IL-8 exist as part of an interconnected regulatory circuit.A, RNA quantification by real-time TaqMan RT-PCR of IL-6 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. B, RNA quantification by real-time TaqMan RT-PCR of IL-8 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. C, RNA quantification by real-time TaqMan RT-PCR of endogenous MMP-8 in naïve, non-transfected MDA-MB-231 cells treated with recombinant human IL-6 and IL-8 for 24 h. *, p < 0.05; **, p < 0.01;***, p < 0.001; ns, not significant.

Mentions: The human breast cancer cell lines we examined (MDA-MB-231, MCF7, and SK-BR-3; Fig. 2A and data not shown) did not express MMP-8 at a level that was detectable by Western blot analysis, and only MDA-MB-231 cells expressed a very small amount of endogenous MMP8 that was detectable by TaqMan quantitative real-time RT-PCR (data not shown, see also Fig. 5C). Using the highly metastatic MDA-MB-231 cells (estrogen receptor-/progesterone receptor-/HER2), we generated stably transfected clones carrying the WT MMP-8, E198A mutant, or EV control vector. Fig. 2A shows that comparable levels of the wild-type and catalytically inactive MMP-8 proteins were expressed and released into the conditioned media and that MMP8 transcript levels originating from the transfected genes were also similar. The levels of MMP-8 protein expression achieved in these transfected clonal lines were similar to that of endogenous MMP-8 in conditioned media from the G361 melanoma cell line, which was included as a control. The major MMP-8 band was 75 kDa, likely corresponding to pro-MMP-8, but a faster migrating band was detected by the MMP-8 antibody, although this could be a C-terminally processed form because it was undetectable by the antibody toward the V5 tag. However, functional collagenolytic activity of the wild-type MMP-8 was confirmed by demonstration of elevated hydroxyproline release compared with the E198A mutant-expressing cells and the empty vector control (Fig. 2B). Analysis of IL-6 and 8 protein production by ELISA of conditioned media from two independent clonal isolates of WT MMP-8 stably transfected MDA-MB-231 cells (Fig. 3, upper panels) showed a 2- to 12-fold increase in IL-6 and an 2- to 8.5-fold increase in IL-8 protein in comparison to EV control cells. Again, the E198A inactive mutant MMP-8-expressing cells showed no increases compared with the EV control cells. Also, as with the transiently transfected MCF-7 cells, we found that in the MDA-MB-231 stably transfected cells, mRNA levels were increased 4-fold for IL-6 and 4- to 12-fold for IL-8 in stable WT MMP-8-expressing MDA-MB-231 clones (Fig. 3, lower panels), mirroring the increased levels of the soluble proteins.


Matrix metalloproteinase 8 (collagenase 2) induces the expression of interleukins 6 and 8 in breast cancer cells.

Thirkettle S, Decock J, Arnold H, Pennington CJ, Jaworski DM, Edwards DR - J. Biol. Chem. (2013)

MMP-8, IL-6, and IL-8 exist as part of an interconnected regulatory circuit.A, RNA quantification by real-time TaqMan RT-PCR of IL-6 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. B, RNA quantification by real-time TaqMan RT-PCR of IL-8 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. C, RNA quantification by real-time TaqMan RT-PCR of endogenous MMP-8 in naïve, non-transfected MDA-MB-231 cells treated with recombinant human IL-6 and IL-8 for 24 h. *, p < 0.05; **, p < 0.01;***, p < 0.001; ns, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675567&req=5

Figure 5: MMP-8, IL-6, and IL-8 exist as part of an interconnected regulatory circuit.A, RNA quantification by real-time TaqMan RT-PCR of IL-6 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. B, RNA quantification by real-time TaqMan RT-PCR of IL-8 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. C, RNA quantification by real-time TaqMan RT-PCR of endogenous MMP-8 in naïve, non-transfected MDA-MB-231 cells treated with recombinant human IL-6 and IL-8 for 24 h. *, p < 0.05; **, p < 0.01;***, p < 0.001; ns, not significant.
Mentions: The human breast cancer cell lines we examined (MDA-MB-231, MCF7, and SK-BR-3; Fig. 2A and data not shown) did not express MMP-8 at a level that was detectable by Western blot analysis, and only MDA-MB-231 cells expressed a very small amount of endogenous MMP8 that was detectable by TaqMan quantitative real-time RT-PCR (data not shown, see also Fig. 5C). Using the highly metastatic MDA-MB-231 cells (estrogen receptor-/progesterone receptor-/HER2), we generated stably transfected clones carrying the WT MMP-8, E198A mutant, or EV control vector. Fig. 2A shows that comparable levels of the wild-type and catalytically inactive MMP-8 proteins were expressed and released into the conditioned media and that MMP8 transcript levels originating from the transfected genes were also similar. The levels of MMP-8 protein expression achieved in these transfected clonal lines were similar to that of endogenous MMP-8 in conditioned media from the G361 melanoma cell line, which was included as a control. The major MMP-8 band was 75 kDa, likely corresponding to pro-MMP-8, but a faster migrating band was detected by the MMP-8 antibody, although this could be a C-terminally processed form because it was undetectable by the antibody toward the V5 tag. However, functional collagenolytic activity of the wild-type MMP-8 was confirmed by demonstration of elevated hydroxyproline release compared with the E198A mutant-expressing cells and the empty vector control (Fig. 2B). Analysis of IL-6 and 8 protein production by ELISA of conditioned media from two independent clonal isolates of WT MMP-8 stably transfected MDA-MB-231 cells (Fig. 3, upper panels) showed a 2- to 12-fold increase in IL-6 and an 2- to 8.5-fold increase in IL-8 protein in comparison to EV control cells. Again, the E198A inactive mutant MMP-8-expressing cells showed no increases compared with the EV control cells. Also, as with the transiently transfected MCF-7 cells, we found that in the MDA-MB-231 stably transfected cells, mRNA levels were increased 4-fold for IL-6 and 4- to 12-fold for IL-8 in stable WT MMP-8-expressing MDA-MB-231 clones (Fig. 3, lower panels), mirroring the increased levels of the soluble proteins.

Bottom Line: In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line.Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity.This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, United Kingdom.

ABSTRACT
Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.

Show MeSH
Related in: MedlinePlus