Limits...
High threshold of β1 integrin inhibition required to block collagen I-induced membrane type-1 matrix metalloproteinase (MT1-MMP) activation of matrix metalloproteinase 2 (MMP-2).

Borrirukwanit K, Pavasant P, Blick T, Lafleur MA, Thompson EW - Cancer Cell Int. (2014)

Bottom Line: All three β1 integrin siRNAs were efficient at β1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins α2 and α3, which are heterodimeric partners of β1, but not αV, which is not.Re-transfection with codon-swapped β1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the β1 integrin target specificity.Together, the data reveals that strong abrogation of β1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of β1 integrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Nursing, Phetchabun Hospital, Phetchabun, Thailand ; Invasion and Metastasis Unit, St. Vincent's Institute, Fitzroy, Victoria 3065 Australia.

ABSTRACT

Background: Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding β1 integrins in collagen-stimulated MMP-2 activation.

Methods: Three β1 integrin siRNAs were used to elucidate the involvement of β1 integrins in the Col I-induced MMP-2 activation mechanism. β1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of β1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography.

Results: All three β1 integrin siRNAs were efficient at β1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins α2 and α3, which are heterodimeric partners of β1, but not αV, which is not. All three β1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of β1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped β1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the β1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by β1 integrin siRNA knockdown.

Conclusion: Together, the data reveals that strong abrogation of β1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of β1 integrin.

No MeSH data available.


Related in: MedlinePlus

Dose response effect of β1 integrin siRNA on MMP-2 activation in MCF-7-MT1 cells. (A): Cells were plated and transfected with β1 integrin siRNA. Serial dilutions of β1/6 siRNA (2.5, 5, 10, 15 and 20 μM) were used in comparison with the 20 μM concentrations of β1/4 and β1/5 siRNAs. Protein concentrations were determined and equal amounts of lysates were electrophoresed with 10% SDS-PAGE under reducing conditions. Western Blot analysis was used to detect β1 integrin and MT1-MMP levels (B). (C): Serial dilutions of β1/6 siRNA (5, 10, and 20 μM) were used in comparison with the 20 μM concentrations of β1/4 and β1/5 siRNAs. Conditioned medium was collected and effects on MMP-2 activation were examined by zymography.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4195858&req=5

Fig4: Dose response effect of β1 integrin siRNA on MMP-2 activation in MCF-7-MT1 cells. (A): Cells were plated and transfected with β1 integrin siRNA. Serial dilutions of β1/6 siRNA (2.5, 5, 10, 15 and 20 μM) were used in comparison with the 20 μM concentrations of β1/4 and β1/5 siRNAs. Protein concentrations were determined and equal amounts of lysates were electrophoresed with 10% SDS-PAGE under reducing conditions. Western Blot analysis was used to detect β1 integrin and MT1-MMP levels (B). (C): Serial dilutions of β1/6 siRNA (5, 10, and 20 μM) were used in comparison with the 20 μM concentrations of β1/4 and β1/5 siRNAs. Conditioned medium was collected and effects on MMP-2 activation were examined by zymography.

Mentions: There are several factors that may influence the efficiency of RNAi in mammalian systems including the choice of the target site of degradation, the transfection method and the turnover rate of the protein. In addition, the mRNA degradation rate is an important factor influencing target gene knockdown by siRNA technology [40,41]. This effect is dependent on the dose response to siRNA concentration. We examined whether β1/4, β1/5 and β1/6 siRNAs had dose-dependent effects on the abrogation of β1 integrin mRNA expression, and whether this was relative to the reduction seen in MMP-2 activation. We hypothesized that the β1/6 siRNA showed a quicker mRNA degradation rate than either β1/4 or β1/5 siRNA. Serial concentrations of β1/6 siRNA (2.5, 5, 10, 15 and 20 μM) were tested. The data showed that at the lowest concentration (2.5 μM), β1/6 siRNA was able to knockdown β1 integrin expression to an extent comparable to the highest concentration (20 μM) of β1/4 or β1/5 siRNA (Figure 4A). The β1 integrin expression was below the threshold of detection 72 hours after transfection with 2.5 and 5 μM of β1/6 siRNA, and this was also seen with β1/4 and β1/5 siRNA at 20 μM. MMP-2 activation reductions were observed only at 20 μM of β1/6 integrin siRNA (Figure 4C), was which relative to reduction of MT1-MMP protein level at this concentration (Figure 4B). These results indicate that the reduction of Col I-induced MMP-2 activation seen only with β1/6 may require the more rapid and complete knock down of β1 integrin seen with this siRNA. A threshold of β1 integrin knockdown that cannot be surpassed by β1/4 or β1/5 siRNAs may exist for the MMP-2 activation effect.Figure 4


High threshold of β1 integrin inhibition required to block collagen I-induced membrane type-1 matrix metalloproteinase (MT1-MMP) activation of matrix metalloproteinase 2 (MMP-2).

Borrirukwanit K, Pavasant P, Blick T, Lafleur MA, Thompson EW - Cancer Cell Int. (2014)

Dose response effect of β1 integrin siRNA on MMP-2 activation in MCF-7-MT1 cells. (A): Cells were plated and transfected with β1 integrin siRNA. Serial dilutions of β1/6 siRNA (2.5, 5, 10, 15 and 20 μM) were used in comparison with the 20 μM concentrations of β1/4 and β1/5 siRNAs. Protein concentrations were determined and equal amounts of lysates were electrophoresed with 10% SDS-PAGE under reducing conditions. Western Blot analysis was used to detect β1 integrin and MT1-MMP levels (B). (C): Serial dilutions of β1/6 siRNA (5, 10, and 20 μM) were used in comparison with the 20 μM concentrations of β1/4 and β1/5 siRNAs. Conditioned medium was collected and effects on MMP-2 activation were examined by zymography.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195858&req=5

Fig4: Dose response effect of β1 integrin siRNA on MMP-2 activation in MCF-7-MT1 cells. (A): Cells were plated and transfected with β1 integrin siRNA. Serial dilutions of β1/6 siRNA (2.5, 5, 10, 15 and 20 μM) were used in comparison with the 20 μM concentrations of β1/4 and β1/5 siRNAs. Protein concentrations were determined and equal amounts of lysates were electrophoresed with 10% SDS-PAGE under reducing conditions. Western Blot analysis was used to detect β1 integrin and MT1-MMP levels (B). (C): Serial dilutions of β1/6 siRNA (5, 10, and 20 μM) were used in comparison with the 20 μM concentrations of β1/4 and β1/5 siRNAs. Conditioned medium was collected and effects on MMP-2 activation were examined by zymography.
Mentions: There are several factors that may influence the efficiency of RNAi in mammalian systems including the choice of the target site of degradation, the transfection method and the turnover rate of the protein. In addition, the mRNA degradation rate is an important factor influencing target gene knockdown by siRNA technology [40,41]. This effect is dependent on the dose response to siRNA concentration. We examined whether β1/4, β1/5 and β1/6 siRNAs had dose-dependent effects on the abrogation of β1 integrin mRNA expression, and whether this was relative to the reduction seen in MMP-2 activation. We hypothesized that the β1/6 siRNA showed a quicker mRNA degradation rate than either β1/4 or β1/5 siRNA. Serial concentrations of β1/6 siRNA (2.5, 5, 10, 15 and 20 μM) were tested. The data showed that at the lowest concentration (2.5 μM), β1/6 siRNA was able to knockdown β1 integrin expression to an extent comparable to the highest concentration (20 μM) of β1/4 or β1/5 siRNA (Figure 4A). The β1 integrin expression was below the threshold of detection 72 hours after transfection with 2.5 and 5 μM of β1/6 siRNA, and this was also seen with β1/4 and β1/5 siRNA at 20 μM. MMP-2 activation reductions were observed only at 20 μM of β1/6 integrin siRNA (Figure 4C), was which relative to reduction of MT1-MMP protein level at this concentration (Figure 4B). These results indicate that the reduction of Col I-induced MMP-2 activation seen only with β1/6 may require the more rapid and complete knock down of β1 integrin seen with this siRNA. A threshold of β1 integrin knockdown that cannot be surpassed by β1/4 or β1/5 siRNAs may exist for the MMP-2 activation effect.Figure 4

Bottom Line: All three β1 integrin siRNAs were efficient at β1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins α2 and α3, which are heterodimeric partners of β1, but not αV, which is not.Re-transfection with codon-swapped β1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the β1 integrin target specificity.Together, the data reveals that strong abrogation of β1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of β1 integrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Nursing, Phetchabun Hospital, Phetchabun, Thailand ; Invasion and Metastasis Unit, St. Vincent's Institute, Fitzroy, Victoria 3065 Australia.

ABSTRACT

Background: Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding β1 integrins in collagen-stimulated MMP-2 activation.

Methods: Three β1 integrin siRNAs were used to elucidate the involvement of β1 integrins in the Col I-induced MMP-2 activation mechanism. β1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of β1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography.

Results: All three β1 integrin siRNAs were efficient at β1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins α2 and α3, which are heterodimeric partners of β1, but not αV, which is not. All three β1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of β1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped β1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the β1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by β1 integrin siRNA knockdown.

Conclusion: Together, the data reveals that strong abrogation of β1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of β1 integrin.

No MeSH data available.


Related in: MedlinePlus