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. 2001 Nov 1;29(21):4361-72.
doi: 10.1093/nar/29.21.4361.

IL-1 induces collagenase-3 (MMP-13) promoter activity in stably transfected chondrocytic cells: requirement for Runx-2 and activation by p38 MAPK and JNK pathways

J A Mengshol  1 , M P Vincenti, C E Brinckerhoff
Affiliations

IL-1 induces collagenase-3 (MMP-13) promoter activity in stably transfected chondrocytic cells: requirement for Runx-2 and activation by p38 MAPK and JNK pathways

J A Mengshol et al. Nucleic Acids Res. .

Abstract

Osteoarthritic chondrocytes secrete matrix metalloproteinase-13 (MMP-13) in response to interleukin-1 (IL-1), causing digestion of type II collagen in cartilage. Using chondrocytic cells, we previously determined that IL-1 induced a strong MMP-13 transcriptional response that requires p38 MAPK, JNK and the transcription factor NF-kappaB. Now, we have studied the tissue-specific transcriptional regulation of MMP-13. Constitutive expression of the transcription factor Runx-2 correlated with the ability of a cell type to express MMP-13 and was required for IL-1 induction; moreover, Runx-2 enhanced IL-1 induction of MMP-13 transcription by synergizing with the p38 MAPK signaling pathway. Transiently transfected MMP-13 promoters were not IL-1 inducible. However, -405 bp of stably integrated promoter was sufficient for 5- to 6-fold IL-1 induction of reporter activity and this integrated reporter required the same p38 MAPK pathway as the endogenous gene. Finally, mutation of the proximal Runx binding site and the proximal AP-1 site blunted the transcriptional response to IL-1, and double mutation synergistically decreased reporter activity. In summary, our data suggest that the transcriptional MMP-13 response to IL-1 is controlled by the p38 pathway interacting at the MMP-13 promoter through the tissue-specific transcription factor Runx-2 and the ubiquitous AP-1 transcription factor.

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Figures

Figure 1
Figure 1
Homology alignment of the proximal MMP-13 promoters from mouse, rabbit and human sequence. The numbering is based on the start of transcription mapped in IL-1-treated OA chondrocytes (36). Conserved AP-1, Runx and Ets sites are in bold; the arrow denotes the human transcriptional start site. The translational start site is highlighted in bold from nucleotide +22 to +24. Alignment was made using GeneInspector (Textco Inc.)
Figure 2
Figure 2
Runx-2 expression correlates with IL-1 inducibility of MMP-13. Total RNA was harvested from SW-1353 cells, rabbit fibroblasts or human foreskin fibroblasts after treatment for the times indicated. Aliquots of 10 µg were analyzed by northern blot analysis using a 32P-labled probe from the 3′-end of the mouse Runx-2 cDNA. rRNA is shown as a loading control. Band intensities were compared with NIH Image software.
Figure 3
Figure 3
Runt domain expression specifically inhibits IL-1 induction of endogenous MMP-13. Samples of 3 × 105 SW-1353 cells were transfected with 1 µg empty pCMV-Tag5A expression plasmid, a CMV driven Runx-2 expression plasmid or pCMV-Tag5A-runt using 10 µl/well Geneporter reagent. After overnight recovery, cells were treated for 24 h with 2 ml of serum-free DMEM/LH or LH and 10 ng/ml IL-1β. Next, 1 ml of supernatant was TCA precipitated and analyzed by SDS–PAGE and western blotting. Polyclonal antibodies to either MMP-13 or MMP-1 were used to visualize protein. Band intensities were compared with NIH Image. A transfection efficiency of 50% was measured by transfection of the green fluorescent protein-expressing vector pEGFP and fluorescence microscopy (9).
Figure 4
Figure 4
(Opposite) ΔMEKK1 and MKK3 synergistically activate 405 bp of MMP-13 reporter through Runx-2. Samples of 2 × 105 SW-1353 cells were transfected with 500 ng of the proximal 405 bp of MMP-13 (A), of 5′-flanking DNA (B) or 4.3 kb of MMP-1 promoter (C), linked to the luciferase reporter gene in pGL3. Cells were co-transfected with 500 ng total of expression plasmid containing combinations of cDNAs for Runx-2, constitutively active ΔMEKK1 (Stratagene) or constitutively active MKK3 (26) driven by a CMV promoter. Total DNA was kept constant by addition of the empty pCMVTag5A expression plasmid. Aliquots of 10 µl of Geneporter (Gene Therapy Systems) were used to transfect the cells. Twenty-four hours after transfection, cells were placed in serum-free medium for 24 h or in 10 µM SB203580, cell extracts were made and assayed for luciferase activity. Units shown are relative light units (RLU). Individual treatments were performed in triplicate; error bars represent standard deviations. Values indicate fold induction over basal activity of the 405 bp construct.
Figure 4
Figure 4
(Opposite) ΔMEKK1 and MKK3 synergistically activate 405 bp of MMP-13 reporter through Runx-2. Samples of 2 × 105 SW-1353 cells were transfected with 500 ng of the proximal 405 bp of MMP-13 (A), of 5′-flanking DNA (B) or 4.3 kb of MMP-1 promoter (C), linked to the luciferase reporter gene in pGL3. Cells were co-transfected with 500 ng total of expression plasmid containing combinations of cDNAs for Runx-2, constitutively active ΔMEKK1 (Stratagene) or constitutively active MKK3 (26) driven by a CMV promoter. Total DNA was kept constant by addition of the empty pCMVTag5A expression plasmid. Aliquots of 10 µl of Geneporter (Gene Therapy Systems) were used to transfect the cells. Twenty-four hours after transfection, cells were placed in serum-free medium for 24 h or in 10 µM SB203580, cell extracts were made and assayed for luciferase activity. Units shown are relative light units (RLU). Individual treatments were performed in triplicate; error bars represent standard deviations. Values indicate fold induction over basal activity of the 405 bp construct.
Figure 4
Figure 4
(Opposite) ΔMEKK1 and MKK3 synergistically activate 405 bp of MMP-13 reporter through Runx-2. Samples of 2 × 105 SW-1353 cells were transfected with 500 ng of the proximal 405 bp of MMP-13 (A), of 5′-flanking DNA (B) or 4.3 kb of MMP-1 promoter (C), linked to the luciferase reporter gene in pGL3. Cells were co-transfected with 500 ng total of expression plasmid containing combinations of cDNAs for Runx-2, constitutively active ΔMEKK1 (Stratagene) or constitutively active MKK3 (26) driven by a CMV promoter. Total DNA was kept constant by addition of the empty pCMVTag5A expression plasmid. Aliquots of 10 µl of Geneporter (Gene Therapy Systems) were used to transfect the cells. Twenty-four hours after transfection, cells were placed in serum-free medium for 24 h or in 10 µM SB203580, cell extracts were made and assayed for luciferase activity. Units shown are relative light units (RLU). Individual treatments were performed in triplicate; error bars represent standard deviations. Values indicate fold induction over basal activity of the 405 bp construct.
Figure 5
Figure 5
(A) pGL3 MMP-13 reporter constructs. The numbering of constructs is based on the start of translation. 5′-Flanking DNA was cloned into pGL3 basic by PCR (181 and 405 bp constructs) and rapid amplification of genomic DNA ends (RAGE) (1.6 and 3.4 kb constructs). The 8.0 kb 5′-flanking region was subcloned from a pBetaGal construct graciously provided by Karen Hasty (University of Tennessee). (B) Transient transfection of 181 and 405 bp of MMP-13 5′-flanking DNA into SW-1353 cells. Samples of 2 × 105 SW-1353 cells were transfected with 1 µg pGL3 luciferase reporter construct containing MMP-13 5′-flanking DNA using 10 µl of Geneporter. (C) Transient transfection of 4.3 kb of MMP-1 5′-flanking DNA. Samples of 2 × 105 SW-1353 cells were transfected with 1 µg pGL3 luciferase reporter construct containing 4.3 kb of MMP-1 5′-flanking DNA (37). Cells were treated with 10 ng/ml IL-1β for 24 h. In all three experiments, luciferase activity was assayed in a luminometer and expressed as RLU. Error bars denote standard deviations.
Figure 5
Figure 5
(A) pGL3 MMP-13 reporter constructs. The numbering of constructs is based on the start of translation. 5′-Flanking DNA was cloned into pGL3 basic by PCR (181 and 405 bp constructs) and rapid amplification of genomic DNA ends (RAGE) (1.6 and 3.4 kb constructs). The 8.0 kb 5′-flanking region was subcloned from a pBetaGal construct graciously provided by Karen Hasty (University of Tennessee). (B) Transient transfection of 181 and 405 bp of MMP-13 5′-flanking DNA into SW-1353 cells. Samples of 2 × 105 SW-1353 cells were transfected with 1 µg pGL3 luciferase reporter construct containing MMP-13 5′-flanking DNA using 10 µl of Geneporter. (C) Transient transfection of 4.3 kb of MMP-1 5′-flanking DNA. Samples of 2 × 105 SW-1353 cells were transfected with 1 µg pGL3 luciferase reporter construct containing 4.3 kb of MMP-1 5′-flanking DNA (37). Cells were treated with 10 ng/ml IL-1β for 24 h. In all three experiments, luciferase activity was assayed in a luminometer and expressed as RLU. Error bars denote standard deviations.
Figure 5
Figure 5
(A) pGL3 MMP-13 reporter constructs. The numbering of constructs is based on the start of translation. 5′-Flanking DNA was cloned into pGL3 basic by PCR (181 and 405 bp constructs) and rapid amplification of genomic DNA ends (RAGE) (1.6 and 3.4 kb constructs). The 8.0 kb 5′-flanking region was subcloned from a pBetaGal construct graciously provided by Karen Hasty (University of Tennessee). (B) Transient transfection of 181 and 405 bp of MMP-13 5′-flanking DNA into SW-1353 cells. Samples of 2 × 105 SW-1353 cells were transfected with 1 µg pGL3 luciferase reporter construct containing MMP-13 5′-flanking DNA using 10 µl of Geneporter. (C) Transient transfection of 4.3 kb of MMP-1 5′-flanking DNA. Samples of 2 × 105 SW-1353 cells were transfected with 1 µg pGL3 luciferase reporter construct containing 4.3 kb of MMP-1 5′-flanking DNA (37). Cells were treated with 10 ng/ml IL-1β for 24 h. In all three experiments, luciferase activity was assayed in a luminometer and expressed as RLU. Error bars denote standard deviations.
Figure 6
Figure 6
Stable transfectants of MMP-13 5′-flanking DNA reporter constructs respond to IL-1β. Stably transfected SW-1353 cells containing 405 bp of MMP-13 5′-flanking DNA were created by co-transfecting cells with a reporter construct and the pCMV-Tag5A vector expressing the neomycin resistance gene, followed by 2 months of selection in G418. To assay the integrated reporter constructs, 2 × 105 cells derived from single cell clones were plated and treated with 10 ng/ml IL-1β for 24 h. Luciferase activity was assayed and plotted as RLU. (A) The 405 bp stably integrated reporter constructs are inhibited by the same MAPK inhibitors as the endogenous MMP-13 gene. A 405 bp clone was treated with 10 ng/ml IL-1β for 24 h with and without 40 µM MEK inhibitor PD98059 or 10 µM p38 inhibitor SB203580. Luciferase activity was assayed and plotted as RLU. (B) IL-1 synergizes with Runx-2 in stable transfectants. Samples of 2 × 105 SW-1353 cells stably transfected with 405 bp of MMP-13 promoter DNA were transiently transfected with 1 µg pCMVTag5A or pCMV Runx-2 using 10 µl of Geneporter. Twenty-four hours after transfection, cells were treated with 10 ng/ml IL-1 for 24 h and/or 10 µM SB203580 in serum-free medium. All treatments were done in triplicate; cells were harvested and assayed for luciferase activity. Luciferase activity is shown as RLU; error bars represent standard deviations. Values indicate fold induction over control.
Figure 6
Figure 6
Stable transfectants of MMP-13 5′-flanking DNA reporter constructs respond to IL-1β. Stably transfected SW-1353 cells containing 405 bp of MMP-13 5′-flanking DNA were created by co-transfecting cells with a reporter construct and the pCMV-Tag5A vector expressing the neomycin resistance gene, followed by 2 months of selection in G418. To assay the integrated reporter constructs, 2 × 105 cells derived from single cell clones were plated and treated with 10 ng/ml IL-1β for 24 h. Luciferase activity was assayed and plotted as RLU. (A) The 405 bp stably integrated reporter constructs are inhibited by the same MAPK inhibitors as the endogenous MMP-13 gene. A 405 bp clone was treated with 10 ng/ml IL-1β for 24 h with and without 40 µM MEK inhibitor PD98059 or 10 µM p38 inhibitor SB203580. Luciferase activity was assayed and plotted as RLU. (B) IL-1 synergizes with Runx-2 in stable transfectants. Samples of 2 × 105 SW-1353 cells stably transfected with 405 bp of MMP-13 promoter DNA were transiently transfected with 1 µg pCMVTag5A or pCMV Runx-2 using 10 µl of Geneporter. Twenty-four hours after transfection, cells were treated with 10 ng/ml IL-1 for 24 h and/or 10 µM SB203580 in serum-free medium. All treatments were done in triplicate; cells were harvested and assayed for luciferase activity. Luciferase activity is shown as RLU; error bars represent standard deviations. Values indicate fold induction over control.
Figure 7
Figure 7
Mutational analysis of the MMP-13 promoter using stable integrants. Pooled stable cell lines were created by transfection with the reporter constructs indicated followed by 1 month of weekly passages and selection in G418. Cells were plated at 2 × 105 per well in 6-well plates and treated with 10 ng/ml IL-1 in serum-free medium for 24 h. All treatments were done in triplicate; cells were harvested and assayed for luciferase activity. Luciferase activity is shown as RLU; error bars represent standard deviations. Values indicate fold IL-1 induction over basal expression for each construct.
Figure 8
Figure 8
IL-1 signaling to AP-1 and Runx-2 at the MMP-13 promoter. The model depicts the expression levels and roles of Runx and AP-1 proteins at the MMP-13 promoter in SW-1353 human chondrosarcoma cells and HFFs human foreskin fibroblasts. Arrows denote activation of signaling pathways or transcriptional activation at the MMP-13 promoter. The question mark refers to unidentified IL-1-responsive elements.
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