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IL-1 Stimulates Cathepsin K Expression in Osteoclasts via the Tyrosine Kinase-NF-B Pathway

¹ Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA; and ² Harvard-Forsyth Department of Oral Biology, The Forsyth Institute & Harvard School of Dental Medicine, Boston, MA 02115, USA;

View larger version (30K): [in this window] [in a new window]   Figure 1. Cathepsin K protein expression and bone resorption activity of osteoclasts are up-regulated by IL-1 in vitro. (A) Application of IL-1 (1.4 nM) to OCL cells resulted in a three- and four-fold increase in cathepsin K protein expression after 10 and 15 hours, respectively, as determined by ELISA assay. Experiments were performed in triplicate (N = 3), and 3 independent experiments yielded similar results (p < 0.05). (B) Demonstration of resorption pits formed by OCLs. Pre-formed OCLs were applied to dentin slices and cultured for 3 days in the absence (a) or presence (b) of IL-1. Cells were then removed and resorption pits stained with an anticollagen type I polyclonal antibody. (C) Application of IL-1 (1.4 nM) to mouse OCLs resulted in a 2.3-fold increase in Pit Area (N = 3).

View larger version (27K): [in this window] [in a new window]   Figure 2. Northern analysis of cathepsin K gene regulation. (A) Up-regulation of cathepsin K expression as determined by Northern blot analysis. IL-1 (1.4 nM) treated (6 hrs) or untreated total RNA (15 µg) from mature osteoclasts was used as the sample. The lower panels in A, C, E, and G represent hybridization of blots to GAPDH to standardize loading. (B) Relative abundance of cathepsin K mRNA on Northern blotting, as evaluated by densitometric scanning. *p < 0.05, compared with control. (C,D) Cathepsin K mRNA expression in response to increasing exposure to IL-1 (1.4 nM), as indicated. Controls cells were cultured for 6 hrs. (E,F) Cathepsin K mRNA expression in MOCP-5 cells after 6 hrs of exposure to increasing amounts of IL-1. (G,H) Northern blot analysis of cathepsin K mRNA expression in mature osteoclast cells (control, lane 1), and following IL-1 treatment for 6 hrs, preceded by 1 hr of pre-incubation with PBS (lane 2), Act D (5 µg/mL; lane 3), or CHX (10 µg/mL; lane 4). These figures represent at least 4 independent experiments (N = 4) that yielded similar results.

View larger version (22K): [in this window] [in a new window]   Figure 3. IL-1 increases cathepsin K promoter-CAT fusion gene activity, and NF-B activation inhibitor and tyrosine kinase inhibitor inhibit IL-1 induction cathepsin K expression. (A) MOCP-5 cells were transiently transfected with pMCCAT by means of the lipofectAMINE reagent, and reporter CAT activity was determined in premature and mature osteoclasts. A three-fold increase in CAT activity was detected after 7 hrs of IL-1 treatment. (B) Northern blot analysis of cathepsin K mRNA expression in mature osteoclast cells (control, lane 1), and following IL-1 (0.7 nM) treatment for 6 hrs preceded by a two-hour incubation with PBS (lane 2), PDTC (100 µM; lane 3), Genistein (100 µM; lane 4), SB 203580 (20 µM; lane 5), or PD 98059 (20 µM; lane 6). We hybridized blots to GAPDH to standardize loading (lower panel). Experiments were performed in triplicate (N = 3). (C) Relative abundance of cathepsin K mRNA on Northern blots, as evaluated by densitometric scanning. *p < 0.05 compared with control pre-treated samples stimulated with IL-1.

View larger version (41K): [in this window] [in a new window]   Figure 4. IL-1 activates NF-B in osteoclasts, and the p65 domain of NF-B mediates IL-1-induced cathepsin K expression. (A) Nuclear extracts from osteoclast cells were analyzed by EMSA for DNA binding to a 32P-labeled NF-B consensus oligonucleotide probe. Lane 1, the probe alone; lane 2, nuclear extract and probe; lane 3, nuclear extract from IL-1-stimulated osteoclast cells and probe; lane 4, nuclear extract from IL-1-stimulated osteoclast cells and probe, plus 10-fold excess unlabeled NF-B consensus oligonucleotide; lane 5, nuclear extract from IL-1-stimulated osteoclast cells and probe, plus 100-fold excess unlabeled NF-B consensus oligonucleotide; lane 6, probe alone. (B) MOCP-5 cells were plated on culture dishes and incubated for 10 hrs in the presence or absence of antisense S-ODNs (p50AS and p65AS) or sense S-ODNs (p50S and p65S) to p50 and p65. We then isolated total RNA from some cultures to determine the expression of mRNAs to p50 and p65 by RT-PCR. The expression of GAPDH mRNA was used as the control. (C) Northern blot analysis of total RNA (15 µg) from mature osteoclasts pre-treated with oligonucleotides sense to p50 (5 µM; lane 3), antisense to p50 (5 µM; lane 4), sense to p65 (10 µM; lane 5), antisense to p65 (10 µM; lane 6), and then stimulated with 0.7 nM IL-1 for 6 hrs. Cells treated with sense oligonucleotides or the same medium without oligonucleotides were used as controls. The lower panel illustrates loading differences as determined by GAPDH hybridization. Experiments were performed in triplicate (N = 4). (D) Relative abundance of cathepsin K mRNA on Northern blots, as evaluated by densitometric scanning. *p < 0.05 compared with control pre-treated with sense oligonucleotides.

Journal of Dental Research, Vol. 83, No. 10, 791-796 (2004)
DOI: 10.1177/154405910408301011


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