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

Effects of various compounds on the synthesis of proMMP-9/CSPG and CSPG.THP-1 cells were incubated in the absence or presence of the compounds shown in serum free medium for 72 h. In (A), the harvested medium was applied to Q-Sepharose chromatography and the presence of proMMP-9/CSPG was detected with gelatin zymography as described in Materials and Methods. Arrowhead shows the border between the separating and stacking gel and arrow shows the position of the 300 kDa proMMP-9/CSPG heteromer. The position of the pro-MMP-9 homodimer (225 kDa) is shown. B: Cells were incubated with [35S]sulphate. The harvested serum free medium (open bars) and the lysed cell preparations (grey bars) were passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The entire pass through fraction that contains the labelled macromolecules was then counted in a liquid scintillation spectrometer. All results (mean ± s.d) were normalized against the controls, i.e. the synthesis in absence of added compounds. In lower panel with ConA and MCSF, the results were in addition normalized against the number of viable cells. The results in (B) are from a typical experiment with four parallels.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105995&req=5

pone-0020616-g008: Effects of various compounds on the synthesis of proMMP-9/CSPG and CSPG.THP-1 cells were incubated in the absence or presence of the compounds shown in serum free medium for 72 h. In (A), the harvested medium was applied to Q-Sepharose chromatography and the presence of proMMP-9/CSPG was detected with gelatin zymography as described in Materials and Methods. Arrowhead shows the border between the separating and stacking gel and arrow shows the position of the 300 kDa proMMP-9/CSPG heteromer. The position of the pro-MMP-9 homodimer (225 kDa) is shown. B: Cells were incubated with [35S]sulphate. The harvested serum free medium (open bars) and the lysed cell preparations (grey bars) were passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The entire pass through fraction that contains the labelled macromolecules was then counted in a liquid scintillation spectrometer. All results (mean ± s.d) were normalized against the controls, i.e. the synthesis in absence of added compounds. In lower panel with ConA and MCSF, the results were in addition normalized against the number of viable cells. The results in (B) are from a typical experiment with four parallels.

Mentions: In order to determine to what extent the heteromer biosynthesis could be induced by other biological factors than PMA, we investigated the effect of factors known to enhance MMP synthesis in various cell lines. Neither of the investigated factors did trigger a significant change in the synthesis of the proMMP-9/CSPG heteromer (Fig. 8A), although most of these compounds (TNF-α, M-CSF, IL-1α, IL-1β, IL-3, IL-6, LPS, PGE2 and ConA) caused an increase in the biosynthesis of proMMP-9 in a concentration dependent manner (data not shown). However, their stimulatory effect on proMMP-9 synthesis was 5–20 times less than the effect of PMA. No effect on the proMMP-9 synthesis was obtained with bFGF (data not shown).


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)

Effects of various compounds on the synthesis of proMMP-9/CSPG and CSPG.THP-1 cells were incubated in the absence or presence of the compounds shown in serum free medium for 72 h. In (A), the harvested medium was applied to Q-Sepharose chromatography and the presence of proMMP-9/CSPG was detected with gelatin zymography as described in Materials and Methods. Arrowhead shows the border between the separating and stacking gel and arrow shows the position of the 300 kDa proMMP-9/CSPG heteromer. The position of the pro-MMP-9 homodimer (225 kDa) is shown. B: Cells were incubated with [35S]sulphate. The harvested serum free medium (open bars) and the lysed cell preparations (grey bars) were passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The entire pass through fraction that contains the labelled macromolecules was then counted in a liquid scintillation spectrometer. All results (mean ± s.d) were normalized against the controls, i.e. the synthesis in absence of added compounds. In lower panel with ConA and MCSF, the results were in addition normalized against the number of viable cells. The results in (B) are from a typical experiment with four parallels.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020616-g008: Effects of various compounds on the synthesis of proMMP-9/CSPG and CSPG.THP-1 cells were incubated in the absence or presence of the compounds shown in serum free medium for 72 h. In (A), the harvested medium was applied to Q-Sepharose chromatography and the presence of proMMP-9/CSPG was detected with gelatin zymography as described in Materials and Methods. Arrowhead shows the border between the separating and stacking gel and arrow shows the position of the 300 kDa proMMP-9/CSPG heteromer. The position of the pro-MMP-9 homodimer (225 kDa) is shown. B: Cells were incubated with [35S]sulphate. The harvested serum free medium (open bars) and the lysed cell preparations (grey bars) were passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The entire pass through fraction that contains the labelled macromolecules was then counted in a liquid scintillation spectrometer. All results (mean ± s.d) were normalized against the controls, i.e. the synthesis in absence of added compounds. In lower panel with ConA and MCSF, the results were in addition normalized against the number of viable cells. The results in (B) are from a typical experiment with four parallels.
Mentions: In order to determine to what extent the heteromer biosynthesis could be induced by other biological factors than PMA, we investigated the effect of factors known to enhance MMP synthesis in various cell lines. Neither of the investigated factors did trigger a significant change in the synthesis of the proMMP-9/CSPG heteromer (Fig. 8A), although most of these compounds (TNF-α, M-CSF, IL-1α, IL-1β, IL-3, IL-6, LPS, PGE2 and ConA) caused an increase in the biosynthesis of proMMP-9 in a concentration dependent manner (data not shown). However, their stimulatory effect on proMMP-9 synthesis was 5–20 times less than the effect of PMA. No effect on the proMMP-9 synthesis was obtained with bFGF (data not shown).

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