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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;

^* corresponding author, ypli{at}forsyth.org

	ABSTRACT

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Interleukin-1 (IL-1) is a powerful activator of osteoclast cells. However, the underlying mechanism for this activation is unknown. In this study, we reveal that IL-1 up-regulates the expression of cathepsin K protein, a key protease in bone resorption, by five-fold. Northern blot analysis and promoter analysis show that this induction occurs at the transcriptional level, in a dose-responsive and time-dependent manner. No increase in expression occurs in the presence of either pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of NF-B, or Genistein, a protein tyrosine kinase inhibitor, suggesting that IL-1 up-regulation may be via the tyrosine kinase-NF-B pathway to regulate cathepsin K expression. Antisense oligonucleotides to p65, but not the p50 subunit of NF-B, suppress the IL-1-induced expression of cathepsin K. We therefore conclude that IL-1 up-regulates cathepsin K gene expression at the transcription level, and this regulation may be via the tyrosine-kinase-NF-B pathway.

KEY WORDS: interleukin-1alpha • cathepsin K • osteoclast • NF-B • tyrosine kinase

	INTRODUCTION

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The bone-resorptive activity of osteoclasts is an essential part of bone remodeling and occurs in response to the bone micro-environment. In normal physiological conditions, stromal and osteoblast cells secrete local factors—such as macrophage-colony-stimulating factor (M-CSF), receptor activator of NF-B ligand (RANKL) (Lacey et al., 1998; Yasuda et al., 1998; Kudo et al., 2002), and TNF (Kudo et al., 2002)—which have been shown to regulate osteoclast differentiation and activity. However, the bone micro-environment is also associated with pathological conditions of bone. For example, under local inflammatory conditions, T-cells secrete interleukin-1-alpha (IL-1), which stimulates bone resorption and results in many diseases related to elevated bone resorption (Romas et al., 2002). Similarly, Wang and Stashenko (1993) reported a role for IL-1 in the pathogenesis of periapical bone destruction. Moreover, since IL-1 increases the expression of RANKL (Romas et al., 2002), these local factors may act in concert to promote osteoclast recruitment, activation, and osteolysis, since they stimulate the same NF-B pathway (Jimi et al., 1998, 1999). However, it is unclear which of the genes downstream of the IL-1-induced activation of the NF-B pathway activate osteoclasts. Cathepsin K has been shown to have a very important role in bone resorption (Gelb et al., 1996). In this study, we sought to characterize the mechanism by which IL-1 induces osteoclast bone resorption through cathepsin K gene expression regulation.

	MATERIALS & METHODS

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Cell Culture and Assay for Dentin Resorption
The osteoclast precursor cell lines, mouse osteoclast precursor cells (MOCP-5), and mouse bone marrow were co-cultured with mouse stromal cell line (MS-12) cells as previously described (Chen and Li, 1998; Kamolmatyakul et al., 2001). For the pure osteoclast population, MOCP-5 were prepared at a density of 4 x 10³ cells/mL in -MEM containing 10% (v/v) FBS. RANKL (50 ng/mL) was added for 5 days, so that mature osteoclasts could be obtained. On day 6, different doses of recombinant mouse IL-1 (Genzyme Corporation, Cambridge, MA, USA) were added. Dentin resorption assays were performed as previously described (Chen and Li, 1998).

Northern Blot Analysis
The total RNA of the osteoclast population was isolated as described previously (Chomczynski and Sacchi, 1987). Northern blot analysis was performed as described previously (Li et al., 1995).

Cathepsin K Protein Determination
The co-culture systems were incubated with IL-1 (250 U/mL or 1.4 nM) for 10 and 15 hrs. We determined cathepsin K protein production by multinucleated osteoclast-like cells (OCLs) by assaying the media using ELISA, as previously described (Kamolmatyakul et al., 2001). In brief, the purified rabbit IgG containing anti-cathepsin K was coated onto 96-well plates (ICN Biomedicals, Aurora, OH, USA). The test supernatants (100–10000x dilution) were applied, followed by HRP-conjugated anti-human cathepsin K. The concentration of cathepsin K in the test solution was interpolated from the standard curve generated from the standard antigen solution.

Cycloheximide and Actinomycin D Study
Mature osteoclasts (approximately 5 x 10⁴) were treated with 10 µg/mL cycloheximide (CHX; Sigma, St. Louis, MO, USA) or 5 µg/mL actinomycin D (Act D; Sigma, St. Louis, MO, USA) and then exposed to IL-1 (1.4 nM; Genzyme Corporation, Cambridge, MA, USA) an hour later. In parallel, control cells were treated with PBS for 1 hr before being exposed to IL-1.

Cathepsin K Promoter Constructs
A cathepsin K promoter-CAT fusion gene (pMCCAT) containing a 1978-bp XbaI/EcoRI fragment (–1400 to +357, numbered relative to the cap site) of the mouse cathepsin K 5'-flanking region was used as to generate the cathepsin K promoter CAT fusion gene construct. The XbaI/EcoRI fragment was subcloned into pCAT-3 Basic reporter vector (Promega, Madison, WI, USA) to produce the cathepsin K promoter constructs and named pMCCAT-1400.

DNA Transfection and CAT (Chloramphenicol acetyltransferase) Assay
Transient transfection of plasmid DNA into MOCP-5 was performed with the lipofectAMINE reagent (Invitrogen, Gaithersburg, MD, USA) as described (Chen and Li, 1998). In all experiments, the effect of IL-1 (250 U/mL or 1.4 nM) on pMCCAT-1400 promoter activity was tested in triplicate, either with or without RANKL (20 ng/mL). CAT assays were performed on lysates as described (Neumann et al., 1987). CAT activity in transfected cultures was standardized by normalization to ß-galactosidase activity of cotransfected pSV-ß-galactosidase constructs (Promega, Madison, WI, USA) (Li and Stashenko, 1993). CAT assay results were standardized to the protein concentration of cell layer extracts by means of a protein assay kit (Bio-Rad, Richmond, CA, USA).

Signaling Pathway Inhibitor Studies
When OCLs were fully developed, the following signaling pathway inhibitors were added: 100 µM PDTC (pyrrolidine dithiocarbamate, from Sigma, St. Louis, MO, USA) dissolved in water, 100 µM Genistein (Sigma, St. Louis, MO, USA), 20 µM SB 203580 (Calbiochem, La Jolla, CA, USA), and 20 µM PD 98059 (Biomol, Plymouth Meeting, PA, USA) dissolved in DMSO (dimethylsulfoxide, Sigma, St. Louis, MO, USA). After 2 hrs of culturing with these inhibitors, IL-1 (125 U/mL or 0.7 nM) was added. The medium was discarded 6 hrs later. The OCL population was washed in PBS, and total RNA was then isolated.

Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extraction was performed as described (Li et al., 1995). The sequence of the NF-B binding oligonucleotide used as a radioactive DNA probe was 5'-AGCTTGGGGACTTTCCGAC-3'.

Antisense Oligodeoxynucleotide Studies
We performed antisense blocking experiments to characterize and confirm that IL-1 acts through NF-B to regulate cathepsin K expression as described (Jimi et al., 1998). In brief, when OCLs were fully developed on day 5, the sense and antisense oligonucleotides were added (see Table for oligo sequences): p50 antisense (5 µM) and p65 antisense (10 µM). Prior to that addition, we mixed 100 nM Tfx^TM-50 (Promega, Madison, WI, USA), polycationic liposome, with these antisense oligonucleotides as the S-ODN (synthetic phosphorothioate oligodeoxynucleotides), as previously described (Inui et al., 1997). After nine-hour incubation with these antisense oligonucleotides, IL-1 (0.7 nM) was added. Control cells received an equal concentration of corresponding sense oligonucleotides or just fresh medium without oligonucleotides. After 6 hrs of culture, the medium was discarded. The OCL population was washed in PBS, total RNA was isolated, and Northern blot analysis was carried out as described above. The expression of p50 or p65 mRNA was detected by RT-PCR with the use of gene-specific PCR primers as described (Jimi et al., 1998).

	RESULTS

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IL-1 Up-regulates Cathepsin K Protein
We examined whether IL-1 up-regulates cathepsin K protein expression in osteoclasts by using osteoclast-like cells (OCLs) which derived from MOCP-5 (Chen and Li, 1998), or mouse bone marrow cells. Using the ELISA technique, we found that incubating OCLs with IL-1 (250 U/mL or 1.4 nM) for 10 and 15 hrs increased cathepsin K protein production by about three- and four-fold, respectively, compared with controls (Fig. 1A). We then examined the effects of IL-1 on inducing bone-resorbing activity of osteoclasts by a dentin resorption assay. Fig. 1B shows the resorption pits formed by OCLs in the absence (a) or presence (b) of IL-1. It has been demonstrated that the resorbed surface area is a suitable parameter in studies of osteoclasts to quantitate pit formation (Chen and Li, 1998). As shown in Fig. 1C, incubating OCLs with IL-1 increased the resorbed surface area by about 2.5-fold, compared with controls, indicating that IL-1 significantly increased the pit-forming activity of OCLs.

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).

IL-1 Up-regulates the mRNA Levels of Cathepsin K in a Dose-responsive and Time-dependent Manner at the Transcription Level

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.

IL-1 Up-regulates the Cathepsin K Promoter-CAT Fusion Gene Only in Mature Osteoclast Cells
To determine whether the cathepsin K promoter is the region that IL-1 uses to regulate cathepsin K transcription, we performed transfection studies with a cathepsin K promoter-CAT fusion gene (pMCCAT). Pre-osteoclast (MOCP-5) and mature osteoclast cells were transiently transfected with pMCCAT by means of the lipofectAMINE reagent, and reporter CAT activity was determined following IL-1 treatment. In mature osteoclast cells, CAT activity was up-regulated approximately three-fold in response to 7 hrs of IL-1 treatment, compared with controls (Fig. 3A), and no effect was observed in pre-osteoclast cells, indicating that the IL-1 signal acted on the cathepsin K promoter region, and this action occurred only in mature osteoclast cells.

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.

NF-B Activation Inhibitor and Tyrosine Kinase Inhibitor Inhibit the Induction of IL-1 on Cathepsin K Gene Expression

IL-1 Activates NF-B in Osteoclasts
The DNA-binding activation of NF-B was strongly detected in nuclear extracts derived from OCLs following 30 min of induction by IL-1 (Fig. 4A, lane 3). The binding activity was partially reduced with 10-fold (lane 4) and almost completely diminished with 100-fold excess unlabeled NF-B binding oligonucleotide (lane 5), signifying specific binding. These findings indicate that NF-B is activated in response to IL-1 stimulation, and that IL-1 activates NF-B in osteoclasts.

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.

p65 but not p50 is Responsible for the Transcription Activation of NF-B.

	DISCUSSION

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IL-1 activates several signaling cascades, including Akt and ERK (Lee et al., 2002), NF-B (Jimi et al., 1996; Miyazaki et al., 2000), and JNK (Jimi et al., 1999) pathways. It has been suggested that ERK is responsible for osteoclast survival, whereas NF-B regulates osteoclast activation for bone resorption (Miyazaki et al., 2000). However, it is not well-known what downstream genes are induced after IL-1 stimulation to promote osteoclastic bone resorption. In the present study, we found that IL-1 up-regulated cathepsin K expression via the NF-B pathway.

In the CAT assay, no increase of CAT activity response to IL-1 was observed in pre-osteoclast cells. This result is consist with our previous finding that cathepsin K mRNA was not detected in pre-osteoclast cells (Li and Chen, 1999). Osteopetrosis in NF-B1 and NF-B2 double-knockout mice has identified an important role for NF-B in osteoclast differentiation (Iotsova et al., 1997). In this study, NF-B was activated in response to IL-1 stimulation, a result consistent with that of Jimi et al.(1996). Furthermore, NF-B activation inhibitor or tyrosine kinase inhibitor completely blocked the increase of cathepsin K mRNA levels induced by IL-1 treatment. We had previously reported that there is an NF-B binding site, GGGACTGCCC, located in -8886 to -8877 of the cathepsin K promoter (Li and Chen, 1999). Taken together, these results indicate that IL-1 up-regulates cathepsin K expression via the NF-B pathway.

The cycloheximide experiment showed that no new protein was needed to induce cathepsin K expression. However, cathepsin K expression could not be induced when p65 subunits of NF-B protein synthesis were blocked in an antisense study. Similar results, in which antisense Hsp60 oligonucleotides reduced Hsp60 expression, while cycloheximide was used to inhibit protein synthesis, did not alter the effect of Hsp60 on IGF-1R signaling, and IGF-1R mRNA levels were not up-regulated by Hsp10 or Hsp60 (Shan et al., 2003). It is possible that CHX inhibited synthesis of all cellular proteins, including proteinases, so p65 is relatively stable to perform IL-1 response stimulation. While cells lack both original p65, due to protein degradation, and new p65 synthesis, due to antisense treatment, blocking failed to respond to IL-1.

Our results also demonstrate that pre-treatment with antisense oligonucleotides to p65, but not p50, suppresses the up-regulation effect of IL-1 on cathepsin K expression in osteoclasts. This indicates that the p65, but not the p50, subunit of NF-B is responsible for the transcriptional regulation of cathepsin K expression induced by IL-1. This finding is consistent with p65, the DNA-binding component of NF-B, having the more critical role in initiating transcription, whereas p50 functions to assist in DNA binding (Schmitz and Baeuerle, 1991) and has a primary structure completely divergent from that of p65 (Nolan et al., 1991). This finding is also consistent with the results of Xing et al.(2003), that p50 and p52 subunits, which are highly homologous, can have overlapping functions in cell activation, as seen in the impaired osteoclast development only in double-knockout mice. Expression of either NF-B p50 or p52 in osteoclast precursors is required for IL-1-mediated formation of mature osteoclasts (Xing et al., 2003).

The results of this study suggest the mechanism by which IL-1 activates bone resorption through its up-regulation on the expression of osteoclast genes. Our findings presented here will add to our understanding of the interplay between the local bone environment and the regulation of expression of osteoclast genes during the normal and pathological bone resorption process, and will help us identify additional targets for therapeutic intervention in bone disorders such as osteoporosis, osteoarthritis, rheumatoid arthritis, periodontal disease, and other bone disorders.

	ACKNOWLEDGMENTS

 
This work was supported by NIH grant AR44741 (Y.-P. Li) and AR-48133-01 (Y.-P. Li). We thank Hong Liang Ci and Qingnin Zhao for manuscript assistance.

Received October 16, 2003; Last revision July 10, 2004; Accepted July 25, 2004

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This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Kamolmatyakul, S. Articles by Li, Y.-P. Search for Related Content PubMed PubMed Citation Articles by Kamolmatyakul, S. Articles by Li, Y.-P.