Limits...
Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway.

Malla N, Berg E, Moens U, Uhlin-Hansen L, Winberg JO - PLoS ONE (2011)

Bottom Line: Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9.Formation of complexes may influence both the specificity and localization of the enzyme.Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway.

ABSTRACT

Background: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG) core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG.

Methodology/principal findings: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3) in both control and PMA exposed cells.

Conclusions/significance: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

Show MeSH

Related in: MedlinePlus

Cell viability in the presence of PMA, ConA, MCSF and Rottlerin.Cell viability was detected by the MTS assay (A, B) and by counting viable cells in a Bürker chamber (C) using the Trypan Blue exclusion test. In (A) 6×104 cells in serum free medium were seeded per well in 96 well plates in the absence or presence of PMA, ConA or MCSF at the indicated concentrations, and incubated for 72 h. In (B) and (C) the cells were incubated with various concentrations of Rottlerin (as indicated in the insert in c) either in the absence or presence of 10−7 M PMA. In (B) 6×104 cells were seeded per well in 96 well plates and in (C) 4×105 cells were added to each well in 12 well plates (0.6 ml/well) and incubated in serum free medium for the indicated time period. The results presented are from a typical experiment where N = 4 in (A), 3 in (B) and 2 in (C); *p<0.05 compared to control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105995&req=5

pone-0020616-g001: Cell viability in the presence of PMA, ConA, MCSF and Rottlerin.Cell viability was detected by the MTS assay (A, B) and by counting viable cells in a Bürker chamber (C) using the Trypan Blue exclusion test. In (A) 6×104 cells in serum free medium were seeded per well in 96 well plates in the absence or presence of PMA, ConA or MCSF at the indicated concentrations, and incubated for 72 h. In (B) and (C) the cells were incubated with various concentrations of Rottlerin (as indicated in the insert in c) either in the absence or presence of 10−7 M PMA. In (B) 6×104 cells were seeded per well in 96 well plates and in (C) 4×105 cells were added to each well in 12 well plates (0.6 ml/well) and incubated in serum free medium for the indicated time period. The results presented are from a typical experiment where N = 4 in (A), 3 in (B) and 2 in (C); *p<0.05 compared to control.

Mentions: Prior to examining the effect of the various compounds on the THP-1 cells synthesis of proMMP-9/CSPG heteromer, proMMP-9 and CSPG, we determined to which extent these compounds affected the cell viability. For this purpose two different methods were used, the MTS and Trypan Blue exclusion test. At the concentrations used (see methods), none of the following compounds (IL-1α, IL-1β, IL-3, IL-6, PGE2, TNF-α, bFGF and LPS) had any effect on the viability based on the MTS test. In contrast to this, a decrease in viability was observed when the cells were exposed to MCSF, ConA and PMA (Fig. 1A). In the case of PMA, the Trypan Blue exclusion test verified that PMA was slightly toxic to the cells. Four independent experiments where the THP-1 cells were cultured in the absence or presence of 10−7 M PMA for 24, 48 and 72 h resulted in 69±12, 55±12 and 56±14% (mean ± SD) of living cells in PMA exposed cultures. The percentage of dead cells (mean ± SD from four independent experiments) after 24, 48 and 72 h were 5±2, 6±3 and 8±5 in control cultures and 31±5, 45±13 and 44±6 in PMA exposed cultures.


Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway.

Malla N, Berg E, Moens U, Uhlin-Hansen L, Winberg JO - PLoS ONE (2011)

Cell viability in the presence of PMA, ConA, MCSF and Rottlerin.Cell viability was detected by the MTS assay (A, B) and by counting viable cells in a Bürker chamber (C) using the Trypan Blue exclusion test. In (A) 6×104 cells in serum free medium were seeded per well in 96 well plates in the absence or presence of PMA, ConA or MCSF at the indicated concentrations, and incubated for 72 h. In (B) and (C) the cells were incubated with various concentrations of Rottlerin (as indicated in the insert in c) either in the absence or presence of 10−7 M PMA. In (B) 6×104 cells were seeded per well in 96 well plates and in (C) 4×105 cells were added to each well in 12 well plates (0.6 ml/well) and incubated in serum free medium for the indicated time period. The results presented are from a typical experiment where N = 4 in (A), 3 in (B) and 2 in (C); *p<0.05 compared to control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105995&req=5

pone-0020616-g001: Cell viability in the presence of PMA, ConA, MCSF and Rottlerin.Cell viability was detected by the MTS assay (A, B) and by counting viable cells in a Bürker chamber (C) using the Trypan Blue exclusion test. In (A) 6×104 cells in serum free medium were seeded per well in 96 well plates in the absence or presence of PMA, ConA or MCSF at the indicated concentrations, and incubated for 72 h. In (B) and (C) the cells were incubated with various concentrations of Rottlerin (as indicated in the insert in c) either in the absence or presence of 10−7 M PMA. In (B) 6×104 cells were seeded per well in 96 well plates and in (C) 4×105 cells were added to each well in 12 well plates (0.6 ml/well) and incubated in serum free medium for the indicated time period. The results presented are from a typical experiment where N = 4 in (A), 3 in (B) and 2 in (C); *p<0.05 compared to control.
Mentions: Prior to examining the effect of the various compounds on the THP-1 cells synthesis of proMMP-9/CSPG heteromer, proMMP-9 and CSPG, we determined to which extent these compounds affected the cell viability. For this purpose two different methods were used, the MTS and Trypan Blue exclusion test. At the concentrations used (see methods), none of the following compounds (IL-1α, IL-1β, IL-3, IL-6, PGE2, TNF-α, bFGF and LPS) had any effect on the viability based on the MTS test. In contrast to this, a decrease in viability was observed when the cells were exposed to MCSF, ConA and PMA (Fig. 1A). In the case of PMA, the Trypan Blue exclusion test verified that PMA was slightly toxic to the cells. Four independent experiments where the THP-1 cells were cultured in the absence or presence of 10−7 M PMA for 24, 48 and 72 h resulted in 69±12, 55±12 and 56±14% (mean ± SD) of living cells in PMA exposed cultures. The percentage of dead cells (mean ± SD from four independent experiments) after 24, 48 and 72 h were 5±2, 6±3 and 8±5 in control cultures and 31±5, 45±13 and 44±6 in PMA exposed cultures.

Bottom Line: Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9.Formation of complexes may influence both the specificity and localization of the enzyme.Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway.

ABSTRACT

Background: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG) core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG.

Methodology/principal findings: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3) in both control and PMA exposed cells.

Conclusions/significance: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

Show MeSH
Related in: MedlinePlus