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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

Reduced functional activity in β1 integrin knockdown cells: (A): Adhesion assay of β1 integrin knockdown MCF-7-MT1 cells for Col I.(B): MDA-MB-231 cells were transfected with the indicated siRNAs. At appropriate time points, cell lysates were collected for analysis of β1 integrin levels by Western blot. (C): Collagen gel contraction by MDA-MB-231 cells after transfection with β1/4, β1/5, β1/6 and control siRNAs. 24 hours after transfection, collagen gels were made at 2 mg/ml and the cells resuspended to a final concentration of 4 × 105 cell/ml. Gels were polymerized and then the gels were released. The relaxed, floating gels were immersed in 1 ml medium with 10% FCS. The gel diameters were measured daily for 5 days. Collagen gel contractions were calculated as mean values in relation to the gel diameters before releasing, as shown in (D). Collagen gel contractions were performed in triplicate independently and mean percent of collagen gel contraction was compared using Student’s t-test.
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Fig2: Reduced functional activity in β1 integrin knockdown cells: (A): Adhesion assay of β1 integrin knockdown MCF-7-MT1 cells for Col I.(B): MDA-MB-231 cells were transfected with the indicated siRNAs. At appropriate time points, cell lysates were collected for analysis of β1 integrin levels by Western blot. (C): Collagen gel contraction by MDA-MB-231 cells after transfection with β1/4, β1/5, β1/6 and control siRNAs. 24 hours after transfection, collagen gels were made at 2 mg/ml and the cells resuspended to a final concentration of 4 × 105 cell/ml. Gels were polymerized and then the gels were released. The relaxed, floating gels were immersed in 1 ml medium with 10% FCS. The gel diameters were measured daily for 5 days. Collagen gel contractions were calculated as mean values in relation to the gel diameters before releasing, as shown in (D). Collagen gel contractions were performed in triplicate independently and mean percent of collagen gel contraction was compared using Student’s t-test.

Mentions: β1 integrin can bind multiple additional partners to form integrins in addition to the primary Col I receptors α1β1, α2β1, α10β1 and α11β1 [35,36]. Integrins α3β1, α6β1, α6β4 and α7β1 are major LM receptors [37]. Integrins α5β1 and α8β1 are major FN receptors. Integrin αIIbβ3 is an alternate FN receptor, while αvβ3 is specific for VN [36]. To examine the functional effects of inhibiting integrin β1 expression, adhesion analysis was performed with ECM substrates including VN, FN, and Col I after β1 integrin siRNA transfection. β1/6 integrin siRNA-treated cells showed significantly less adhesion (p < 0.05) to Col I (Figure 2A). Decreased adhesion to FN was observed for β1/4, β1/5 and β1/6 siRNAs compared to the control siRNA cells (p < 0.05, 0.05 and 0.01 respectively), and again β1/6 integrin siRNA showed a stronger effect than β1/4 and β1/5 (data not shown). None of the β1 siRNA treated cells showed any reduction in adhesion to VN (ligand for αvβ3), confirming that the β1 effects were specific (data not shown).Figure 2


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)

Reduced functional activity in β1 integrin knockdown cells: (A): Adhesion assay of β1 integrin knockdown MCF-7-MT1 cells for Col I.(B): MDA-MB-231 cells were transfected with the indicated siRNAs. At appropriate time points, cell lysates were collected for analysis of β1 integrin levels by Western blot. (C): Collagen gel contraction by MDA-MB-231 cells after transfection with β1/4, β1/5, β1/6 and control siRNAs. 24 hours after transfection, collagen gels were made at 2 mg/ml and the cells resuspended to a final concentration of 4 × 105 cell/ml. Gels were polymerized and then the gels were released. The relaxed, floating gels were immersed in 1 ml medium with 10% FCS. The gel diameters were measured daily for 5 days. Collagen gel contractions were calculated as mean values in relation to the gel diameters before releasing, as shown in (D). Collagen gel contractions were performed in triplicate independently and mean percent of collagen gel contraction was compared using Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4195858&req=5

Fig2: Reduced functional activity in β1 integrin knockdown cells: (A): Adhesion assay of β1 integrin knockdown MCF-7-MT1 cells for Col I.(B): MDA-MB-231 cells were transfected with the indicated siRNAs. At appropriate time points, cell lysates were collected for analysis of β1 integrin levels by Western blot. (C): Collagen gel contraction by MDA-MB-231 cells after transfection with β1/4, β1/5, β1/6 and control siRNAs. 24 hours after transfection, collagen gels were made at 2 mg/ml and the cells resuspended to a final concentration of 4 × 105 cell/ml. Gels were polymerized and then the gels were released. The relaxed, floating gels were immersed in 1 ml medium with 10% FCS. The gel diameters were measured daily for 5 days. Collagen gel contractions were calculated as mean values in relation to the gel diameters before releasing, as shown in (D). Collagen gel contractions were performed in triplicate independently and mean percent of collagen gel contraction was compared using Student’s t-test.
Mentions: β1 integrin can bind multiple additional partners to form integrins in addition to the primary Col I receptors α1β1, α2β1, α10β1 and α11β1 [35,36]. Integrins α3β1, α6β1, α6β4 and α7β1 are major LM receptors [37]. Integrins α5β1 and α8β1 are major FN receptors. Integrin αIIbβ3 is an alternate FN receptor, while αvβ3 is specific for VN [36]. To examine the functional effects of inhibiting integrin β1 expression, adhesion analysis was performed with ECM substrates including VN, FN, and Col I after β1 integrin siRNA transfection. β1/6 integrin siRNA-treated cells showed significantly less adhesion (p < 0.05) to Col I (Figure 2A). Decreased adhesion to FN was observed for β1/4, β1/5 and β1/6 siRNAs compared to the control siRNA cells (p < 0.05, 0.05 and 0.01 respectively), and again β1/6 integrin siRNA showed a stronger effect than β1/4 and β1/5 (data not shown). None of the β1 siRNA treated cells showed any reduction in adhesion to VN (ligand for αvβ3), confirming that the β1 effects were specific (data not shown).Figure 2

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