Fig 1.
IL-1+OSM-induced MMP1 mRNA expression is preceded by nascent hnRNA formation.
Chondrocytes were stimulated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated durations. Total RNA was isolated, reverse transcribed and subjected to qPCR for (A) MMP1 mRNA or (B) MMP1 nascent hnRNA as described in the Materials and Methods. Data are expressed relative to 18S rRNA and presented as fold increase compared to basal expression. qPCR data (n = 4) are representative of at least three separate chondrocyte populations. All data are presented as mean (± SD), where ***, p<0.001; **, p<0.01; *, p<0.05 IL-1+OSM-treated compared to control; ANOVA.
Fig 2.
ChIP analyses of the MMP1 proximal promoter.
Human chondrocytes were treated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated durations. Cells were then subject to DNA-protein cross-linking, lysis and DNA shearing. Immuno-precipitation for cFOS (A,B), pRNA Pol II (C,D) or AcH3 (E) was followed by isolation of complexed genomic DNA and qPCR for the proximal (closed bars) and 3’-UTR (used for normalization; open bars) regions of MMP1 as indicated. Data (mean ± SD, n = 4) are pooled from at least three separate chondrocyte populations from different donors. Statistical comparisons are: #, p<0.05 (1 h IL-1+OSM stimulation versus basal); *, p<0.05 (24 h IL-1+OSM stimulation versus basal).
Fig 3.
IL-1+OSM-induced MMP1 expression is dependent on new protein synthesis.
Chondrocytes were treated with IL-1 (0.05 ng/mL) and OSM (10 ng/mL) for 24 h. Emetine (10 μM final concentration) was added at the indicated times after initiation of IL-1+OSM stimulation and qPCR performed on extracted RNA. Relative expression levels of MMP1 mRNA were normalized to 18S rRNA, where ###, p<0.001 (IL-1+OSM vs basal); ***, p<0.001 (IL-1+OSM+emetine vs basal). Data (mean ± SD, n = 6) are representative of three separate experiments each using chondrocyte cultures from different donors.
Fig 4.
IL-1+OSM-induced MMP1 expression is maximal at 24 h post-stimulation.
Human chondrocytes were treated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated time points and total RNA isolated. (A) RNA from three separate populations (#1, #2 and #3) was profiled using the Human HT-12v4 Expression Beadchip. The expression profile for MMP1 from the microarray dataset for each chondrocyte population is shown. (B) RNA from six separate chondrocyte populations was subjected to qPCR for MMP1, and relative expression levels (normalized to 18S rRNA) at the indicated times determined. The plots show mean ± SD (n = 6). Data from the populations used in the microarray analyses are highlighted by the solid symbols (#1, ▲; #2 ■; #3, ●). (C) Prior to stimulation with IL-1 (0.05 ng/ml) in combination with OSM (10 ng/ml), human chondrocytes were transfected with siRNA specific for the indicated genes, or a non-targeting control (siCon; all 100 nM), and mRNA expression levels (mean ± S.D., n = 6) of MMP1 measured 24 h post-stimulation by qPCR, relative to siCon treated cells, normalized to 18S rRNA. Statistical comparisons are versus IL-1 + OSM + siCon (Student’s two-tailed unpaired t test), where ***, p<0.001; **, p <0.01; *, p<0.05; ns = not significant. The data are pooled from three separate experiments, each using chondrocyte cultures from different donors.
Fig 5.
IL-1+OSM induces sustained expression of CSRNP1 mRNA.
Human chondrocytes were treated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated time points and total RNA isolated. (A) RNA from three separate populations (#1, #2 and #3) was profiled using the Human HT-12v4 Expression Beadchip. The expression profile for CSRNP1 from the microarray dataset for each chondrocyte population is shown. RNA from 6 (B) or 3 (C) separate chondrocyte populations was subjected to qPCR for FOS (C) or CSRNP1 (B and C), and relative expression levels (normalized to 18S rRNA or GAPDH as indicated) at the indicated times determined. The plots show mean ± SD. Data from the populations used in the microarray analyses in (A) are highlighted by the solid symbols (#1, ▲; #2 ■; #3, ●). (C) Statistical comparisons are: ***, p<0.001; **, p<0.01; *, p<0.05 versus basal.
Fig 6.
IL-1+OSM induces nuclear expression of CSRNP1.
Human chondrocytes were stimulated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated durations. Protein from either whole cell lysates (A) or subcellular fractions (C: a, cytosolic; b, membrane-bound; c, soluble nuclear; d, chromatin-bound; e, cytoskeletal) were resolved using SDS-PAGE and immunoblotted with an antibody to CSRNP1. Combined densitometric scans of separate blots for CSRNP1 in cell lysates (n = 3; B) or pooled soluble and chromatin-bound nuclear fractions (n = 5; D), relative to t = 0 h, are shown and were obtained from separate chondrocyte populations. All data are presented as mean (± SD), where **, p<0.01, IL-1+OSM-treated compared to control; ANOVA.
Fig 7.
CSRNP1 selectively binds the proximal MMP1 AP-1 motif at a transcriptionally active time-point in IL-1+OSM-stimulated chondrocytes.
Human chondrocytes were treated with IL-1 (0.2 ng/mL) in combination with OSM (10 ng/mL) for 3 h. Nuclear lysates were prepared and subjected to either Western blotting alone (input) or DAPA with CSRNP1 or ATF3 antibodies, as described in the Materials and Methods. Non-biotinylated AP-1 oligonucleotides (50x excess; Comp) were used to confirm specificity of binding in the DAPAs. The data are representative of 3–5 separate experiments each using chondrocyte cultures from different donors.