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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 and CSPG in the absence and presence of PMA.A–C: Typical gelatin zymographies of 20 times diluted conditioned medium from THP-1 cells. A: Cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. B: Cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M and 10−5 M PMA. 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. A–C: The position of proMMP-9 homodimer (225 kDa), monomer (92 kDa) and proMMP-2 (72 kDa) standards is indicated. D and E: Conditioned medium from THP-1 cells incubated with [35S]sulphate was passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The amount of labelled macromolecules was determined by counting the entire pass through fraction in a liquid scintillation spectrometer. D: Cells were incubated in the absence or presence of various concentrations of PMA for 72 h as indicated and in (E) the cells were incubated for various time periods in the absence or presence of 10−7 M PMA. (D: upper and lower panel) Results (mean ± s.d) were normalized against the control (without PMA). Lower panel, results were in addition normalized against the number of viable cells. E: Results (mean ± s.d) are presented as cpm (upper panel) and cpm/viable cells (lower panel). The results in (D) and (E) are from a typical experiment with four parallels in (D) and three parallels in (E), where *p<0.05 compared to control without PMA.
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pone-0020616-g003: Synthesis of proMMP-9 and CSPG in the absence and presence of PMA.A–C: Typical gelatin zymographies of 20 times diluted conditioned medium from THP-1 cells. A: Cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. B: Cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M and 10−5 M PMA. 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. A–C: The position of proMMP-9 homodimer (225 kDa), monomer (92 kDa) and proMMP-2 (72 kDa) standards is indicated. D and E: Conditioned medium from THP-1 cells incubated with [35S]sulphate was passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The amount of labelled macromolecules was determined by counting the entire pass through fraction in a liquid scintillation spectrometer. D: Cells were incubated in the absence or presence of various concentrations of PMA for 72 h as indicated and in (E) the cells were incubated for various time periods in the absence or presence of 10−7 M PMA. (D: upper and lower panel) Results (mean ± s.d) were normalized against the control (without PMA). Lower panel, results were in addition normalized against the number of viable cells. E: Results (mean ± s.d) are presented as cpm (upper panel) and cpm/viable cells (lower panel). The results in (D) and (E) are from a typical experiment with four parallels in (D) and three parallels in (E), where *p<0.05 compared to control without PMA.

Mentions: PMA stimulation of the THP-1 cells resulted in a concentration (Fig. 3A) and time (Fig. 3B) dependent increase of proMMP-9 in the cell conditioned media. Likewise, the PMA induced synthesis of proMMP-9 continued after the removal of the phorbol ester (Fig. 3C). Whether pre-incubation of the cells with PMA occurred in serum or serum-free media had no detectable effect on the biosynthesis of the proMMP-9 monomer and homodimer (Fig. 3C).


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 and CSPG in the absence and presence of PMA.A–C: Typical gelatin zymographies of 20 times diluted conditioned medium from THP-1 cells. A: Cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. B: Cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M and 10−5 M PMA. 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. A–C: The position of proMMP-9 homodimer (225 kDa), monomer (92 kDa) and proMMP-2 (72 kDa) standards is indicated. D and E: Conditioned medium from THP-1 cells incubated with [35S]sulphate was passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The amount of labelled macromolecules was determined by counting the entire pass through fraction in a liquid scintillation spectrometer. D: Cells were incubated in the absence or presence of various concentrations of PMA for 72 h as indicated and in (E) the cells were incubated for various time periods in the absence or presence of 10−7 M PMA. (D: upper and lower panel) Results (mean ± s.d) were normalized against the control (without PMA). Lower panel, results were in addition normalized against the number of viable cells. E: Results (mean ± s.d) are presented as cpm (upper panel) and cpm/viable cells (lower panel). The results in (D) and (E) are from a typical experiment with four parallels in (D) and three parallels in (E), where *p<0.05 compared to control without PMA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020616-g003: Synthesis of proMMP-9 and CSPG in the absence and presence of PMA.A–C: Typical gelatin zymographies of 20 times diluted conditioned medium from THP-1 cells. A: Cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. B: Cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M and 10−5 M PMA. 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. A–C: The position of proMMP-9 homodimer (225 kDa), monomer (92 kDa) and proMMP-2 (72 kDa) standards is indicated. D and E: Conditioned medium from THP-1 cells incubated with [35S]sulphate was passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The amount of labelled macromolecules was determined by counting the entire pass through fraction in a liquid scintillation spectrometer. D: Cells were incubated in the absence or presence of various concentrations of PMA for 72 h as indicated and in (E) the cells were incubated for various time periods in the absence or presence of 10−7 M PMA. (D: upper and lower panel) Results (mean ± s.d) were normalized against the control (without PMA). Lower panel, results were in addition normalized against the number of viable cells. E: Results (mean ± s.d) are presented as cpm (upper panel) and cpm/viable cells (lower panel). The results in (D) and (E) are from a typical experiment with four parallels in (D) and three parallels in (E), where *p<0.05 compared to control without PMA.
Mentions: PMA stimulation of the THP-1 cells resulted in a concentration (Fig. 3A) and time (Fig. 3B) dependent increase of proMMP-9 in the cell conditioned media. Likewise, the PMA induced synthesis of proMMP-9 continued after the removal of the phorbol ester (Fig. 3C). Whether pre-incubation of the cells with PMA occurred in serum or serum-free media had no detectable effect on the biosynthesis of the proMMP-9 monomer and homodimer (Fig. 3C).

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