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New Insights into the Regulation of Cathepsin K Gene Expression by Osteoprotegerin Ligand
Biochemical and Biophysical Research Communications 285, 335–339 (2001) doi:10.1006/bbrc.2001.5127, available online at http://www.idealibrary.com on
New Insights into the Regulation of Cathepsin K Gene Expression by Osteoprotegerin Ligand Susanne Corisdeo, Michael Gyda, Mone Zaidi, Baljit S. Moonga, and Bruce R. Troen 1 Geriatric Research Education and Clinical Center, Bronx Veterans Administration Medical Center, Bronx, New York 10468; and Department of Geriatrics and Adult Development and Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029
Received May 30, 2001
Cathepsin K plays a key role in bone resorption. We provide the first evidence that osteoprotegerin ligand (OPGL), a critical pro-resorptive cytokine, acutely stimulates the expression of cathepsin K in osteoclasts. We used in situ RT-PCR and real time quantitative RT-PCR to analyze cathepsin K gene expression. OPGL enhanced cathepsin K mRNA levels in mature osteoclasts isolated from rat neonatal long bones. OPGL together with macrophage colony-stimulating factor (M-CSF) also stimulated cathepsin K gene expression in monocytic cells and multinucleate osteoclasts in bone marrow cultures. Real time quantitative RT-PCR demonstrated high levels of cathepsin K mRNA in bone marrow cultures, paralleling the degree of osteoclastogenesis. We therefore suggest that OPGL enhances bone resorption, at least in part, by inducing cathepsin K gene expression. © 2001 Academic Press Key Words: osteoclast; osteoprotegerin ligand; macrophage colony-stimulating factor; cathepsin K; rat.
Bone resorption occurs within the resorption lacuna, a tightly sealed zone beneath the ruffled border of the osteoclast where it has attached to the bone surface. Resorption depends upon acidification of this extracellular compartment leading to demineralization and subsequent removal of organic matrix by cysteine proteases, such as cathepsin K (1– 4). Earlier reports have suggested the involvement of several cathepsins in bone resorption (5–9). More recent data, however, strongly implicate cathepsin K as the predominant effector of matrix degradation. Northern analysis shows that cathepsin K mRNA is preferentially expressed in rabbit osteoclasts at high levels (10). Cathepsin K is expressed in human osteoclasts, whereas
1 To whom correspondence should be addressed at 526/GRECC, Bronx VA Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468. Fax: (718) 741-4211. E-mail: [email protected].
cathepsins B, S, and L are not (11). During mouse embryogenesis, cathepsin K expression is restricted to osteoclasts at sites of active cartilage and bone modeling (12). In actively resorbing osteoclasts, the enzyme is found at the ruffled border. Osteoblasts and osteocytes do not express either the transcript or the protein (12, 13). Inhibition of cathepsin K gene expression by an antisense oligonucleotide suppresses bone resorption by rabbit osteoclasts (14). Xia et al. have localized cathepsin K in rat osteoclasts to the resorption pit and shown that specific inhibitors reduced bone resorption (15). In nonhuman primates, specific inhibitors of the cathepsin K enzyme markedly depressed bone remodeling (16). Nonsense, missense, and stop codon mutations in the cathepsin K gene have been identified in patients with pycnodysostosis, an autosomal recessive osteochondrodysplasia characterized by osteosclerosis and short stature (17–19). Cathepsin K null mice develop osteopetrosis and display features characteristic of pycnodysostosis, (20, 21). Matrix degradation is significantly diminished in these mice (21). Furthermore, cathepsin K null osteoclasts exhibit impaired bone resorption in vitro (20). Since cathepsin K plays a critical and limiting role in matrix degradation during bone resorption, we have chosen to study the regulation of cathepsin K expression in mature osteoclasts and during osteoclastogenesis in vitro. METHODS Animals. Osteoclasts were isolated freshly from the long bones of 2- to 3-day-old Wistar rats (Harlan, Indianapolis, IN). Bone marrow cultures were established from 2-month-old Sprague–Dawley rats (Harlan, Indianapolis, IN). The animals were sacrificed by decapitation. Isolated authentic osteoclasts. Osteoclasts were isolated from the tibiae and femora of the neonatal rats by a method of mechanical disaggregation (22, 23). The femora and tibiae were freed of adherent soft tissues and cut across their epiphyses in Hepes buffered medium 199 supplemented with 10% fetal calf serum, penicillin (100,000 units/liter), and streptomycin (100 mg/liter) (LifeTechnologies, Rock-
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ville, MD). The bones of each rat were curetted with a scalpel blade into 1 ml of the above medium, gently agitated, dispersed onto glass coverslips, and placed into the wells of a multiwell dish. After a 15-min incubation at 37°C, other cells were removed by rinsing the coverslips with the medium 199. Osteoclasts were then treated as described and cathepsin K expression subsequently determined. Establishment of primary bone marrow cultures for the induction of osteoclasts. After sacrifice, the femora and tibiae were freed of adherent soft tissues and cut across their epiphyses while in the tissue culture hood. Using a syringe and an 18-gauge needle, approximately 3.5 ml of ␣-MEM/long bone was used to flush out the bone marrow into a 50-ml conical tube. The cells were centrifuged at 1000 rpm for 4 min, resuspended in medium, and washed two more times. After final resuspension, 1 ⫻ 10 6 cells/0.5 ml were placed into each well of a 24-well plate. For in situ RT-PCR, 2 ⫻ 10 6 cells/1iter ml were placed on top of a glass coverslip into each well of a 12-well plate. The next day (Day 1), 500 l of fresh media with twice the final concentration of the indicated agent was added. Every 2 days, 500 l was carefully aspirated and discarded prior to the addition of 500 l fresh media with twice the final concentration of the treatment agent. On days 3–16, TRAP staining was performed to assess osteoclast formation (number of multinucleated stained cells) and cells were harvested for RNA. Northern analyses. 10 g samples of RNA were electrophoresed on 1% formaldehyde–Mops gel and subjected to Northern analysis as we have described (24, 25). Cathepsin K mRNA was normalized to GAPDH mRNA expression. Cells were lysed with TRIzol reagent (LifeTechnologies, Rockville, MD). Extraction was performed after the addition of 0.1 vol chloroform followed by centrifugation. RNA was recovered from the aqueous phase by precipitation with 0.5 vol of isopropyl alcohol. In situ RT-PCR. In situ RT-PCR was performed as we have previously described (26). Freshly isolated osteoclasts were dispersed onto glass coverslips and fixed with 4% paraformaldehyde for 20 min at 4°C and then washed with cold PBS. After immersion in 0.2 N HCl for 20 min at room temperature, cells were washed and then treated with proteinase-K (5 mg/liter in 10 mM Tris–HCl, pH 8.0) for 15 min at 37°C, 0.1 M triethanolamine for 2 min, 0.25% acetic anhydride for 10 min, and paraformaldehyde for 30 min at 4°C. Osteoclasts were dehydrated by immersion in a series of aqueous ethanol solutions (70, 80, 90, and 100%) for 1 min each and then air dried. Cells were then incubated overnight at 37°C with RNase-free, DNase I (25 units/ml) to remove genomic DNA. The enzyme was removed and inactivated by washing with DEPC-treated water and heated at 90°C for 10 minutes. First-strand cDNA was synthesized by incubating with 50 l of RT mixture (10 mM dNTPs, 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 100 mM DTT, 400 fM gene-specific antisense primer, 200 units Superscript RT II) at 42°C for 90 min. Subsequently, the coverslips were washed with DEPC-treated water and dried. PCR amplification was performed with 50 l of PCR mixture (10 mM dNTPs, 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 25 mM MgCl 2, 2 units Taq polymerase, 400 fM sense and antisense primers, 1 mM digoxigenin (DIG) labeled-11-dUTP, and DEPCtreated water). The cathepsin K primer sequences are: forward: 5⬘-CCCAGACTCCATCGACTATCG-3⬘, reverse: 5⬘-CTGTACCCTCTGCACTTAGCTGCC-3⬘. PCR was performed as follows: 4 min soak at 94°C; 20 to 60 cycles of 94°C for 1 min, 65°C for 2 min, and 72°C for 3 min. Incorporated DIG-11-dUTP in the PCR product was detected using a DIG nucleic acid detection kit (Boehringer Mannheim). Negative controls, in which primers were omitted, were run in parallel. A blinded observer scored osteoclasts for the intensity of staining. The results from multiple experiments were plotted as a frequency histogram. This permits determination of the proportion of cells that lay in a certain intensity range. The few retracted osteoclasts were discarded from the analysis to prevent a biased intensity assessment.
Reverse transcription and Taqman PCR. Duplex cathepsin K/18 S Taqman assays were performed to assess both cathepsin K mRNA and 18S RNA expression in 1 ng total RNA for each sample. All values in the chart (Fig. 3, right panel) are expressed as the ratio of cathepsin K to 18S expression and then further normalized to the no treatment (no Rx) day 3 sample. This provides an internal control for each sample, eliminates variability due to pipetting imprecision, and permits accurate quantitative comparisons between samples. Depending upon the standard error of determinations in a given experiment, as little as a 50% change in expression can be assessed. All samples were analyzed in duplicate. Reverse transcription was performed according to according to manufacturer’s instructions (Promega, Madison, WI): M-MLV RT 5 U/l, 0.5 mM dNTPs, rRNasin 1 U/l, 0.5 g of random hexamer/g RNA, and incubated in a final volume of 25 l at 37°C for 1 h. The rat cathepsin K primers are: forward: 5⬘-ACCCACGGGAAGCAGTACAA-3⬘ (anneals between residues 136 and 155 with a T m of 59°C), reverse: 5⬘-GGCCTCAAGATTATGGACAGAAA-3⬘ (anneals between residues 234 and 212 with a T m of 58°), and Taqman Probe: 5⬘-FAM-ACGCCGAGAGATTTCATCCACCTTGCT-TAMRA-3⬘ (anneals between residues 157 and 183 with a T m of 70°C). The 18S RNA primers and Taqman probe (with the VIC fluor) are those supplied by Perkin–Elmer. Our conditions for the Taqman duplex reaction for amplification of both cathepsin K mRNA and 18S RNA were: 1⫻ Taqman Universal PCR master mix, cDNA, 300 nM cathepsin K forward primer, 300 nM cathepsin K reverse primer, 100 nM cathepsin K Taqman probe, 100 nM GAPDH forward primer, 100 nM GAPDH reverse primer, 200 nM GAPDH Taqman probe in a total volume of 25 l. Thermocycler conditions: Stage 1, 50°C for 2 min; Stage 2, 95°C for 10 min; Stage 3, 95°C for 15 s followed by 60°C for 1 min, Stage 3 repeated a total of 40 times. Data were processed with the SDDS 1.6 software supplied by Perkin–Elmer and further analyzed by spreadsheet.
RESULTS AND DISCUSSION Figure 1 shows that OPGL increases cathepsin K mRNA expression acutely in isolated mature osteoclasts. We have previously quantitated CD38 and IL-6 gene expression in isolated rat osteoclasts by in situ RT-PCR cytoimaging (26, 27). The histostained images in the upper row of the left panel are representative of untreated cells. Osteoclasts in the lower row of the left panel are representative of cells treated with 100 ng/ml OPGL for 12 h. These exhibit more intense staining, reflecting greater amounts cathepsin K mRNA. Semiquantitative estimates of staining for cathepsin K were obtained by a blinded observer who assigned an arbitrary intensity level as a number from 0 to 3 (absent to intense staining). Frequency histograms relating the number of cells to their assigned intensity score are shown in the right panel of Fig. 1. We restricted our assessment to characteristic multinucleated spread osteoclasts. Osteoclasts without treatment that underwent in situ RT-PCR (control) exhibited a skewed distribution to the left. The data became significantly skewed to the right upon OPGL treatment (P ⬍ 0.05). In contrast, the expression of mRNA for GAPDH followed a similar distribution in both control and treated cells (data not shown). The histogram scoring analysis reflects the heterogeneity of gene expression in these mature osteoclasts. The spectrum of intensities, rather than more homogenous staining pattern, suggests that
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FIG. 1. OPGL acutely stimulates cathepsin K mRNA expression in mature osteoclasts. Left panel: cathepsin K was analyzed by in situ RT-PCR cytoimaging. Top row, no treatment; bottom row, treated with 100 ng OPGL for 12 h. Right panel: intensity score of histostained osteoclasts after in situ RT-PCR for cathepsin K mRNA.
the amplification is occurring within the log linear range. To assess cathepsin K expression during osteoclast formation, we established primary bone marrow cultures from 2 month-old rat long bones as described previously (28, 29) (see Methods). After plating 1–2.4 ⫻ 10 6 bone marrow cells per well in a 24 well plate, we treated these cultures for 7 days with either 10 nM calcitriol (D3) or a combination of 50 ng/ml human macrophage colony stimulating factory (M-CSF) and 100 ng/ml OPGL (30). We then examined cathepsin K mRNA via in situ RT-PCR (Fig. 2). Almost no cathepsin K mRNA was detected after 7 days of calcitriol. In contrast, M-CSF plus OPGL treatment markedly increased cathepsin K mRNA expression in both monocytic osteoclast precursors and multinucleate osteoclasts. We next examined the time course of osteoclast formation with multiple treatments (Fig. 3A) and subse-
FIG. 2. Cathepsin K mRNA expression in bone marrow-derived osteoclasts. In situ RT-PCR was performed on bone marrow cultures treated for the indicated times with either 10 nM calcitriol (D3) or 50 ng/ml M-CSF plus 100 ng/ml OPGL. CTSK, cathepsin K primers; Ø, no primers used for negative control.
quently assessed cathepsin K mRNA expression (Fig. 3B). Treatment with vehicle failed to yield any tartrate resistant acid phosphatase (TRAP) staining multinucleate osteoclasts at 3 and 7 days. Increasing numbers of osteoclasts developed between 10 and 16 days. Treatment with 50 ng M-CSF alone was unable to induce osteoclast formation throughout the experiment. Treatment with 10 nM calcitriol (D3) alone resulted in occasional osteoclasts at 7 and 10 days and numerous multinucleated cells at 13 and 16 days. Treatment with 100 ng/ml OPGL alone resulted in many osteoclasts by 16 days. However, combined treatment with M-CSF and OPGL resulted in abundant multinucleate osteoclasts as early as 7 days. Interestingly, osteoclast numbers subsequently declined between 10 and 16 days. Combined treatment with D3 plus OPGL also resulted in numerous osteoclasts by day 10. To quantitate cathepsin K mRNA expression accurately in a small number of cells, we performed real time quantitative RT-PCR using the 5⬘-nuclease (Taqman) (see Methods). We performed duplex Taqman assays for cathepsin K mRNA (Fam-labeled probe) and 18S RNA (Vic-labeled probe) (Fig. 3B). The assay was linear over four orders of magnitude (Fig. 4). The slope of the curves in multiple experiments was between 0.03 and 0.08 over four orders of magnitude (Fig. 4 and data not shown). A slope of less than 0.1 indicates that the assay is linear over the range of measurement. We could measure as little as 2.5 pg of total RNA, which comprises as little as 1/10 the RNA found in a single osteoclast. Duplex cathepsin K/18 S Taqman assays were performed to assess both cathepsin K mRNA and 18S RNA expression in 1 ng total RNA for each sample. All values in Fig. 3B are expressed as the ratio of cathepsin K to 18S expression and then further normalized to the no treatment (no Rx) day 3 sample. The
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FIG. 3. Osteoclastogenesis and cathepsin K mRNA expression in bone marrow cultures. (A) Bone marrow cultures were treated for the indicated times with the various agents (Ø Rx, vehicle; MCSF, 50 ng/ml M-CSF; D3, 10 nM calcitriol; OPGL, 100 ng/ml OPGL; MCSF/OPGL, 50 ng/ml M-CSF plus 100 ng/ml OPGL; D3/OPGL, 10 nM calcitriol plus 100 ng/ml OPGL) and stained for TRAP. (B) Duplex real time quantitative RT-PCR (Taqman assay) was performed using RNA from bone marrow cultures treated for the same times and with the same agents as indicated in A. Both cathepsin K and 18 S RNA were simultaneously quantitated. All values in the chart are expressed as the ratio of cathepsin K to 18S expression and then further normalized to the no treatment (no Rx) day 3 sample.
marked increases in cathepsin K mRNA in the various treatment protocols were concordant with the level of osteoclast formation seen in Fig. 3A. M-CSF treatment alone increased cathepsin K expression, consistent with the presence of osteoclast precursors (despite the lack of mature multi-nucleated osteoclasts). In situ RT-PCR confirmed that monocytic osteoclast precursors were responsible for this M-CSF mediated increase in cathepsin K expression. The M-CSF treated cultures also lacked mature osteoblasts (Gyda et al., see companion paper, this issue (33)), suggesting that M-CSF regulated cathepsin K gene expression directly. As shown above, bone marrow cultures treated with M-CSF plus OPGL yielded large numbers of osteoclasts. We therefore pooled four wells (of a 24-well
plate) for each condition, harvested RNA, and performed a Northern analysis for cathepsin K mRNA (Fig. 5). There was a marked increase in expression of cathepsin K mRNA after treatment for 3 and 6 days with 50 ng/ml M-CSF plus 100 ng/ml OPGL (3 and 6 days), consistent with the real time quantitative RTPCR. To our knowledge, little work on cathepsin K has been done in mature osteoclasts harvested from rat long bones or in primary rat bone marrow cultures. Retinoic acid increases cathepsin K gene expression in isolated mature rabbit osteoclasts (2). OPGL stimulates cathepsin K mRNA and protein expression in osteoclasts derived from human peripheral blood mononuclear cell precursors (31). OPGL also enhances
FIG. 4. Primer efficiency of Taqman assay. The relative primer efficiency of the cathepsin K and 18S oligonucleotide pairs was plotted as delta Ct (⫽ cathepsin K Ct ⫺ 18S Ct) versus the log of the total RNA concentration. The Ct (cycle threshold) value reflects the relative amount of the original mRNA and cDNA. Greater amounts of initial transcript result in lower Ct values.
FIG. 5. Northern analysis of bone marrow culture RNA. RNA was harvested from 3 day untreated cultures and from cultures treated for 3 and 6 days with 50 ng/ml M-CSF plus 100 ng/ml OPGL and probed with radiolabeled cathepsin K cDNA or radiolabeled 18S ribosomal cDNA.
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cathepsin K mRNA expression in murine myeloid RAW 264.7 cells concomitant with induction toward an osteoclastic phenotype (32). Here we show that, in mature rat osteoclasts, OPGL exerts an acute effect upon cathepsin K gene expression within 12 h. Since OPGL acts only upon osteoclasts and since cathepsin K is expressed only in osteoclasts (in bone), it is highly unlikely that osteoblasts or stromal cells mediate OPGL induced cathepsin K expression. In conclusion, we provide new data demonstrating dramatic effects of OPGL upon cathepsin K gene expression. We also show that M-CSF enhances cathepsin K expression. The stimulation of cathepsin K expression resulting in matrix degradation may in part explain the pro-resorptive action of OPGL (and M-CSF). Further studies on the regulation of cathepsin K expression by OPGL and other cytokines and hormones may therefore have a direct physiological and therapeutic relevance. ACKNOWLEDGMENTS This study was supported in full by the National Institutes of Health (RO3-AG15628 to B.R.T.) and the Lankenau Institute for Medical Research. B.R.T. acknowledges the support of Dr. Vincent Cristofalo. M.Z. acknowledges support from the NIH (RO1-AG14917) and the Department of Veterans Affairs (Merit Review). B.R.T. and M.Z. together acknowledge the support of Dr. Christine Cassel, the Bronx VA GRECC, and the Brookdale Department of Geriatrics and Adult Development at the Mount Sinai School of Medicine.
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New Insights into the Regulation of Cathepsin K Gene Expression by Osteoprotegerin Ligand Susanne Corisdeo, Michael Gyda, Mone Zaidi, Baljit S. Moonga, and Bruce R. Troen 1 Geriatric Research Education and Clinical Center, Bronx Veterans Administration Medical Center, Bronx, New York 10468; and Department of Geriatrics and Adult Development and Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029
Received May 30, 2001
Cathepsin K plays a key role in bone resorption. We provide the first evidence that osteoprotegerin ligand (OPGL), a critical pro-resorptive cytokine, acutely stimulates the expression of cathepsin K in osteoclasts. We used in situ RT-PCR and real time quantitative RT-PCR to analyze cathepsin K gene expression. OPGL enhanced cathepsin K mRNA levels in mature osteoclasts isolated from rat neonatal long bones. OPGL together with macrophage colony-stimulating factor (M-CSF) also stimulated cathepsin K gene expression in monocytic cells and multinucleate osteoclasts in bone marrow cultures. Real time quantitative RT-PCR demonstrated high levels of cathepsin K mRNA in bone marrow cultures, paralleling the degree of osteoclastogenesis. We therefore suggest that OPGL enhances bone resorption, at least in part, by inducing cathepsin K gene expression. © 2001 Academic Press Key Words: osteoclast; osteoprotegerin ligand; macrophage colony-stimulating factor; cathepsin K; rat.
Bone resorption occurs within the resorption lacuna, a tightly sealed zone beneath the ruffled border of the osteoclast where it has attached to the bone surface. Resorption depends upon acidification of this extracellular compartment leading to demineralization and subsequent removal of organic matrix by cysteine proteases, such as cathepsin K (1– 4). Earlier reports have suggested the involvement of several cathepsins in bone resorption (5–9). More recent data, however, strongly implicate cathepsin K as the predominant effector of matrix degradation. Northern analysis shows that cathepsin K mRNA is preferentially expressed in rabbit osteoclasts at high levels (10). Cathepsin K is expressed in human osteoclasts, whereas
1 To whom correspondence should be addressed at 526/GRECC, Bronx VA Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468. Fax: (718) 741-4211. E-mail: [email protected].
cathepsins B, S, and L are not (11). During mouse embryogenesis, cathepsin K expression is restricted to osteoclasts at sites of active cartilage and bone modeling (12). In actively resorbing osteoclasts, the enzyme is found at the ruffled border. Osteoblasts and osteocytes do not express either the transcript or the protein (12, 13). Inhibition of cathepsin K gene expression by an antisense oligonucleotide suppresses bone resorption by rabbit osteoclasts (14). Xia et al. have localized cathepsin K in rat osteoclasts to the resorption pit and shown that specific inhibitors reduced bone resorption (15). In nonhuman primates, specific inhibitors of the cathepsin K enzyme markedly depressed bone remodeling (16). Nonsense, missense, and stop codon mutations in the cathepsin K gene have been identified in patients with pycnodysostosis, an autosomal recessive osteochondrodysplasia characterized by osteosclerosis and short stature (17–19). Cathepsin K null mice develop osteopetrosis and display features characteristic of pycnodysostosis, (20, 21). Matrix degradation is significantly diminished in these mice (21). Furthermore, cathepsin K null osteoclasts exhibit impaired bone resorption in vitro (20). Since cathepsin K plays a critical and limiting role in matrix degradation during bone resorption, we have chosen to study the regulation of cathepsin K expression in mature osteoclasts and during osteoclastogenesis in vitro. METHODS Animals. Osteoclasts were isolated freshly from the long bones of 2- to 3-day-old Wistar rats (Harlan, Indianapolis, IN). Bone marrow cultures were established from 2-month-old Sprague–Dawley rats (Harlan, Indianapolis, IN). The animals were sacrificed by decapitation. Isolated authentic osteoclasts. Osteoclasts were isolated from the tibiae and femora of the neonatal rats by a method of mechanical disaggregation (22, 23). The femora and tibiae were freed of adherent soft tissues and cut across their epiphyses in Hepes buffered medium 199 supplemented with 10% fetal calf serum, penicillin (100,000 units/liter), and streptomycin (100 mg/liter) (LifeTechnologies, Rock-
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ville, MD). The bones of each rat were curetted with a scalpel blade into 1 ml of the above medium, gently agitated, dispersed onto glass coverslips, and placed into the wells of a multiwell dish. After a 15-min incubation at 37°C, other cells were removed by rinsing the coverslips with the medium 199. Osteoclasts were then treated as described and cathepsin K expression subsequently determined. Establishment of primary bone marrow cultures for the induction of osteoclasts. After sacrifice, the femora and tibiae were freed of adherent soft tissues and cut across their epiphyses while in the tissue culture hood. Using a syringe and an 18-gauge needle, approximately 3.5 ml of ␣-MEM/long bone was used to flush out the bone marrow into a 50-ml conical tube. The cells were centrifuged at 1000 rpm for 4 min, resuspended in medium, and washed two more times. After final resuspension, 1 ⫻ 10 6 cells/0.5 ml were placed into each well of a 24-well plate. For in situ RT-PCR, 2 ⫻ 10 6 cells/1iter ml were placed on top of a glass coverslip into each well of a 12-well plate. The next day (Day 1), 500 l of fresh media with twice the final concentration of the indicated agent was added. Every 2 days, 500 l was carefully aspirated and discarded prior to the addition of 500 l fresh media with twice the final concentration of the treatment agent. On days 3–16, TRAP staining was performed to assess osteoclast formation (number of multinucleated stained cells) and cells were harvested for RNA. Northern analyses. 10 g samples of RNA were electrophoresed on 1% formaldehyde–Mops gel and subjected to Northern analysis as we have described (24, 25). Cathepsin K mRNA was normalized to GAPDH mRNA expression. Cells were lysed with TRIzol reagent (LifeTechnologies, Rockville, MD). Extraction was performed after the addition of 0.1 vol chloroform followed by centrifugation. RNA was recovered from the aqueous phase by precipitation with 0.5 vol of isopropyl alcohol. In situ RT-PCR. In situ RT-PCR was performed as we have previously described (26). Freshly isolated osteoclasts were dispersed onto glass coverslips and fixed with 4% paraformaldehyde for 20 min at 4°C and then washed with cold PBS. After immersion in 0.2 N HCl for 20 min at room temperature, cells were washed and then treated with proteinase-K (5 mg/liter in 10 mM Tris–HCl, pH 8.0) for 15 min at 37°C, 0.1 M triethanolamine for 2 min, 0.25% acetic anhydride for 10 min, and paraformaldehyde for 30 min at 4°C. Osteoclasts were dehydrated by immersion in a series of aqueous ethanol solutions (70, 80, 90, and 100%) for 1 min each and then air dried. Cells were then incubated overnight at 37°C with RNase-free, DNase I (25 units/ml) to remove genomic DNA. The enzyme was removed and inactivated by washing with DEPC-treated water and heated at 90°C for 10 minutes. First-strand cDNA was synthesized by incubating with 50 l of RT mixture (10 mM dNTPs, 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 100 mM DTT, 400 fM gene-specific antisense primer, 200 units Superscript RT II) at 42°C for 90 min. Subsequently, the coverslips were washed with DEPC-treated water and dried. PCR amplification was performed with 50 l of PCR mixture (10 mM dNTPs, 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 25 mM MgCl 2, 2 units Taq polymerase, 400 fM sense and antisense primers, 1 mM digoxigenin (DIG) labeled-11-dUTP, and DEPCtreated water). The cathepsin K primer sequences are: forward: 5⬘-CCCAGACTCCATCGACTATCG-3⬘, reverse: 5⬘-CTGTACCCTCTGCACTTAGCTGCC-3⬘. PCR was performed as follows: 4 min soak at 94°C; 20 to 60 cycles of 94°C for 1 min, 65°C for 2 min, and 72°C for 3 min. Incorporated DIG-11-dUTP in the PCR product was detected using a DIG nucleic acid detection kit (Boehringer Mannheim). Negative controls, in which primers were omitted, were run in parallel. A blinded observer scored osteoclasts for the intensity of staining. The results from multiple experiments were plotted as a frequency histogram. This permits determination of the proportion of cells that lay in a certain intensity range. The few retracted osteoclasts were discarded from the analysis to prevent a biased intensity assessment.
Reverse transcription and Taqman PCR. Duplex cathepsin K/18 S Taqman assays were performed to assess both cathepsin K mRNA and 18S RNA expression in 1 ng total RNA for each sample. All values in the chart (Fig. 3, right panel) are expressed as the ratio of cathepsin K to 18S expression and then further normalized to the no treatment (no Rx) day 3 sample. This provides an internal control for each sample, eliminates variability due to pipetting imprecision, and permits accurate quantitative comparisons between samples. Depending upon the standard error of determinations in a given experiment, as little as a 50% change in expression can be assessed. All samples were analyzed in duplicate. Reverse transcription was performed according to according to manufacturer’s instructions (Promega, Madison, WI): M-MLV RT 5 U/l, 0.5 mM dNTPs, rRNasin 1 U/l, 0.5 g of random hexamer/g RNA, and incubated in a final volume of 25 l at 37°C for 1 h. The rat cathepsin K primers are: forward: 5⬘-ACCCACGGGAAGCAGTACAA-3⬘ (anneals between residues 136 and 155 with a T m of 59°C), reverse: 5⬘-GGCCTCAAGATTATGGACAGAAA-3⬘ (anneals between residues 234 and 212 with a T m of 58°), and Taqman Probe: 5⬘-FAM-ACGCCGAGAGATTTCATCCACCTTGCT-TAMRA-3⬘ (anneals between residues 157 and 183 with a T m of 70°C). The 18S RNA primers and Taqman probe (with the VIC fluor) are those supplied by Perkin–Elmer. Our conditions for the Taqman duplex reaction for amplification of both cathepsin K mRNA and 18S RNA were: 1⫻ Taqman Universal PCR master mix, cDNA, 300 nM cathepsin K forward primer, 300 nM cathepsin K reverse primer, 100 nM cathepsin K Taqman probe, 100 nM GAPDH forward primer, 100 nM GAPDH reverse primer, 200 nM GAPDH Taqman probe in a total volume of 25 l. Thermocycler conditions: Stage 1, 50°C for 2 min; Stage 2, 95°C for 10 min; Stage 3, 95°C for 15 s followed by 60°C for 1 min, Stage 3 repeated a total of 40 times. Data were processed with the SDDS 1.6 software supplied by Perkin–Elmer and further analyzed by spreadsheet.
RESULTS AND DISCUSSION Figure 1 shows that OPGL increases cathepsin K mRNA expression acutely in isolated mature osteoclasts. We have previously quantitated CD38 and IL-6 gene expression in isolated rat osteoclasts by in situ RT-PCR cytoimaging (26, 27). The histostained images in the upper row of the left panel are representative of untreated cells. Osteoclasts in the lower row of the left panel are representative of cells treated with 100 ng/ml OPGL for 12 h. These exhibit more intense staining, reflecting greater amounts cathepsin K mRNA. Semiquantitative estimates of staining for cathepsin K were obtained by a blinded observer who assigned an arbitrary intensity level as a number from 0 to 3 (absent to intense staining). Frequency histograms relating the number of cells to their assigned intensity score are shown in the right panel of Fig. 1. We restricted our assessment to characteristic multinucleated spread osteoclasts. Osteoclasts without treatment that underwent in situ RT-PCR (control) exhibited a skewed distribution to the left. The data became significantly skewed to the right upon OPGL treatment (P ⬍ 0.05). In contrast, the expression of mRNA for GAPDH followed a similar distribution in both control and treated cells (data not shown). The histogram scoring analysis reflects the heterogeneity of gene expression in these mature osteoclasts. The spectrum of intensities, rather than more homogenous staining pattern, suggests that
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FIG. 1. OPGL acutely stimulates cathepsin K mRNA expression in mature osteoclasts. Left panel: cathepsin K was analyzed by in situ RT-PCR cytoimaging. Top row, no treatment; bottom row, treated with 100 ng OPGL for 12 h. Right panel: intensity score of histostained osteoclasts after in situ RT-PCR for cathepsin K mRNA.
the amplification is occurring within the log linear range. To assess cathepsin K expression during osteoclast formation, we established primary bone marrow cultures from 2 month-old rat long bones as described previously (28, 29) (see Methods). After plating 1–2.4 ⫻ 10 6 bone marrow cells per well in a 24 well plate, we treated these cultures for 7 days with either 10 nM calcitriol (D3) or a combination of 50 ng/ml human macrophage colony stimulating factory (M-CSF) and 100 ng/ml OPGL (30). We then examined cathepsin K mRNA via in situ RT-PCR (Fig. 2). Almost no cathepsin K mRNA was detected after 7 days of calcitriol. In contrast, M-CSF plus OPGL treatment markedly increased cathepsin K mRNA expression in both monocytic osteoclast precursors and multinucleate osteoclasts. We next examined the time course of osteoclast formation with multiple treatments (Fig. 3A) and subse-
FIG. 2. Cathepsin K mRNA expression in bone marrow-derived osteoclasts. In situ RT-PCR was performed on bone marrow cultures treated for the indicated times with either 10 nM calcitriol (D3) or 50 ng/ml M-CSF plus 100 ng/ml OPGL. CTSK, cathepsin K primers; Ø, no primers used for negative control.
quently assessed cathepsin K mRNA expression (Fig. 3B). Treatment with vehicle failed to yield any tartrate resistant acid phosphatase (TRAP) staining multinucleate osteoclasts at 3 and 7 days. Increasing numbers of osteoclasts developed between 10 and 16 days. Treatment with 50 ng M-CSF alone was unable to induce osteoclast formation throughout the experiment. Treatment with 10 nM calcitriol (D3) alone resulted in occasional osteoclasts at 7 and 10 days and numerous multinucleated cells at 13 and 16 days. Treatment with 100 ng/ml OPGL alone resulted in many osteoclasts by 16 days. However, combined treatment with M-CSF and OPGL resulted in abundant multinucleate osteoclasts as early as 7 days. Interestingly, osteoclast numbers subsequently declined between 10 and 16 days. Combined treatment with D3 plus OPGL also resulted in numerous osteoclasts by day 10. To quantitate cathepsin K mRNA expression accurately in a small number of cells, we performed real time quantitative RT-PCR using the 5⬘-nuclease (Taqman) (see Methods). We performed duplex Taqman assays for cathepsin K mRNA (Fam-labeled probe) and 18S RNA (Vic-labeled probe) (Fig. 3B). The assay was linear over four orders of magnitude (Fig. 4). The slope of the curves in multiple experiments was between 0.03 and 0.08 over four orders of magnitude (Fig. 4 and data not shown). A slope of less than 0.1 indicates that the assay is linear over the range of measurement. We could measure as little as 2.5 pg of total RNA, which comprises as little as 1/10 the RNA found in a single osteoclast. Duplex cathepsin K/18 S Taqman assays were performed to assess both cathepsin K mRNA and 18S RNA expression in 1 ng total RNA for each sample. All values in Fig. 3B are expressed as the ratio of cathepsin K to 18S expression and then further normalized to the no treatment (no Rx) day 3 sample. The
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FIG. 3. Osteoclastogenesis and cathepsin K mRNA expression in bone marrow cultures. (A) Bone marrow cultures were treated for the indicated times with the various agents (Ø Rx, vehicle; MCSF, 50 ng/ml M-CSF; D3, 10 nM calcitriol; OPGL, 100 ng/ml OPGL; MCSF/OPGL, 50 ng/ml M-CSF plus 100 ng/ml OPGL; D3/OPGL, 10 nM calcitriol plus 100 ng/ml OPGL) and stained for TRAP. (B) Duplex real time quantitative RT-PCR (Taqman assay) was performed using RNA from bone marrow cultures treated for the same times and with the same agents as indicated in A. Both cathepsin K and 18 S RNA were simultaneously quantitated. All values in the chart are expressed as the ratio of cathepsin K to 18S expression and then further normalized to the no treatment (no Rx) day 3 sample.
marked increases in cathepsin K mRNA in the various treatment protocols were concordant with the level of osteoclast formation seen in Fig. 3A. M-CSF treatment alone increased cathepsin K expression, consistent with the presence of osteoclast precursors (despite the lack of mature multi-nucleated osteoclasts). In situ RT-PCR confirmed that monocytic osteoclast precursors were responsible for this M-CSF mediated increase in cathepsin K expression. The M-CSF treated cultures also lacked mature osteoblasts (Gyda et al., see companion paper, this issue (33)), suggesting that M-CSF regulated cathepsin K gene expression directly. As shown above, bone marrow cultures treated with M-CSF plus OPGL yielded large numbers of osteoclasts. We therefore pooled four wells (of a 24-well
plate) for each condition, harvested RNA, and performed a Northern analysis for cathepsin K mRNA (Fig. 5). There was a marked increase in expression of cathepsin K mRNA after treatment for 3 and 6 days with 50 ng/ml M-CSF plus 100 ng/ml OPGL (3 and 6 days), consistent with the real time quantitative RTPCR. To our knowledge, little work on cathepsin K has been done in mature osteoclasts harvested from rat long bones or in primary rat bone marrow cultures. Retinoic acid increases cathepsin K gene expression in isolated mature rabbit osteoclasts (2). OPGL stimulates cathepsin K mRNA and protein expression in osteoclasts derived from human peripheral blood mononuclear cell precursors (31). OPGL also enhances
FIG. 4. Primer efficiency of Taqman assay. The relative primer efficiency of the cathepsin K and 18S oligonucleotide pairs was plotted as delta Ct (⫽ cathepsin K Ct ⫺ 18S Ct) versus the log of the total RNA concentration. The Ct (cycle threshold) value reflects the relative amount of the original mRNA and cDNA. Greater amounts of initial transcript result in lower Ct values.
FIG. 5. Northern analysis of bone marrow culture RNA. RNA was harvested from 3 day untreated cultures and from cultures treated for 3 and 6 days with 50 ng/ml M-CSF plus 100 ng/ml OPGL and probed with radiolabeled cathepsin K cDNA or radiolabeled 18S ribosomal cDNA.
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cathepsin K mRNA expression in murine myeloid RAW 264.7 cells concomitant with induction toward an osteoclastic phenotype (32). Here we show that, in mature rat osteoclasts, OPGL exerts an acute effect upon cathepsin K gene expression within 12 h. Since OPGL acts only upon osteoclasts and since cathepsin K is expressed only in osteoclasts (in bone), it is highly unlikely that osteoblasts or stromal cells mediate OPGL induced cathepsin K expression. In conclusion, we provide new data demonstrating dramatic effects of OPGL upon cathepsin K gene expression. We also show that M-CSF enhances cathepsin K expression. The stimulation of cathepsin K expression resulting in matrix degradation may in part explain the pro-resorptive action of OPGL (and M-CSF). Further studies on the regulation of cathepsin K expression by OPGL and other cytokines and hormones may therefore have a direct physiological and therapeutic relevance. ACKNOWLEDGMENTS This study was supported in full by the National Institutes of Health (RO3-AG15628 to B.R.T.) and the Lankenau Institute for Medical Research. B.R.T. acknowledges the support of Dr. Vincent Cristofalo. M.Z. acknowledges support from the NIH (RO1-AG14917) and the Department of Veterans Affairs (Merit Review). B.R.T. and M.Z. together acknowledge the support of Dr. Christine Cassel, the Bronx VA GRECC, and the Brookdale Department of Geriatrics and Adult Development at the Mount Sinai School of Medicine.
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