Cathepsin G Recruits Osteoclast Precursors Via Proteolytic Activation of Protease-Activated Receptor-1

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Published OnlineFirst March 17, 2009; DOI: 10.1158/0008-5472.CAN-08-1956 Published Online First on March 17, 2009 as 10.1158/0008-5472.CAN-08-1956

Research Article

Cathepsin G Recruits Osteoclast Precursors via Proteolytic Activation of Protease-Activated Receptor-1

Thomas J. Wilson, Kalyan C. Nannuru, and Rakesh K. Singh

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska

Abstract As malignant tumor cells enter the bone microenvironment, Metastatic breast cancer shows extreme tropism for the bone they release signaling molecules that initiate a vicious cycle that microenvironment, leading to the establishment of osteolytic promotes both tumor cell growth and osteolysis (2, 3). The rate- metastases. Perpetuation of tumor-induced osteolysis requires limiting step of this cycle is the signaling that occurs between osteoblasts and osteoclasts because this requires cell to cell contact a continuous supply of osteoclast precursors migrating into n the bone microenvironment that can subsequently differenti- to bring receptor activator of nuclear factor- B (RANK) ligand ate into mature osteoclasts and resorb bone. Thus, identifi- (RANKL) on the surface of osteoblasts and RANK on the surface cation and subsequent targeting of chemoattractants of of osteoclast precursors into contact. Our recent data show that osteoclast precursors that are up-regulated at the tumor-bone this requirement can be bypassed via cleavage of RANKL from the interface represents a potential avenue to interrupt osteolysis. cell surface of osteoblasts by cathepsin G, generating soluble We report that cathepsin G, a serine protease, plays a vital RANKL (sRANKL) that relieves the necessity of cell-cell contact and allows widespread osteoclast activation and enhanced osteo- role in the bone microenvironment by modulating tumor- stromal interaction in a manner that favors tumor establish- lysis (4). Cathepsin G expression is up-regulated at the tumor-bone ment and regulates chemotaxis of monocytes, a subset of interface of mammary tumor–induced osteolytic lesions (4). which has the potential to differentiate into osteoclasts. Our However, even with the increased generation of sRANKL, there data show that cathepsin G–induced chemotaxis of monocytes must be continuous recruitment of osteoclast precursors that can is mediated by proteolytic activation of protease-activated become activated for osteolysis to continue to occur. Identification receptor-1 (PAR-1). Attenuation of PAR-1 activation abrogates of a chemoattractant that is up-regulated at the tumor-bone cathepsin G–mediated induction of monocyte chemotaxis. interface that is responsible for recruiting osteoclast precursors We also show that in vivo inhibition of cathepsin G reduces would represent a point in the cycle that could potentially be dis- the number of CD11b+ osteoclast precursors and mature osteo- rupted with therapeutic targeting. Cathepsin G has previously been clasts at the tumor-bone interface. Together, these data sug- shown to be a chemoattractant for monocytes, and a subset of gest that therapeutic targeting of both PAR-1 signaling in monocytes serves as osteoclast precursors (5). Thus, up-regulation osteoclast precursors as well as cathepsin G at the tumor-bone of cathepsin G at the tumor-bone interface is potentially important interface has the potential to reduce osteolysis by inhibiting in perpetuating osteolysis via recruitment of monocytes. Conse- the recruitment, differentiation, and activation of osteoclast quently, in this report, we sought to confirm that cathepsin G is a chemoattractant for osteoclast precursors and then sought to precursors. [Cancer Res 2009;69(7):OF1–8] delineate the signaling pathway used by cathepsin G in the chemoattraction of osteoclast precursors. Introduction One receptor family that has been previously implicated in protease-induced chemoattraction is the protease-activated recep- Nearly 200,000 women in the United States are diagnosed with tors (PAR). These G protein–coupled receptors (GPCR) are breast cancer annually and over 40,000 women every year activated when the NH2 terminus is cleaved by proteases ultimately succumb to breast cancer (1). Of those that succumb generating a tethered ligand that can bind intramolecularly and to their disease, nearly all are found to have bone metastases that initiate signaling (6). The mammalian genome encodes four are predominantly osteolytic in nature (2). These osteolytic lesions members of the PAR family (6). This family of receptors is involved carry significant consequences, including hypercalcemia, increased both directly and indirectly in the chemotaxis of a variety of cell risk of pathologic fracture, and intractable bone pain (2), which types. Direct activation of the PARs has been shown to affect the both dramatically decrease the quality of life and increase mortality migration of eosinophils (7), monocytes (8), and breast cancer cells for breast cancer patients. Thus, understanding the complex (9–12). The PAR family of receptors has also been shown to dialogue that occurs between tumor cells and cells of the bone indirectly influence the migration of prostate cancer cells and microenvironment that distort normal bone physiology in a way monocytes through increased production of monocyte chemo- that favors osteolysis becomes important for designing new attractant protein-1 (MCP-1; refs. 13, 14). Here, we report that therapeutic strategies to combat this serious health problem. cathepsin G–mediated recruitment of osteoclast precursors requires proteolytic activation of PAR-1, which potentially increases osteolysis by indirectly increasing the number of activated osteoclasts. Requests for reprints: Rakesh K. Singh, Department of Pathology and Microbiology, University of Nebraska Medical Center, 985845 Nebraska Medical Center, Omaha, NE 68198-5845. Phone: 402-559-9949; Fax: 402-559-4077; E-mail: Materials and Methods [email protected]. I2009 American Association for Cancer Research. Cathepsin G chemotaxis assay. Human peripheral blood mononu- doi:10.1158/0008-5472.CAN-08-1956 clear cells (PBMC) were isolated from blood from healthy donors using www.aacrjournals.org OF1 Cancer Res 2009; 69: (7). April 1, 2009

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Figure 1. Cathepsin G is a chemoattractant for monocytes via a proteolytic mechanism. A, cathepsin G showed a concentration-dependent induction of monocyte chemotaxis. At the highest concentrations, it induced chemotaxis similar to MCP-1, a known monocyte chemoattractant. Bars, SD. ***, significantly different from cells incubated with medium alone by post hoc analysis using Bonferroni’s correction. B, representative fields from HBSS alone, MCP-1 (10 ng/mL), and cathepsin G (1 Ag/mL), respectively. C, TPCK, an inhibitor of the proteolytic activity of cathepsin G, effectively eliminated cathepsin G–induced chemotaxis of monocytes. Bars, SD. ***, significantly different by post hoc analysis using Bonferroni’s correction.

Fico/Lite-Monocytes (Atlanta Biologicals) per standard procedures. The generated using oligo(dT)18 (Fermentas) and SuperScript II RT (Invitrogen). blood samples were obtained using protocols approved by the Institutional The resulting cDNA (2 AL; 1:10dilution) was used in the reactions with Review Board of the University of Nebraska Medical Center. A chemotaxis gene-specific primers. The following gene-specific primers were used: PAR- assay was set up using a three-tiered chemotaxis chamber (Neuro Probe) 1,5¶-GTGATTGGCAGTTTGGGTCT-3¶ and 5¶-GCCAGACAAGTGAAG- and the following chemoattractants: HBSS alone (negative control); 10and GAAGC-3¶; PAR-2,5¶-TGCTAGCAGCCTCTCTCTCC-3¶ and 5¶-CCAGTGAG- 100 ng/mL of MCP-1 (positive control); and 100 ng/mL, 1 Ag/mL, 5 Ag/mL, GACAGATGCAGA-3¶; PAR-3,5¶-GGCACAGGAGAGCAAACTTC-3¶ and 5¶- and 10 Ag/mL of cathepsin G. Each chemoattractant was plated in CTGCAAAGATGAGGGCTTTC-3¶; PAR-4,5¶-CAGACTTCCTGGAGGAGTGG- triplicate. For each well, ten 250-Am fields were counted. 3¶ and 5¶-GTTGTTGTTGAGCTCGTTGG-4¶; and glyceraldehyde-3-phosphate N-tosyl-L-phenylalanine chloromethyl ketone inhibition chemotaxis dehydrogenase,5¶-AGCCTCGTCCCGTAGACAAAA-3¶ and 5¶-GATGA- assay. To test whether the proteolytic activity of cathepsin G is necessary CAAGCTTCCCATCTCG-3¶. PCRs were carried out using a PTC-100 for its ability to serve as a chemoattractant, the chemotaxis assay was Programmable Thermal Controller (MJ Research) under optimal conditions. repeated using HBSS, 1 Ag/mL cathepsin G, and 1 Ag/mL cathepsin G PCR fragments were separated on 2% ethidium bromide containing agarose preincubated with 1 mmol/L N-tosyl-L-phenylalanine chloromethyl ketone gel and analyzed using a MultiImage Light Cabinet (Alpha Innotech Corp.). (TPCK; Sigma-Aldrich) for 30min at 37 jC. TPCK is an inhibitor of the PAR activation chemotaxis assay. To determine if PAR-1 activation chymotrypsin-like group of proteases and is a potent inhibitor of cathepsin leads to chemotaxis of monocytes, the chemotaxis assay was repeated G (4, 15, 16). TPCK has also previously been shown to be nontoxic to using HBSS alone (negative control), 10and 100ng/mL of MCP-1 (positive osteoclast precursors/osteoclasts (4). control), 1 and 5 Ag/mL of cathepsin G, and 1 and 5 Ag/mL of PAR-1 PAR expression in monocytes. Human PBMCs were isolated as agonist (AnaSpec). Because PBMCs do not express PAR-2, PAR-2 agonist described. Total RNA was isolated using Trizol reagent (Invitrogen). Total (AnaSpec) was also used as a negative control at concentrations of 1 and RNA (5 Ag) was used for reverse transcription. First-strand cDNA was 5 Ag/mL.

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Cathepsin G and Osteoclast Recruitment

Figure 2. PAR-1 activation results in induction of monocyte chemotaxis. A, human monocytes expressed PAR-1 and PAR-3 at the mRNA level but not PAR-2 or PAR-4. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, PAR-1 peptide agonist induced chemotaxis of monocytes but less than cathepsin G at similar concentrations. PAR-2 peptide agonist had no effect on monocyte chemotaxis because monocytes do not express PAR-2. Bars, SD. ***, significantly different from cells incubated with medium alone by post hoc analysis using Bonferroni’s correction.

PAR-1 inhibition chemotaxis assay. To evaluate the role of PAR-1 in were incubated overnight. The following day, the concentration of FBS was cathepsin G–mediated chemoattraction, the chemotaxis assay was repeated reduced to 1%. The cells were then allowed to grow for 48 h, at which time using HBSS (negative control), 5 Ag/mL cathepsin G, 5 Ag/mL cathepsin G conditioned medium was collected for each of the cell lines. plus blocking anti–PAR-1 antibody, and 5 Ag/mL cathepsin G plus RAW 264.7 cells were then seeded onto six-well plates at a density nonblocking anti–PAR-1 antibody. The blocking antibody (Santa Cruz of 5,000/cm2. The cells were then grown in DMEM complete medium plus Biotechnology) and the nonblocking antibody (Santa Cruz Biotechnology) 10%, 25%, or 50% Cl66-conditioned medium. Simultaneously, Cl66 cells were both used at a final dilution of 1:200 (17). were seeded onto six-well plates at a density of 5,000/cm2. The cells were Pertussis toxin/cholera toxin inhibition chemotaxis assay. To then grown in DMEM complete medium plus 10%, 25%, or 50% conditioned determine what subunits of the GPCR are activated during cathepsin medium from RAW 264.7 cells. At the end of 4 d, total RNA was isolated as G–mediated chemotaxis, the chemotaxis assay was repeated using HBSS, previously described and quantitative reverse transcription-PCR was done 5 Ag/mL cathepsin G, and 5 Ag/mL PAR-1 agonist using monocytes that for cathepsin G using gene-specific primers. had been preincubated with 100 ng/mL pertussis toxin (PTX; Sigma- In vivo inhibition of cathepsin G using TPCK. Cathepsin G function Aldrich) for 2 h at 37jC, monocytes that had been preincubated with was inhibited using TPCK in a murine bone invasion model as previously 10 Ag/mL cholera toxin (CTX) for 2 h at 37jC, or monocytes not pretreated. described (4). Cl66 tumor cells (1 Â 105) mixed with growth factor–reduced Conditioned medium treatment of RAW 264.7 and Cl66 cells. RAW Matrigel were implanted on the dorsal skin flap over the calvaria of female 264.7, a murine monocyte/macrophage cell line, and Cl66 mammary tumor BALB/c mice. Tumor growth was monitored twice a week. Beginning 7 d cells were seeded onto 100-mm dishes at a density of 1 Â 106 per dish. Cells after tumor implantation, mice were injected s.c. with TPCK at 50mg/kg/d were grown in DMEM plus 10% fetal bovine serum (FBS), 1% vitamins, 1% (n =4)or50ALDMSO(n = 4) for 21 d. Mice were sacrificed at day 31 after L-glutamine, and 0.08% gentamicin (DMEM complete medium). The cells implantation and necropsied for examination of osteolytic lesions. All studies were done in accordance with the Institutional Animal Use and Care Committee of the University of Nebraska Medical Center. Immunohistochemistry for CD11b. CD11b is a marker for osteoclast precursors (18–25). Sections from TPCK-treated animals or control (DMSO)– treated animals were rehydrated and endogenous peroxidase activity was quenched. Antigen retrieval was then performed by boiling sections in 10mmol/L sodium citrate buffer (pH 6.0)for 11 min. Sections were blocked using goat serum. Sections were then incubated overnight at 4jCwith antibody directed against CD11b (Caltag Laboratories) diluted 1:20in blocking solution. After washing, sections were incubated for 1 h at room temperature with biotinylated anti-rat IgG diluted 1:200. After washing again, sections were incubated with avidin-biotin complex (Vectastain ABC, Vector Laboratories) for 45 min at room temperature. Sections were then washed and developed using 3,3¶-diaminobenzidine (Vector Laboratories) substrate. The sections were then counterstained with hematoxylin. Species-specific IgG isotype was added in lieu of primary antibody as a negative control, and these sections showed no detectable staining. For each section, f8,000 cells were counted. The CD11b staining index was calculated by dividing the number of Figure 3. Cathepsin G induces chemotaxis of monocytes via proteolytic labeled cells by the total number of cells and multiplying by 100. The CD11b activation of PAR-1. Blocking antibody to PAR-1 reduced but did not eliminate staining index was calculated for each section. cathepsin G–induced monocyte chemotaxis. A nonblocking antibody also directed against PAR-1 had no effect. Bars, SD. ***, significantly different by post Tartrate-resistant acid phosphatase staining. Sections from TPCK- hoc analysis using Bonferroni’s correction. treated animals or control (DMSO)–treated animals were rehydrated using www.aacrjournals.org OF3 Cancer Res 2009; 69: (7). April 1, 2009

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from 100 ng/mL to 10 Ag/mL (Fig. 1A and B). Even in response to as little as 100 ng/mL cathepsin G, induction of chemotaxis was statistically significant compared with control. The highest concentration used, 10 Ag/mL, induced chemotaxis at a level similar to 100 ng/mL MCP-1 (Fig. 1A and B). Cathepsin G–induced chemotaxis of monocytes occurs via a proteolytic mechanism. Next, we used TPCK, an inhibitor of the proteolytic activity of cathepsin G, to examine whether proteolytic activity is necessary for induction of chemotaxis. We observed a

Figure 4. Cathepsin G–mediated activation of PAR-1 signals through the Gai/o subunits. A, PTX, an inhibitor of the Gai/o subunits of the GPCR, eliminated the ability of cathepsin G to induce monocyte chemotaxis, whereas CTX, an inhibitor of the Gas subunit, had no effect. ***, significantly different by post hoc analysis using Bonferroni’s correction. B, PTX reduced PAR-1 agonist–induced monocyte chemotaxis, whereas CTX had no effect. Bars, SD. ***, significantly different by post hoc analysis using Bonferroni’s correction. a series of xylenes and ethanols. Then, the slides were incubated in tartrate- resistant acid phosphatase (TRAP) staining solution (Sigma-Aldrich) at 37jC for 2 h. After washing with deionized water for 10min, counter- staining was done with Gill Hematoxylin 3 solution for 2 min. Slides were washed with running tap water for 10min and then mounted with aqueous mount. The total number of TRAP-positive, multinucleated osteoclasts over the entire length of the tumor-bone interface was then counted for each section using a light microscope. The total number of osteoclasts was then divided by the length of the tumor-bone interface to get the number of osteoclasts per millimeter of tumor-bone interface. Statistical analysis. ANOVA was performed for statistical comparison of multiple groups. P < 0.05 was considered significant. If significance was found in the ANOVA, then post hoc analysis was performed to compare specific groups using Bonferroni’s correction, where the significance level was determined by dividing 0.05 by the number of comparisons to be made. Figure 5. Source of cathepsin G. A, cathepsin G expression was tested by lane 1 Where only two groups were tested, the Student’s t test was used. P < 0.05 PCR in human PBMCs ( ), RAW 264.7 (a murine monocyte/macrophage cell line; lane 2), 4T1 (lane 3), Cl66 (lane 4), and Cl66M2 (murine breast was considered significant. adenocarcinoma cell lines; lane 5). All cell lines expressed cathepsin G at the RNA level. B, Cl66 cells were cultured with conditioned medium from RAW 264.7 cells and the expression of cathepsin G was compared with Cl66 baseline Results expression. RAW 264.7–conditioned medium (CM) increased expression of cathepsin G in Cl66. Bars, SD. *, P < 0.01. C, RAW 264.7 cells were cultured Cathepsin G is a chemoattractant for monocytes. MCP-1 is with conditioned medium from Cl66 cells and the expression of cathepsin G was known to induce monocyte chemoattraction. It was used in a compared with RAW 264.7 baseline expression. Cl66-conditioned medium monocyte chemotaxis assay as a positive control, whereas HBSS markedly increased expression of cathepsin G in RAW 264.7. Bars, SD. *, P < 0.05; **, P < 0.01. D, for each of the treatment groups and at baseline, alone was used as a negative control. Cathepsin G showed a expression of cathepsin G was higher in RAW 264.7 cells (dotted columns) concentration-dependent induction of monocyte chemotaxis than in Cl66 cells (striped columns).

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Cathepsin G and Osteoclast Recruitment

Figure 6. Inhibition of cathepsin G reduces osteoclast precursors and mature osteoclasts at the tumor-bone interface. A, sections stained with anti-CD11b antibody, a marker for osteoclast precursors. Scale bars, 0.01 mm. B, the CD11b staining index was significantly decreased in TPCK-treated animals, showing that there are fewer osteoclast precursors at the tumor-bone interface. Bars, SD. *, P < 0.05; **, P < 0.01. C, serial sections stained for TRAP, a marker of osteoclasts (arrows). Scale bars, 0.01 mm. D, the number of mature, multinucleated, TRAP+ osteoclasts at the tumor-bone interface was counted and then divided by the total length of the tumor-bone interface to calculate the number of osteoclasts per millimeter of tumor-bone interface. TPCK treatment significantly reduced the number of osteoclasts present at the tumor-bone interface compared with control treatment. Bars, SD. *, P < 0.05; **, P < 0.01.

significant inhibition of cathepsin G–induced chemotaxis of mono- PAR-2. As expected, the PAR-2 agonist did not induce chemotaxis cytes following TPCK treatment (Fig. 1C). These data show that of monocytes and did not differ from HBSS alone (Fig. 2B). The induction of chemotaxis of monocytes by cathepsin G occurs via a PAR-1 agonist did induce chemotaxis of monocytes at a level that proteolytic mechanism. was statistically significant compared with control but did not Monocytes express PAR-1 and PAR-3. PCR was used to exam- induce the same level of chemotaxis as cathepsin G at similar ine the expression of the PARs by monocytes. Monocytes express concentrations (Fig. 2B). both PAR-1 and PAR-3 but do not express PAR-2 or PAR-4 at Cathepsin G induces chemotaxis of monocytes via proteo- the mRNA level (Fig. 2A). PAR-1 was expressed at a higher level lytic cleavage of PAR-1. To attempt to show that the mechanism than PAR-3. by which cathepsin G induces chemotaxis of monocytes is via PAR-1 activation induces chemotaxis of monocytes. To proteolytic cleavage of PAR-1, blocking antibody to PAR-1 was determine if PAR-1 activation is potentially involved in cathepsin used. Blocking antibody to PAR-1 significantly reduced cathepsin G–induced chemotaxis of monocytes, a PAR-1 peptide agonist G–mediated chemotaxis, although it did not eliminate it (Fig. 3). was used to evaluate chemotaxis. A PAR-2 peptide agonist was A nonblocking antibody to PAR-1 had no effect on the ability of also used as a negative control because monocytes do not express cathepsin G to induce chemotaxis of monocytes (Fig. 3), confirming www.aacrjournals.org OF5 Cancer Res 2009; 69: (7). April 1, 2009

Cancer Research that proteolytic cleavage of PAR-1 mediates cathepsin G–induced enhances RANKL-mediated osteoclast activation (4). In this study, chemotaxis of monocytes. we have identified an additional role for cathepsin G in the Cathepsin G–mediated activation of PAR-1 results in induction of chemotaxis of osteoclast precursors to the site of signaling via GAi/o. The PARs are GPCRs that are known to signal metastasis. We hypothesized that as tumor cells enter the bone through a variety of pathways (26, 27). PTX is a known inhibitor of microenvironment, a cycle thus ensues whereby osteoclasts pro- the Gai/o subunits of GPCRs, whereas CTX is a known inhibitor of duce increased cathepsin G as they interact with tumor cells, which the Gas subunit of GPCRs (28). PTX eliminated the ability of the recruits additional osteoclast precursors that can differentiate PAR-1 agonist to induce chemotaxis, whereas CTX had no effect, and produce more cathepsin G, leading to further recruitment suggesting that PAR-1 is inducing chemotaxis via the Gai/o sub- of osteoclast precursors. The amplification of cathepsin G produc- units (Fig. 4). Similarly, PTX reduced cathepsin G–induced tion and continuous recruitment of osteoclast precursors ensures chemotaxis, whereas CTX had no effect, suggesting that cathepsin perpetuation of the cycle. G–induced chemotaxis occurs, at least partially, through a Gai/o Induction of chemotaxis of monocytes is dependent on the subunit–mediated mechanism (Fig. 4). proteolytic activity of cathepsin G. One family of receptors Interaction of mammary tumor cells with osteoclast that requires proteolytic cleavage to be activated and that has precursors regulates cathepsin G expression. To determine previously been shown to play a role in chemotaxis is the PARs. which cells up-regulate the expression of cathepsin G during the We found that monocytes express both PAR-1 and PAR-3. Be- migration of breast tumor cells into the bone microenvironment, cause PAR-1 is extensively involved in chemotaxis (7, 12, 14) and we examined whether conditioned medium from Cl66 cells mod- it was expressed at the highest level by monocytes, we first ulates cathepsin G expression by osteoclast precursors and vice examined whether cathepsin G–induced chemotaxis occurs via versa. First, cathepsin G baseline mRNA expression was examined PAR-1 activation. in human PBMCs and RAW 264.7 cells, a murine monocyte/ Activation of PAR-1 by a small peptide, PAR-1 agonist, induced macrophage cell line, as well as in the mammary tumor cell lines chemotaxis of monocytes but to a lesser extent than similar 4T1, Cl66, and Cl66M2. All cell lines expressed cathepsin G at the concentrations of cathepsin G. Because monocytes do not express mRNA level (Fig. 5A). Cl66 cells increased expression of cathepsin PAR-2, we used a small peptide, PAR-2 agonist, as a negative G when treated with RAW 264.7–conditioned medium (Fig. 5B). control. To link cathepsin G–mediated induction of chemotaxis to Similarly, RAW 264.7 cells increased cathepsin G expression PAR-1 activation, we used two different anti–PAR-1 antibodies: a when treated with Cl66-conditioned medium (Fig. 5C). Although blocking antibody that prevents proteolytic activation and a Cl66 cells showed more marked up-regulation of cathepsin G nonblocking antibody that recognizes PAR-1 but does not prevent expression, the baseline expression was higher in RAW 264.7 cells proteolytic activation. The blocking antibody significantly reduced (Fig. 5D). Thus, RAW 264.7 cells treated with Cl66-conditioned induction of chemotaxis. Conversely, the nonblocking antibody medium showed higher overall expression of cathepsin G than Cl66 had no effect. Taken together, these results confirm that cathepsin cells treated with RAW 264.7–conditioned medium. G–mediated induction of chemotaxis is partially mediated by Inhibition of cathepsin G in vivo reduces osteoclast proteolytic activation of PAR-1. precursors at the tumor-bone interface. We used CD11b as a PAR-1 is a GPCR that initiates signaling through several different marker for osteoclast precursors (18–25). We stained sections from pathways. Because PAR-1 mediates numerous effects through a the tumor-bone interface of mice bearing Cl66 tumors treated with myriad of signaling pathways, we sought to determine if Gas and/ either control treatment (DMSO) or TPCK, a cathepsin G inhibitor. or Gai/o are involved in cathepsin G–mediated induction of + There were significantly less CD11b osteoclast precursors at the chemotaxis. PTX constitutively activates Gai/o subunits, which tumor-bone interface of TPCK-treated animals (Fig. 6A and B). prevents further activation, whereas CTX constitutively activates in vivo Inhibition of cathepsin G reduces osteoclasts at the the Gas subunit, preventing further activation (28). Monocytes tumor-bone interface. TRAP staining was used to identify mature, pretreated with CTX did not show reduced chemoattraction in multinucleated osteoclasts at the tumor-bone interface. We stained response to cathepsin G. PTX significantly reduced cathepsin sections from Cl66 tumor-bearing mice that were treated with G–mediated induction of chemotaxis, which shows the involve- either control treatment (DMSO) or TPCK. Treatment with TPCK ment of the Gai/o subunits. significantly reduced the number of osteoclasts at the tumor-bone We have previously reported that cathepsin G is up-regulated at interface (Fig. 6C and D). the tumor-bone interface of mammary tumor–induced osteolytic lesions (4). To evaluate the source of cathepsin G in the bone microenvironment, we looked at expression in 4T1, Cl66, and Discussion Cl66M2 (murine breast cancer cell lines), RAW 264.7 cells (murine As mammary tumor cells enter the bone microenvironment, monocyte/macrophage cell line), and human PBMCs. All cell they secrete soluble factors that initiate a vicious cycle leading to lines tested expressed cathepsin G at the mRNA level. We observed both tumor growth and osteolysis (2). Continuation of the vicious up-regulation of cathepsin G in both RAW 264.7 cells treated cycle depends on a continuous supply of osteoclast precursors that with Cl66-conditioned medium and Cl66 cells treated with RAW can be activated by RANKL. Without this influx of osteoclast 264.7–conditioned medium, suggesting that both osteoclasts and precursors to the site of metastasis, the cycle would be disrupted. tumor cells likely contribute to the observed up-regulation of A subset of the hematopoietic lineage of monocytes/macrophages cathepsin G at the tumor-bone interface. Although the most serves as osteoclast precursors. To date, the chemoattractant significant up-regulation was observed in the Cl66 cells treated responsible for ensuring this continuous supply of osteoclast with RAW 264.7–conditioned medium, the baseline expression precursors remains unclear. of cathepsin G was higher in RAW 264.7 cells. Consequently, Cathepsin G plays an important role in amplifying the vicious RAW 264.7 cells treated with Cl66-conditioned medium showed cycle by shedding the extracellular domain of RANKL, which higher expression of cathepsin G than did Cl66 cells treated

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Cathepsin G and Osteoclast Recruitment with RAW 264.7–conditioned medium. Hence, although both observe significantly fewer mature osteoclasts at the tumor-bone Cl66 cells and osteoclasts up-regulate cathepsin G expression as interface of TPCK-treated animals. This suggests that inhibition tumor cells interact with the bone microenvironment, osteoclasts of cathepsin G does, in fact, reduce the number of osteoclast likely make the most significant contribution. This agrees with our precursors migrating to the tumor-bone interface, which in turn previously reported immunohistochemistry data that showed depletes the pool of osteoclast precursors and reduces the number moderate positivity for cathepsin G in Cl66 cells at the tumor- of osteoclasts. bone interface and strong positivity for cathepsin G in osteo- Little is known about cathepsin G in the physiologic bone clasts (4). microenvironment. In fact, cathepsin G knockout mice have no Serine proteases such as cathepsin G are stored in azurophilic morphologic defects in bone development (38), suggesting that granules of neutrophils, and the major physiologic function of cathepsin G may be an ideal target in mammary tumor–induced these proteinases is commonly thought to be the intralysosomal bone lesions as side effects would likely be minimal. Furthermore, degradation of engulfed debris or microorganisms. However, it has despite apparently normal bone development in cathepsin G become evident that cathepsin G plays a crucial role in extra- knockout mice and no known physiologic roles for cathepsin cellular proteolytic processes at sites of inflammation (4, 29–36). G in the bone microenvironment, other pathophysiologic roles in Our immunohistochemical analysis shows diffused cytoplasmic the bone microenvironment have been established, including cathepsin G expression in osteoclasts and malignant cells at the periodontitis (39), rheumatoid arthritis (40), and tumor-induced tumor-bone interface (4). In our in vitro coculture experiments, osteolysis (4). we observed cathepsin G activity in cell-free supernatant (data not In conclusion, we report that cathepsin G serves as a chemo- shown). A previous report has shown that cathepsin G is found attractant for osteoclast precursors via proteolytic activation of in gingival tissue during periodontal disease (37). Furthermore, PAR-1. The up-regulation of cathepsin G at the tumor-bone inter- activation of neutrophils with inflammatory cytokines induced face of mammary tumor–induced osteolytic lesions thus serves to release of cathepsin G into the extracellular space and increased increase osteoclast differentiation via the generation of sRANKL the membrane-bound form of catalytically active cathepsin G (4) as well as by ensuring there is a continuous supply of osteo- (5, 32, 34–37). clast precursors that can be differentiated by the newly generated These data, taken as a whole, suggested that inhibition of sRANKL. Other factors, including interleukin-8, are also likely cathepsin G in vivo would potentially reduce the number of involved. However, for the first time, we report the involvement osteoclast precursors at the tumor-bone interface, which would of cathepsin G and have delineated portions of its downstream have the potential to reduce osteolysis. We have previously signaling pathway. reported that inhibition of cathepsin G in vivo does, in fact, significantly reduce the bone destruction index (4). Here, we show Disclosure of Potential Conflicts of Interest that one potential mechanism for the observed reduction in the bone destruction index, in addition to the reduction in sRANKL No potential conflicts of interest were disclosed. that we previously reported (4), is a reduction in the number of osteoclast precursors at the tumor-bone interface. We used CD11b Acknowledgments as a marker for osteoclast precursors (18–25) and observed + Received 5/23/08; revised 1/9/09; accepted 2/4/09; published OnlineFirst 3/17/09. significantly fewer CD11b osteoclast precursors in TPCK-treated Grant support: NIH grant CA72781 and Cancer Center Support Grant animals, suggesting that cathepsin G is responsible for the P30CA036727 (R.K. Singh), Nebraska Department of Health and Human Services, Nebraska Research initiative (R.K. Singh), and Howard Hughes Medical Institute recruitment of osteoclast precursors to the site of mammary Research Training Fellowship (T.J. Wilson). T.J. Wilson is a Howard Hughes Medical tumor–induced osteolysis. Institute Research Training Fellow. The question remaining to be answered was whether reduc- The costs of publication of this article were defrayed in part by the payment of page + charges. This article must therefore be hereby marked advertisement in accordance tion of CD11b cells at the tumor-bone interface would translate with 18 U.S.C. Section 1734 solely to indicate this fact. into a reduction in the number of mature osteoclasts. We did We thank Dr. Dhirendra P. Singh for careful review and critique of the manuscript.

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Cancer Res 2009; 69: (7). April 1, 2009 OF8 www.aacrjournals.org

Cathepsin G Recruits Osteoclast Precursors via Proteolytic Activation of Protease-Activated Receptor-1

Thomas J. Wilson, Kalyan C. Nannuru and Rakesh K. Singh

Cancer Res Published OnlineFirst March 17, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-08-1956

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