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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.

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Effect of PKC inhibitors on THP-1 cells synthesis of proMMP-9/CSPG heteromer and proMMP-9.Shown is a typical experiment where cells in the absence (−) and presence (+) of PMA (10−7 M) were incubated with various concentrations of the PKC inhibitors Gö6976 and Gö6983 (A, B) and Rottlerin (C, D). In the presence of Gö6976 and Gö6983 (A, B), cells were incubated for 72 h, while in the presence of Rottlerin (C, D) the cells were incubated for 12 h in serum free medium. To detect the effect of the PKC inhibitors on the synthesis of the proMMP-9/CSPG heteromer (A, C), the harvested media was applied to Q-Sepharose chromatography as described in Materials and Methods prior to gelatin zymography. To determine the effect of the inhibitors on the synthesised proMMP-9 (B, D), the harvested medium was diluted 20 times and then applied to gelatin zymography. Arrowhead shows the border between the separating and stacking gel and the position of purified proMMP-9 monomer (92 kDa) and proMMP-9 homodimer (225 kDa) used as a standard (Std) is shown.
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pone-0020616-g005: Effect of PKC inhibitors on THP-1 cells synthesis of proMMP-9/CSPG heteromer and proMMP-9.Shown is a typical experiment where cells in the absence (−) and presence (+) of PMA (10−7 M) were incubated with various concentrations of the PKC inhibitors Gö6976 and Gö6983 (A, B) and Rottlerin (C, D). In the presence of Gö6976 and Gö6983 (A, B), cells were incubated for 72 h, while in the presence of Rottlerin (C, D) the cells were incubated for 12 h in serum free medium. To detect the effect of the PKC inhibitors on the synthesis of the proMMP-9/CSPG heteromer (A, C), the harvested media was applied to Q-Sepharose chromatography as described in Materials and Methods prior to gelatin zymography. To determine the effect of the inhibitors on the synthesised proMMP-9 (B, D), the harvested medium was diluted 20 times and then applied to gelatin zymography. Arrowhead shows the border between the separating and stacking gel and the position of purified proMMP-9 monomer (92 kDa) and proMMP-9 homodimer (225 kDa) used as a standard (Std) is shown.

Mentions: We tested to what extent inhibitors of various PKC isoforms could block the PMA induced biosynthesis of the proMMP-9/CSPG complex. Neither 25 nM Gö6976 (inhibits PKC α, β1 and μ) nor 25–100 nM Gö6983 (inhibits PKC α, β1, γ, δ and ζ) affected the PMA induced biosynthesis of the proMMP-9/CSPG complex (Fig. 5A). Furthermore, these two PKC inhibitors did not affect the biosynthesis of the proMMP-9 monomer and homodimer (Fig. 5B).


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)

Effect of PKC inhibitors on THP-1 cells synthesis of proMMP-9/CSPG heteromer and proMMP-9.Shown is a typical experiment where cells in the absence (−) and presence (+) of PMA (10−7 M) were incubated with various concentrations of the PKC inhibitors Gö6976 and Gö6983 (A, B) and Rottlerin (C, D). In the presence of Gö6976 and Gö6983 (A, B), cells were incubated for 72 h, while in the presence of Rottlerin (C, D) the cells were incubated for 12 h in serum free medium. To detect the effect of the PKC inhibitors on the synthesis of the proMMP-9/CSPG heteromer (A, C), the harvested media was applied to Q-Sepharose chromatography as described in Materials and Methods prior to gelatin zymography. To determine the effect of the inhibitors on the synthesised proMMP-9 (B, D), the harvested medium was diluted 20 times and then applied to gelatin zymography. Arrowhead shows the border between the separating and stacking gel and the position of purified proMMP-9 monomer (92 kDa) and proMMP-9 homodimer (225 kDa) used as a standard (Std) is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020616-g005: Effect of PKC inhibitors on THP-1 cells synthesis of proMMP-9/CSPG heteromer and proMMP-9.Shown is a typical experiment where cells in the absence (−) and presence (+) of PMA (10−7 M) were incubated with various concentrations of the PKC inhibitors Gö6976 and Gö6983 (A, B) and Rottlerin (C, D). In the presence of Gö6976 and Gö6983 (A, B), cells were incubated for 72 h, while in the presence of Rottlerin (C, D) the cells were incubated for 12 h in serum free medium. To detect the effect of the PKC inhibitors on the synthesis of the proMMP-9/CSPG heteromer (A, C), the harvested media was applied to Q-Sepharose chromatography as described in Materials and Methods prior to gelatin zymography. To determine the effect of the inhibitors on the synthesised proMMP-9 (B, D), the harvested medium was diluted 20 times and then applied to gelatin zymography. Arrowhead shows the border between the separating and stacking gel and the position of purified proMMP-9 monomer (92 kDa) and proMMP-9 homodimer (225 kDa) used as a standard (Std) is shown.
Mentions: We tested to what extent inhibitors of various PKC isoforms could block the PMA induced biosynthesis of the proMMP-9/CSPG complex. Neither 25 nM Gö6976 (inhibits PKC α, β1 and μ) nor 25–100 nM Gö6983 (inhibits PKC α, β1, γ, δ and ζ) affected the PMA induced biosynthesis of the proMMP-9/CSPG complex (Fig. 5A). Furthermore, these two PKC inhibitors did not affect the biosynthesis of the proMMP-9 monomer and homodimer (Fig. 5B).

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