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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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Synthesis of proMMP-9/CSPG in the absence and presence of PMA.THP-1 cells were incubated in the absence or presence of PMA in serum free medium. Harvested medium was thereafter applied to Q-Sepharose chromatography and the presence of proMMP-9/CSPG was detected with gelatin zymography as described in Materials and Methods. In (A), cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. In (B), cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M PMA. The samples (containing CSPG and proMMP-9/CSPG complex) from cells not exposed to PMA were five times more concentrated than the samples from the PMA treated cells when applied to the gel. In (C), cells were either incubated for 72 h in the absence (−) or presence (+) of 10−7 M PMA at serum free conditions, or pre-incubated for 3 h in the absence (−) or presence (+) of 10−7 M PMA and/or 10% fetal calf serum. After the pre-incubation, cells were washed three times in PBS and thereafter incubated in serum free medium for 72 h. Arrowhead shows the border between the separating and stacking gel, and arrows show the position of the proMMP-9/CSPG complexes. Purified proMMP-9 was used as a standard and the position of the 225 kDa homodimer form is shown at the left. The gels are representative for several similar experiments.
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pone-0020616-g002: Synthesis of proMMP-9/CSPG in the absence and presence of PMA.THP-1 cells were incubated in the absence or presence of PMA in serum free medium. Harvested medium was thereafter applied to Q-Sepharose chromatography and the presence of proMMP-9/CSPG was detected with gelatin zymography as described in Materials and Methods. In (A), cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. In (B), cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M PMA. The samples (containing CSPG and proMMP-9/CSPG complex) from cells not exposed to PMA were five times more concentrated than the samples from the PMA treated cells when applied to the gel. In (C), cells were either incubated for 72 h in the absence (−) or presence (+) of 10−7 M PMA at serum free conditions, or pre-incubated for 3 h in the absence (−) or presence (+) of 10−7 M PMA and/or 10% fetal calf serum. After the pre-incubation, cells were washed three times in PBS and thereafter incubated in serum free medium for 72 h. Arrowhead shows the border between the separating and stacking gel, and arrows show the position of the proMMP-9/CSPG complexes. Purified proMMP-9 was used as a standard and the position of the 225 kDa homodimer form is shown at the left. The gels are representative for several similar experiments.

Mentions: PMA is known to stimulate monocytic cell lines to increase the synthesis of MMPs [46]–[49]. We therefore determined to what extent PMA could stimulate the biosynthesis of the proMMP-9/CSPG heteromer by THP-1 cells. In several independent experiments the biosynthesis of the proMMP-9/CSPG heteromers increased with increasing PMA concentrations with a peak at 10−7 M, and thereafter decreases with increasing PMA concentrations (Fig. 2A).


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)

Synthesis of proMMP-9/CSPG in the absence and presence of PMA.THP-1 cells were incubated in the absence or presence of PMA in serum free medium. Harvested medium was thereafter applied to Q-Sepharose chromatography and the presence of proMMP-9/CSPG was detected with gelatin zymography as described in Materials and Methods. In (A), cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. In (B), cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M PMA. The samples (containing CSPG and proMMP-9/CSPG complex) from cells not exposed to PMA were five times more concentrated than the samples from the PMA treated cells when applied to the gel. In (C), cells were either incubated for 72 h in the absence (−) or presence (+) of 10−7 M PMA at serum free conditions, or pre-incubated for 3 h in the absence (−) or presence (+) of 10−7 M PMA and/or 10% fetal calf serum. After the pre-incubation, cells were washed three times in PBS and thereafter incubated in serum free medium for 72 h. Arrowhead shows the border between the separating and stacking gel, and arrows show the position of the proMMP-9/CSPG complexes. Purified proMMP-9 was used as a standard and the position of the 225 kDa homodimer form is shown at the left. The gels are representative for several similar experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020616-g002: Synthesis of proMMP-9/CSPG in the absence and presence of PMA.THP-1 cells were incubated in the absence or presence of PMA in serum free medium. Harvested medium was thereafter applied to Q-Sepharose chromatography and the presence of proMMP-9/CSPG was detected with gelatin zymography as described in Materials and Methods. In (A), cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. In (B), cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M PMA. The samples (containing CSPG and proMMP-9/CSPG complex) from cells not exposed to PMA were five times more concentrated than the samples from the PMA treated cells when applied to the gel. In (C), cells were either incubated for 72 h in the absence (−) or presence (+) of 10−7 M PMA at serum free conditions, or pre-incubated for 3 h in the absence (−) or presence (+) of 10−7 M PMA and/or 10% fetal calf serum. After the pre-incubation, cells were washed three times in PBS and thereafter incubated in serum free medium for 72 h. Arrowhead shows the border between the separating and stacking gel, and arrows show the position of the proMMP-9/CSPG complexes. Purified proMMP-9 was used as a standard and the position of the 225 kDa homodimer form is shown at the left. The gels are representative for several similar experiments.
Mentions: PMA is known to stimulate monocytic cell lines to increase the synthesis of MMPs [46]–[49]. We therefore determined to what extent PMA could stimulate the biosynthesis of the proMMP-9/CSPG heteromer by THP-1 cells. In several independent experiments the biosynthesis of the proMMP-9/CSPG heteromers increased with increasing PMA concentrations with a peak at 10−7 M, and thereafter decreases with increasing PMA concentrations (Fig. 2A).

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