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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 biosynthesis and molecular size of CSPG and the CS-chains.A, B: Cells in the absence (control) or presence of 10−7 M PMA were incubated with increasing concentrations of Rottlerin for 8 h and 23 h in serum free medium containing either [35S]sulphate (A) or [3H]glucosamine (B). A typical experiment is shown, where the results (total cpm not adjusted to the amount of living cells) are presented as mean ± s.d. (n = 2). C–F: Cells were incubated for 24 h in serum free medium containing either [35S]sulphate or [3H]glucosamine. C: [35S]sulphate and [3H]glucosamine labelled macromolecules were applied to Q-Sepharose chromatography and the bound CSPG were eluted with a 0.15–1.5 M NaCl gradient (----) as shown in the upper graph. ○, absence of Rottlerin; Δ, presence of 1 µM Rottlerin. D, E: Eluted CSPG from the Q-Sepharose column was either untreated (solid line) or treated with cABC (---) or 0.5 M NaOH (…….) prior to application on a Superose 6 gel chromatography column as described in Material and Methods. Cont., control without Rottlerin; Rotl., presence of 1 µM Rottlerin and Fr.No., fraction number. F: [35S]CSPG (•) and free [35S]CS-chains (○) from control and Rottlerin-treated cells were subjected to Q-Sepharose chromatography as in (C). Arrow shows the elution position of shark cartilage CS. G: [35S]Sulphate labelled CSPG (isolated from control, PMA (10−7 M) and Rottlerin treated cells) was subjected to SDS-PAGE (upper panel: 4% stacking gel and 7.5% separating gel; lower panel: 4–12% gradient gel) followed by autoradiography (see Materials and Methods). An equal amount of radioactivity (based on scintillation counting) was loaded to each well in order to be able to compare the bands. Arrowhead shows the border between the separating and stacking gel and the position of molecular size markers are shown. Small arrow shows the bottom of the application well.
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pone-0020616-g006: Effect of Rottlerin on biosynthesis and molecular size of CSPG and the CS-chains.A, B: Cells in the absence (control) or presence of 10−7 M PMA were incubated with increasing concentrations of Rottlerin for 8 h and 23 h in serum free medium containing either [35S]sulphate (A) or [3H]glucosamine (B). A typical experiment is shown, where the results (total cpm not adjusted to the amount of living cells) are presented as mean ± s.d. (n = 2). C–F: Cells were incubated for 24 h in serum free medium containing either [35S]sulphate or [3H]glucosamine. C: [35S]sulphate and [3H]glucosamine labelled macromolecules were applied to Q-Sepharose chromatography and the bound CSPG were eluted with a 0.15–1.5 M NaCl gradient (----) as shown in the upper graph. ○, absence of Rottlerin; Δ, presence of 1 µM Rottlerin. D, E: Eluted CSPG from the Q-Sepharose column was either untreated (solid line) or treated with cABC (---) or 0.5 M NaOH (…….) prior to application on a Superose 6 gel chromatography column as described in Material and Methods. Cont., control without Rottlerin; Rotl., presence of 1 µM Rottlerin and Fr.No., fraction number. F: [35S]CSPG (•) and free [35S]CS-chains (○) from control and Rottlerin-treated cells were subjected to Q-Sepharose chromatography as in (C). Arrow shows the elution position of shark cartilage CS. G: [35S]Sulphate labelled CSPG (isolated from control, PMA (10−7 M) and Rottlerin treated cells) was subjected to SDS-PAGE (upper panel: 4% stacking gel and 7.5% separating gel; lower panel: 4–12% gradient gel) followed by autoradiography (see Materials and Methods). An equal amount of radioactivity (based on scintillation counting) was loaded to each well in order to be able to compare the bands. Arrowhead shows the border between the separating and stacking gel and the position of molecular size markers are shown. Small arrow shows the bottom of the application well.

Mentions: We have previously shown that ∼0.1% of the total amount of CSPGs sythesized by the THP-1 cells are proMMP-9/CSPG heteromers [39]. Therefore, biosynthetic labelling of the CS-chains with either [35S]sulphate or [3H]glucosamine in the absence or presence of PMA and Rottlerin will reflect the latter compounds effect on the cells biosynthesis of CSPGs. The results revealed that the biosynthesis of the second component of the complex, the CSPG, was also inhibited by Rottlerin in a concentration dependent manner. As shown in figures 6(A) and 6(B), the conditioned culture medium from cells exposed to Rottlerin contained less amounts of the [35S]sulphate and [3H]glucosamine labelled macromolecules than medium from cells not treated with Rottlerin. This effect of Rottlerin was seen in both the untreated and PMA treated cells. Since PGs are highly negatively charged molecules, the radioactively labelled macromolecules were subjected to Q-Sepharose chromatography. As shown in Figure 6C (upper panel), all the 35S-labelled material was PGs as it was eluted with 0.7 M NaCl. Rottlerin did not affect the position of the eluted material, indicating that it did not affect the sulphate density of the GAG-chains. The use of 3H-labelled glucosamine showed that the majority of the radioactive sugar is incorporated in the PGs (Fig. 6C, lower panel), but a small amount is also eluted at around 0.3 M NaCl which is most likely hyaluronan and non-PG glycoproteins. The fractions eluted from the Q-Sepharose column at the position of PGs (∼0.7 M NaCl) were pooled and divided in three parts. One was treated with cABC to degrade CS-chains, another was treated with NaOH to liberate the CS-chains from the core protein and the third part was untreated (control). These fractions were applied to a Superose 6 column. As seen in Figures 6(D) and 6(E), cABC totally degraded the GAG-chains to disaccharides showing that the radioactively labelled PGs were substituted with CS-chains. Furthermore, Rottlerin did not affect the type of GAG-chains synthesized. However, the size of the PGs synthesized by Rottlerin exposed cells was smaller than the PGs synthesized by the control cells. NaOH treatment of the material shows that also the size of the CS-chains was smaller in the Rottlerin treated material. Since free CS-chains with a molecular size less than 15 kDa would be expected to elute from the Q-Sepharose column at a lower NaCl concentration than CS-chains with equal charge density but with molecular size >20 kDa [54], [55], several experiments were performed with material from both the control and Rottlerin-exposed cells where the CSPG had been treated or not treated with NaOH. Figure 6F shows that the elution profile of the free CS-chains of the Rottlerin-treated and untreated materials superimposed on the corresponding intact PGs. In all cases, gel filtration on a Superose 6 column revealed that the CSPG from the Rottlerin exposed cells had a reduced size compared to the CSPG from the untreated cells, although the reduction in size varied from experiment to experiment. Thus, if there were some free CS-chains in the Rottlerin-treated material it was not possible to separate these chains from intact CSPGs by Q-Sepharose column chromatography at least during the conditions used in the present work. The reduced size of the CSPG from Rottlerin exposed cells compared to CSPG from the control cells was not detected when [35S]CSPG was subjected to SDS-PAGE (7.5% acrylamide) followed by autoradiography (Fig. 6F, upper panel). Both bands appeared at the top of the separating gel at a position that corresponded with the proMMP-9/CSPG complex seen in gelatin zymography. However when a 4–12% gradient gel was used, the reduced size of the CSPG from Rottlerin exposed cells was detected in spite of the smeared bands that appear due to the heterogenous size of the CS-chains (Fig. 6F, lower panel). In summary, Rottlerin treatment of the THP-1 cells was followed by a reduced synthesis of the proMMP-9/CSPG heteromer, both components in the heteromer as well as a reduced size of the CSPG and its CS chains.


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 biosynthesis and molecular size of CSPG and the CS-chains.A, B: Cells in the absence (control) or presence of 10−7 M PMA were incubated with increasing concentrations of Rottlerin for 8 h and 23 h in serum free medium containing either [35S]sulphate (A) or [3H]glucosamine (B). A typical experiment is shown, where the results (total cpm not adjusted to the amount of living cells) are presented as mean ± s.d. (n = 2). C–F: Cells were incubated for 24 h in serum free medium containing either [35S]sulphate or [3H]glucosamine. C: [35S]sulphate and [3H]glucosamine labelled macromolecules were applied to Q-Sepharose chromatography and the bound CSPG were eluted with a 0.15–1.5 M NaCl gradient (----) as shown in the upper graph. ○, absence of Rottlerin; Δ, presence of 1 µM Rottlerin. D, E: Eluted CSPG from the Q-Sepharose column was either untreated (solid line) or treated with cABC (---) or 0.5 M NaOH (…….) prior to application on a Superose 6 gel chromatography column as described in Material and Methods. Cont., control without Rottlerin; Rotl., presence of 1 µM Rottlerin and Fr.No., fraction number. F: [35S]CSPG (•) and free [35S]CS-chains (○) from control and Rottlerin-treated cells were subjected to Q-Sepharose chromatography as in (C). Arrow shows the elution position of shark cartilage CS. G: [35S]Sulphate labelled CSPG (isolated from control, PMA (10−7 M) and Rottlerin treated cells) was subjected to SDS-PAGE (upper panel: 4% stacking gel and 7.5% separating gel; lower panel: 4–12% gradient gel) followed by autoradiography (see Materials and Methods). An equal amount of radioactivity (based on scintillation counting) was loaded to each well in order to be able to compare the bands. Arrowhead shows the border between the separating and stacking gel and the position of molecular size markers are shown. Small arrow shows the bottom of the application well.
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Related In: Results  -  Collection

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

pone-0020616-g006: Effect of Rottlerin on biosynthesis and molecular size of CSPG and the CS-chains.A, B: Cells in the absence (control) or presence of 10−7 M PMA were incubated with increasing concentrations of Rottlerin for 8 h and 23 h in serum free medium containing either [35S]sulphate (A) or [3H]glucosamine (B). A typical experiment is shown, where the results (total cpm not adjusted to the amount of living cells) are presented as mean ± s.d. (n = 2). C–F: Cells were incubated for 24 h in serum free medium containing either [35S]sulphate or [3H]glucosamine. C: [35S]sulphate and [3H]glucosamine labelled macromolecules were applied to Q-Sepharose chromatography and the bound CSPG were eluted with a 0.15–1.5 M NaCl gradient (----) as shown in the upper graph. ○, absence of Rottlerin; Δ, presence of 1 µM Rottlerin. D, E: Eluted CSPG from the Q-Sepharose column was either untreated (solid line) or treated with cABC (---) or 0.5 M NaOH (…….) prior to application on a Superose 6 gel chromatography column as described in Material and Methods. Cont., control without Rottlerin; Rotl., presence of 1 µM Rottlerin and Fr.No., fraction number. F: [35S]CSPG (•) and free [35S]CS-chains (○) from control and Rottlerin-treated cells were subjected to Q-Sepharose chromatography as in (C). Arrow shows the elution position of shark cartilage CS. G: [35S]Sulphate labelled CSPG (isolated from control, PMA (10−7 M) and Rottlerin treated cells) was subjected to SDS-PAGE (upper panel: 4% stacking gel and 7.5% separating gel; lower panel: 4–12% gradient gel) followed by autoradiography (see Materials and Methods). An equal amount of radioactivity (based on scintillation counting) was loaded to each well in order to be able to compare the bands. Arrowhead shows the border between the separating and stacking gel and the position of molecular size markers are shown. Small arrow shows the bottom of the application well.
Mentions: We have previously shown that ∼0.1% of the total amount of CSPGs sythesized by the THP-1 cells are proMMP-9/CSPG heteromers [39]. Therefore, biosynthetic labelling of the CS-chains with either [35S]sulphate or [3H]glucosamine in the absence or presence of PMA and Rottlerin will reflect the latter compounds effect on the cells biosynthesis of CSPGs. The results revealed that the biosynthesis of the second component of the complex, the CSPG, was also inhibited by Rottlerin in a concentration dependent manner. As shown in figures 6(A) and 6(B), the conditioned culture medium from cells exposed to Rottlerin contained less amounts of the [35S]sulphate and [3H]glucosamine labelled macromolecules than medium from cells not treated with Rottlerin. This effect of Rottlerin was seen in both the untreated and PMA treated cells. Since PGs are highly negatively charged molecules, the radioactively labelled macromolecules were subjected to Q-Sepharose chromatography. As shown in Figure 6C (upper panel), all the 35S-labelled material was PGs as it was eluted with 0.7 M NaCl. Rottlerin did not affect the position of the eluted material, indicating that it did not affect the sulphate density of the GAG-chains. The use of 3H-labelled glucosamine showed that the majority of the radioactive sugar is incorporated in the PGs (Fig. 6C, lower panel), but a small amount is also eluted at around 0.3 M NaCl which is most likely hyaluronan and non-PG glycoproteins. The fractions eluted from the Q-Sepharose column at the position of PGs (∼0.7 M NaCl) were pooled and divided in three parts. One was treated with cABC to degrade CS-chains, another was treated with NaOH to liberate the CS-chains from the core protein and the third part was untreated (control). These fractions were applied to a Superose 6 column. As seen in Figures 6(D) and 6(E), cABC totally degraded the GAG-chains to disaccharides showing that the radioactively labelled PGs were substituted with CS-chains. Furthermore, Rottlerin did not affect the type of GAG-chains synthesized. However, the size of the PGs synthesized by Rottlerin exposed cells was smaller than the PGs synthesized by the control cells. NaOH treatment of the material shows that also the size of the CS-chains was smaller in the Rottlerin treated material. Since free CS-chains with a molecular size less than 15 kDa would be expected to elute from the Q-Sepharose column at a lower NaCl concentration than CS-chains with equal charge density but with molecular size >20 kDa [54], [55], several experiments were performed with material from both the control and Rottlerin-exposed cells where the CSPG had been treated or not treated with NaOH. Figure 6F shows that the elution profile of the free CS-chains of the Rottlerin-treated and untreated materials superimposed on the corresponding intact PGs. In all cases, gel filtration on a Superose 6 column revealed that the CSPG from the Rottlerin exposed cells had a reduced size compared to the CSPG from the untreated cells, although the reduction in size varied from experiment to experiment. Thus, if there were some free CS-chains in the Rottlerin-treated material it was not possible to separate these chains from intact CSPGs by Q-Sepharose column chromatography at least during the conditions used in the present work. The reduced size of the CSPG from Rottlerin exposed cells compared to CSPG from the control cells was not detected when [35S]CSPG was subjected to SDS-PAGE (7.5% acrylamide) followed by autoradiography (Fig. 6F, upper panel). Both bands appeared at the top of the separating gel at a position that corresponded with the proMMP-9/CSPG complex seen in gelatin zymography. However when a 4–12% gradient gel was used, the reduced size of the CSPG from Rottlerin exposed cells was detected in spite of the smeared bands that appear due to the heterogenous size of the CS-chains (Fig. 6F, lower panel). In summary, Rottlerin treatment of the THP-1 cells was followed by a reduced synthesis of the proMMP-9/CSPG heteromer, both components in the heteromer as well as a reduced size of the CSPG and its CS chains.

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