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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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PKC and PKD isoenzymes expressed in THP-1 cells.RT-PCR was used to detect the PKC and PKD isoenzyme mRNA present in cells incubated in the absence (−) or presence (+) of 10-7 M PMA. To ensure that equal amounts of cDNA has been used in samples from control and PMA stimulated cells, rRNA was used as a standard. The probes used are described in Materials and Methods. Ladder is a marker with DNA fragments of known size (in bp).
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pone-0020616-g004: PKC and PKD isoenzymes expressed in THP-1 cells.RT-PCR was used to detect the PKC and PKD isoenzyme mRNA present in cells incubated in the absence (−) or presence (+) of 10-7 M PMA. To ensure that equal amounts of cDNA has been used in samples from control and PMA stimulated cells, rRNA was used as a standard. The probes used are described in Materials and Methods. Ladder is a marker with DNA fragments of known size (in bp).

Mentions: Previously it has been shown that undifferentiated THP-1 cells express the classical PKC isoforms α and β, novel PKC isoforms δ and ε and the atypical PKC ζ [50]. The same authors showed that PMA changed the expression of some of these PKC isoforms. As PMA is known to induce activation of various classical and novel PKC isoforms [51], we have reinvestigated at the mRNA level which isoforms are produced by THP-1 cells in the presence and absence of PMA. This has extended the list of PKC isoforms present in these cells. As shown in Figure 4, both untreated and PMA treated THP-1 cells contained mRNA for classical PKC isoforms α, βI and βII, novel PKC isoforms δ, ε and θ, atypical PKC ζ and ι as well as for PKD3 (PKCυ). The signals for PKC isoforms θ and γ were weak, and no mRNA could be detected for the PKC η, and the PKD isoforms 1 (PKC μ) and 2 (Fig. 4). As also shown in Figure 4, exposure to PMA did not seem to affect the mRNA levels of the PKC isoforms in THP-1 cells.


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)

PKC and PKD isoenzymes expressed in THP-1 cells.RT-PCR was used to detect the PKC and PKD isoenzyme mRNA present in cells incubated in the absence (−) or presence (+) of 10-7 M PMA. To ensure that equal amounts of cDNA has been used in samples from control and PMA stimulated cells, rRNA was used as a standard. The probes used are described in Materials and Methods. Ladder is a marker with DNA fragments of known size (in bp).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020616-g004: PKC and PKD isoenzymes expressed in THP-1 cells.RT-PCR was used to detect the PKC and PKD isoenzyme mRNA present in cells incubated in the absence (−) or presence (+) of 10-7 M PMA. To ensure that equal amounts of cDNA has been used in samples from control and PMA stimulated cells, rRNA was used as a standard. The probes used are described in Materials and Methods. Ladder is a marker with DNA fragments of known size (in bp).
Mentions: Previously it has been shown that undifferentiated THP-1 cells express the classical PKC isoforms α and β, novel PKC isoforms δ and ε and the atypical PKC ζ [50]. The same authors showed that PMA changed the expression of some of these PKC isoforms. As PMA is known to induce activation of various classical and novel PKC isoforms [51], we have reinvestigated at the mRNA level which isoforms are produced by THP-1 cells in the presence and absence of PMA. This has extended the list of PKC isoforms present in these cells. As shown in Figure 4, both untreated and PMA treated THP-1 cells contained mRNA for classical PKC isoforms α, βI and βII, novel PKC isoforms δ, ε and θ, atypical PKC ζ and ι as well as for PKD3 (PKCυ). The signals for PKC isoforms θ and γ were weak, and no mRNA could be detected for the PKC η, and the PKD isoforms 1 (PKC μ) and 2 (Fig. 4). As also shown in Figure 4, exposure to PMA did not seem to affect the mRNA levels of the PKC isoforms in THP-1 cells.

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