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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 Rottlerin on cytosolic and plasma membrane bound PKC isoenzymes.Isolated cytosol and plasma membranes from untreated (−) or PMA and Rottlerin treated (+) THP-1 cells were subjected to Western blotting using PKC specific antibodies. The amount of total protein loaded (PL) to each well is shown. As an additional loading control, ERK2 was used. The diagram under each blot shows the relative amounts (mean ±s.d.) of the PKC isoenzymes in cytosol and membranes, where the results were normalised against the untreated cytosol and membrane controls. All results are based on equal protein loading and in all cases n = 2 except for PKC δ and υ (PKD3) where n = 3. The arrowhead shows the active PKD3 at 100 kDa, while the two bands with reduced molecular size may be truncated variants of PKD3. The position of the molecular mass markers at 100 and 80 kDa are shown at the left. In order to show the increased level of PKC ε in the cytosol from PMA treated cells (353 µg/well) in the presence of Rottlerin compared to the absence of this compound, a largely increased developmental exposure time of the membrane in the presence of the Luminol substrate was needed.
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pone-0020616-g007: Effect of Rottlerin on cytosolic and plasma membrane bound PKC isoenzymes.Isolated cytosol and plasma membranes from untreated (−) or PMA and Rottlerin treated (+) THP-1 cells were subjected to Western blotting using PKC specific antibodies. The amount of total protein loaded (PL) to each well is shown. As an additional loading control, ERK2 was used. The diagram under each blot shows the relative amounts (mean ±s.d.) of the PKC isoenzymes in cytosol and membranes, where the results were normalised against the untreated cytosol and membrane controls. All results are based on equal protein loading and in all cases n = 2 except for PKC δ and υ (PKD3) where n = 3. The arrowhead shows the active PKD3 at 100 kDa, while the two bands with reduced molecular size may be truncated variants of PKD3. The position of the molecular mass markers at 100 and 80 kDa are shown at the left. In order to show the increased level of PKC ε in the cytosol from PMA treated cells (353 µg/well) in the presence of Rottlerin compared to the absence of this compound, a largely increased developmental exposure time of the membrane in the presence of the Luminol substrate was needed.

Mentions: The inhibitory effect of Rottlerin on the biosynthesis of proMMP-9, CSPG and proMMP-9/CSPG heteromer may be due to inhibition of PKC activation or factors downstream to PKC. We have therefore determined the amount of PKC in the cytosol and the plasma membrane fraction of untreated and PMA treated THP-1 cells. To ensure equal loading, the total amount of cytosol and membrane proteins were determined as described in Materials and Methods. As shown in figure 7, PMA treatment of the cells resulted in decreased amounts of the classical and the novel PKCs in the cytosol as expected. PMA treatment resulted also in increased amounts of some PKCs to the plasma membranes. The presence of 5.0 µM Rottlerin resulted in a slight decrease in the amounts of the PKC isoenzymes δ, ε, θ and υ (PKD3; 100 kDa) bound to the plasma membranes in the PMA treated cells (Fig. 7). In the presence of Rottlerin, the PKC isoenzymes ε and θ showed a small increase in the cytosol of the PMA treated cells. In cells not exposed to PMA, the presence of Rottlerin resulted in a slightly increased amount of the PKC isoenzymes δ, ε and θ in the cytosol and a minor decrease in the plasma membrane (Fig. 7). MAPKAPK-2 is a mitogen-activated protein kinase expressed in THP-1 cells [56]. Because PMA also can stimulate the activity of MAPKAPK-2 [57], [58], and Rottlerin is a potent inhibitor of MAPKAPK-2 (IC50 = 5.4 µM; [59]), we examined whether PMA activated MAPKAPK-2 in THP-1 cells. No phosphorylation of MAPKAPK-2 at Thr-222 was detected in control cells or cell exposed to PMA, indicating that PMA did not activate MAPKAPK-2 in these cells (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)

Effect of Rottlerin on cytosolic and plasma membrane bound PKC isoenzymes.Isolated cytosol and plasma membranes from untreated (−) or PMA and Rottlerin treated (+) THP-1 cells were subjected to Western blotting using PKC specific antibodies. The amount of total protein loaded (PL) to each well is shown. As an additional loading control, ERK2 was used. The diagram under each blot shows the relative amounts (mean ±s.d.) of the PKC isoenzymes in cytosol and membranes, where the results were normalised against the untreated cytosol and membrane controls. All results are based on equal protein loading and in all cases n = 2 except for PKC δ and υ (PKD3) where n = 3. The arrowhead shows the active PKD3 at 100 kDa, while the two bands with reduced molecular size may be truncated variants of PKD3. The position of the molecular mass markers at 100 and 80 kDa are shown at the left. In order to show the increased level of PKC ε in the cytosol from PMA treated cells (353 µg/well) in the presence of Rottlerin compared to the absence of this compound, a largely increased developmental exposure time of the membrane in the presence of the Luminol substrate was needed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105995&req=5

pone-0020616-g007: Effect of Rottlerin on cytosolic and plasma membrane bound PKC isoenzymes.Isolated cytosol and plasma membranes from untreated (−) or PMA and Rottlerin treated (+) THP-1 cells were subjected to Western blotting using PKC specific antibodies. The amount of total protein loaded (PL) to each well is shown. As an additional loading control, ERK2 was used. The diagram under each blot shows the relative amounts (mean ±s.d.) of the PKC isoenzymes in cytosol and membranes, where the results were normalised against the untreated cytosol and membrane controls. All results are based on equal protein loading and in all cases n = 2 except for PKC δ and υ (PKD3) where n = 3. The arrowhead shows the active PKD3 at 100 kDa, while the two bands with reduced molecular size may be truncated variants of PKD3. The position of the molecular mass markers at 100 and 80 kDa are shown at the left. In order to show the increased level of PKC ε in the cytosol from PMA treated cells (353 µg/well) in the presence of Rottlerin compared to the absence of this compound, a largely increased developmental exposure time of the membrane in the presence of the Luminol substrate was needed.
Mentions: The inhibitory effect of Rottlerin on the biosynthesis of proMMP-9, CSPG and proMMP-9/CSPG heteromer may be due to inhibition of PKC activation or factors downstream to PKC. We have therefore determined the amount of PKC in the cytosol and the plasma membrane fraction of untreated and PMA treated THP-1 cells. To ensure equal loading, the total amount of cytosol and membrane proteins were determined as described in Materials and Methods. As shown in figure 7, PMA treatment of the cells resulted in decreased amounts of the classical and the novel PKCs in the cytosol as expected. PMA treatment resulted also in increased amounts of some PKCs to the plasma membranes. The presence of 5.0 µM Rottlerin resulted in a slight decrease in the amounts of the PKC isoenzymes δ, ε, θ and υ (PKD3; 100 kDa) bound to the plasma membranes in the PMA treated cells (Fig. 7). In the presence of Rottlerin, the PKC isoenzymes ε and θ showed a small increase in the cytosol of the PMA treated cells. In cells not exposed to PMA, the presence of Rottlerin resulted in a slightly increased amount of the PKC isoenzymes δ, ε and θ in the cytosol and a minor decrease in the plasma membrane (Fig. 7). MAPKAPK-2 is a mitogen-activated protein kinase expressed in THP-1 cells [56]. Because PMA also can stimulate the activity of MAPKAPK-2 [57], [58], and Rottlerin is a potent inhibitor of MAPKAPK-2 (IC50 = 5.4 µM; [59]), we examined whether PMA activated MAPKAPK-2 in THP-1 cells. No phosphorylation of MAPKAPK-2 at Thr-222 was detected in control cells or cell exposed to PMA, indicating that PMA did not activate MAPKAPK-2 in these cells (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