Role of interleukin-8 in PiT-1 expression and CXCR1-mediated inorganic phosphate uptake in chondrocytes.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 1, January 2005, pp 144–154 DOI 10.1002/art.20748 © 2005, American College of Rheumatology Role of Interleukin-8 in PiT-1 Expression and CXCR1-Mediated Inorganic Phosphate Uptake in Chondrocytes Denise L. Cecil, David M. Rose, Robert Terkeltaub, and Ru Liu-Bryan Objective. The proinflammatory chemokine interleukin-8 (IL-8) induces chondrocyte hypertrophy. Moreover, chondrocyte hypertrophy develops in situ in osteoarthritic (OA) articular cartilage and promotes dysregulated matrix repair and calcification. Growth plate chondrocyte hypertrophy is associated with expression of the type III sodium-dependent inorganic phosphate (Pi) cotransporter phosphate transporter/ retrovirus receptor 1 (PiT-1). This study was undertaken to test the hypothesis that IL-8 promotes chondrocyte hypertrophy by modulating chondrocyte PiT-1 expression and sodium-dependent Pi uptake, and to assess differential roles in this activity. Methods. The selective IL-8 receptor CXCR1 and the promiscuous chemokine receptor CXCR2 were used. Human knee OA cartilage, cultured normal bovine knee chondrocytes, and immortalized human articular chondrocytic CH-8 cells were transfected with CXCR1/ CXCR2 chimeric receptors in which the 40–amino acid C-terminal cytosolic tail domains were swapped and site mutants of a CXCR1-specific region were generated. Results. Up-regulated PiT-1 expression was detected in OA cartilage. IL-8, but not IL-1 or the CXCR2 ligand growth-related oncogene ␣, induced PiT-1 ex- pression and increased sodium-dependent Pi uptake by >40% in chondrocytes. The sodium/phosphate cotransport inhibitor phosphonoformic acid blocked IL-8– induced chondrocyte hypertrophic differentiation. Signaling mediated by kinase Pyk-2 was essential for IL-8 induction of PitT-1 expression and Pi uptake. Signaling through the TSYT346–349 region of the CXCR1 cytosolic tail, a region divergent from the CXCR2 cytosolic tail, was essential for IL-8 to induce Pi uptake. Conclusion. Our results link low-grade IL-8– mediated cartilaginous inflammation in OA to altered chondrocyte differentiation and disease progression through PiT-1 expression and sodium-dependent Pi uptake mediated by CXCR1 signaling. Low-grade inflammation in both cartilage and synovium regulates pathogenesis and disease progression in osteoarthritis (OA) (1). Interleukin-1 (IL-1) stimulates chondrocyte oxidative stress and matrix degradation (1). Moreover, IL-1 has been demonstrated to be a significant contributor to the development of experimental OA (1). However, up-regulated expression of other proinflammatory cytokines within cartilage also can contribute to the pathogenesis of OA (1). One such cytokine is the CXC chemokine subfamily member IL-8 (CXCL8) (2–4), which was initially recognized to actively modulate leukocyte-driven inflammation, primarily by regulating the differentiation and the migratory, adhesive, and killing functions of hematopoietic-lineage cells (5). Recently, we demonstrated that IL-8 also affects the differentiation and function of articular chondrocytes, cells of mesenchymal origin (2). Specifically, IL-8 (but not IL-1␤) stimulates the maturation of resting chondrocytes to a state of hypertrophic differentiation in vitro (2). Chondrocyte hypertrophy, first characterized as a central physiologic event in growth plate development (6), also develops in OA cartilage, where it can contrib- Dr. Rose’s work was supported by a Department of Veterans Affairs Merit Review Entry Program award, by the Arthritis Foundation, and by the NIH (grant P30-AR-47360). Dr. Terkeltaub’s work was supported by a Department of Veterans Affairs Merit Review award and by the NIH (grants P01-AG0-7996, AR-47347, and R01HL-077360). Dr. Liu-Bryan’s work was supported by the NIH (grant R03-AR-49416-10). Denise L. Cecil, BS, David M. Rose, DVM, PhD, Robert Terkeltaub, MD, Ru Liu-Bryan, PhD: Veterans Affairs Medical Center, University of California, San Diego. Drs. Terkeltaub and Liu-Bryan contributed equally to this work. Address correspondence and reprint requests to Robert Terkeltaub, MD, VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail: firstname.lastname@example.org. Submitted for publication June 24, 2004; accepted in revised form September 21, 2004. 144 IL-8 AND PiT-1 IN CHONDROCYTE PHOSPHATE UPTAKE ute to dysregulation of matrix repair, partly via alterations in collagen subtype and increased matrix metalloproteinase (MMP) expression (7–10). Chondrocytes that have undergone hypertrophic differentiation in OA cartilage also promote pathologic calcification (9), analogous to the critical role of resting chondrocyte maturation to hypertrophy in physiologic growth plate mineralization (6,8,10). IL-8 is a ligand of both the promiscuous chemokine receptor CXCR2 and the highly selective IL-8 receptor CXCR1 (5,11). We previously observed that growth-related oncogene ␣ (GRO␣) (CXCL1), a CXCR2-selective ligand, shares the capacity of IL-8 to induce chondrocyte hypertrophy (2). In addition, our findings have implicated p38 MAPK signaling and upregulated activity of the transglutaminase (TGase) isoenzyme TG2 (8) as essential mediators of chondrocyte hypertrophy in response to IL-8 and to GRO␣ (2). A primary objective of the present study was to identify further effects of IL-8–induced signaling involved in chondrocyte differentiation, and in doing so, to test the hypothesis that CXCR1 and CXCR2 have differential roles in IL-8–induced chondrocyte hypertrophy. CXCR1 and CXCR2 are 76% identical “7-membrane spanning” G protein–coupled receptors, with the principal divergences in sequence homology at their ligand-binding N-terminal domains and their signal-transducing cytosolic C-terminal domains (11). They are expressed in situ in normal and OA cartilage (12,13). Furthermore, differential signaling and functions via CXCR1 and CXCR2 have been observed in leukocytes that express both receptors concurrently (14–16). We previously demonstrated that IL-8 induces alkaline phosphatase (2), an ectoenzyme in chondrocytes (6) that generates inorganic phosphate (Pi). Importantly, alkaline phosphatase up-regulation occurs in association with chondrocyte hypertrophy (2,6). Moreover, exogenous Pi promotes maturation of growth plate chondrocytes in vitro (17,18). The uptake of Pi modulates cell differentiation via signals that produce regulatory effects on expression of certain Pi-sensitive genes, on ATP synthesis, on the nuclear export of proteins including cbfa1, and on mitochondrial function (17–23). Sodium-dependent phosphate cotransporters exert major effects on the cellular uptake of Pi in cells, including growth plate chondrocytes (21,24). Furthermore, chondrocytes, like osteoblasts, selectively express sodium-dependent phosphate cotransporters of the type III class, including phosphate transporter/retrovirus receptor 1 (PiT-1; also known as Glvr-1) (25–30). Our results in the present study revealed up-regulated PiT-1 145 expression in OA cartilage. Because PiT-1 has been observed to regulate differentiation and calcification in mineralization-competent cells in vitro (25–30), we then elected to test the hypothesis that IL-8 promotes chondrocyte hypertrophy partly by PiT-1 expression and sodium-dependent Pi uptake. We demonstrated CXCR1-dependent effects on chondrocyte differentiation and function that are mediated through regulation of PiT-1 expression and sodium-dependent Pi uptake. In addition, we found that a TSYT motif unique to the CXCR1 cytosolic tail was a critical mediator of IL-8– induced sodium-dependent Pi uptake in chondrocytic cells. MATERIALS AND METHODS Reagents. Recombinant human IL-1␤, IL-8, and GRO␣ were purchased from R&D Systems (Minneapolis, MN), and rabbit antibody to type X collagen from Calbiochem (San Diego, CA). The human PiT-1 complementary DNA (cDNA) construct in the vector pCMV-SPORT6 (ResGen clone 3918690) was obtained from Invitrogen (Carlsbad, CA) and verified, by sequencing, to be full-length PiT-1. Myctagged wild-type Pyk-2 and an inhibitory mutant of Pyk-2 (Pyk-2–Y402F) in pcDNA3.1 vector were generously provided by Dr. D. Schlaepfer (Scripps Research Institute, La Jolla, CA). Unless otherwise indicated, all other reagents were from Sigma (St. Louis, MO). Chondrocytic cells used and culture and transfection methods. Human knee articular chondrocytes and normal adult bovine knee articular chondrocytes (which were more consistently available to us than normal human articular chondrocytes) were isolated and cultured as previously described (2). Briefly, primary chondrocytes were obtained from articular cartilage slices from the medial and lateral condyles, the patellar groove, and the tibial plateau, with primary cells placed in monolayer culture in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% fetal calf serum (FCS), 1% L-glutamine, 100 units/ml penicillin, and 50 g/ml streptomycin at 37°C with 5% CO2. For treatment of first-passage chondrocytes under nonadherent conditions, aliquots of 0.1 ⫻ 106 cells were plated on polyHEME-coated 96-well round-bottomed plates in DMEM high glucose supplemented with 1% FCS, 100 units/ml penicillin, and 50 g/ml streptomycin (2). CH-8 cells, a previously described line of immortalized normal human knee articular chondrocytes (31), were generously provided by Dr. M. Hiramoto (Nihon University School of Medicine, Tokyo, Japan). The CH-8 cells were studied between passages 5 and 15 under the same culture conditions described above, under which maintenance of type II collagen and aggrecan expression was confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) as described (32). For transfection studies, aliquots of 1.2 ⫻ 106 bovine chondrocytes were plated on 100-mm dishes, or aliquots of 0.5 ⫻ 106 CH-8 chondrocytes on 60-mm dishes were grown for 18 hours in DMEM high glucose containing 10% serum. Then, transfection of human PiT-1 cDNA was performed in bovine 146 chondrocytes using a previously described FuGENE 6– (Roche Applied Science, Indianapolis, IN) and hyaluronidasebased method (33). The chondrocytic CH-8 cells were transfected using Lipofectamine Plus, according to the instructions of the manufacturer (Invitrogen). Transfection efficiency, assessed as a control in each experiment via ␤-galactosidase transfection and staining (34), was consistently between 20% and 25% in bovine chondrocytes and averaged 84.2 ⫾ 3.4% (mean ⫾ SD; n ⫽ 20) in CH-8 cells. To verify expression of CXCR1 and CXCR2, we assessed CXCR1 and CXCR2 expression by fluorescence-activated cell sorter (FACS) analysis, using murine monoclonal anti-CXCR1 (BioSource International, Camarillo, CA) and anti-CXCR2 (PharMingen, San Diego, CA), as described previously (2). Assays of Pi uptake. To quantify sodium-dependent 33 Pi uptake in chondrocytic cells, we modified a previously described method (35). Briefly, cells were washed with buffer A (1 mM CaCl2, 1.8 mM MgCl2, 10 mM HEPES [pH 7.4]) and were phosphate starved for 2 hours at 37°C in 200 l of the same buffer. We then added 200 l of buffer A containing 150 mM NaCl and 3 Ci/ml carrier-free 33P orthophosphate (ICN Biomedicals, Costa Mesa, CA) to the cells for 5 minutes. The time frame for sodium-dependent Pi uptake was terminated by adding 1 ml ice-cold sodium-free buffer A, and the absence of 33 Pi uptake was verified in the total absence of added sodium. Extensively washed cells were solubilized with 0.2N NaOH, and cell-associated 33Pi (counts per minute) was normalized per microgram cell DNA, measured spectrophotometrically (32). Immunohistochemistry studies for PiT-1. Rabbit polyclonal antibodies recognizing the human PiT-1–specific peptide CDSFRAKEGEQKGEE were generated at Zymed (South San Francisco, CA). Specimens of normal and OA human articular cartilage were obtained as full-thickness blocks, as previously described (33). For immunohistologic analyses of PiT-1, frozen sections (5) were fixed for 20 minutes using 4% paraformaldehyde. After washing with phosphate buffered saline, the sections were incubated with 1% Triton X-100 and microwaved for 1 minute, blocked with 10% goat serum for 20 minutes, and incubated for 24 hours at 4oC with rabbit anti–PiT-1. The primary antibody was detected using the avidin–biotin conjugate method with the Rabbit ExtrAvidin Peroxidase Staining kit, according to the instructions of the manufacturer (Sigma). Peroxidase activity was detected using the Fast DAB staining kit, according to the instructions of the manufacturer (Sigma). In control studies, we mixed antibodies to PiT-1 with a 5-fold excess of the immunogenic PiT-1 polypeptide and incubated for 18 hours at 4oC before applying this mixture to sections. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting, Pyk-2 activation assay, and RT-PCR analyses. For preparation of cell lysates, cell pellets were resuspended in lysis buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin, 1 g/ml aprotinin, 1 g/ml leupeptin). The cell pellets were then sonicated, incubated on ice for 15 minutes, and centrifuged to release cell proteins. Aliquots of 30 g of cell protein were separated by 10% CECIL ET AL SDS-PAGE and then transferred onto nitrocellulose (BioRad, Hercules, CA) and studied by Western blotting as previously described, using horseradish peroxidase (HRP)– conjugated secondary antibody (32). Immunoreactive products were detected using enhanced chemiluminescence (ECL, Amersham Pharmacia, Piscataway, NJ). Primary and secondary antibody dilutions were 1:2,000. Antibodies to type X collagen were obtained from Calbiochem, and antibodies to tubulin were from Sigma. HRP-conjugated goat anti-rabbit IgG and anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). For kinase Pyk-2 immunoprecipitation assays, aliquots of cell lysates (200 g protein) were precleared with rabbit IgG before incubation with 2 g of Pyk-2 polyclonal antibodies (Upstate Biotechnology, Lake Placid, NY) at 4oC overnight, and were then mixed with 20 l of Protein A/G Plus-Agarose beads (1:1) at 4oC for 3 hours. The beads were washed 4 times prior to sample separation by 10% SDS PAGE and immunoblotting with antiphosphotyrosine monoclonal antibody (pY99; Santa Cruz Biotechnology). For RT-PCR analyses, total RNA was prepared and reversed transcribed as previously described (32). Primers and RT-PCR conditions for the ribosomal housekeeping gene L30 were as described (36). PiT-1 primers were 5⬘-CAATGAGAAAGTTGGTGCAA-3⬘ (sense) and 5⬘-CCAGCTGTTTCTCAGAAGTAATTTA-3⬘ (antisense), which amplified a 224-bp product in chondrocytes that was confirmed, by sequencing, to be PiT-1. Assays of TGase activity. To assay TGase transamidation activity, we used a spectrophotometric protocol for 5-biotinamidopentylamine binding to dimethylcasein, as previously described (8). One unit of TGase was designated as 1 M substrate catalyzed per hour. Construction of chimeric receptors and site-directed mutagenesis. The cDNA for human CXCR1 and CXCR2 in the plasmid pSFFV.neo were the generous gift of Dr. Ingrid Schraufstatter (La Jolla Institute for Molecular Medicine, San Diego, CA). The 2 receptor cDNA were subcloned into the Eco RI site of pcDNA3.1(⫺) (Invitrogen) containing a previously deleted Xba I site. A silent Xba I restriction site was introduced by QuickChange site-directed mutagenesis (Stratagene, La Jolla, CA) in the vicinity of the coding region for the alanine 286–leucine 287 residues of CXCR1 and the conserved region of CXCR2, which were identified by BLAST analysis. Primers for CXCR1 were 5⬘-CATCGGGGCGGCTCTAGATGCCACTGAGATTCTG-3⬘ (sense) and 5⬘-CAGAATCTCAGTGGCATCTAGAGCCCGGCCGATG-3⬘ (antisense); primers for CXCR2 were 5⬘-CACATCGACCGGGCTCTAGATGCCACCGAGATTCTG-3⬘ (sense) and 5⬘-CAGAATCTCGGTGGCATCTAGAGCCCGGTCGATTGT-3⬘ (antisense). Each construct was cut with Nhe I and Xba I. The resulting fragment encoding the N-terminal 285 amino acids of CXCR1 was ligated to the plasmid encoding the C-terminal 66 amino acids of CXCR2, and the fragment encoding the N-terminal 294 amino acids of CXCR2 was ligated into the plasmid encoding the C-terminal 65 amino acids of CXCR1. The wild-type CXCR1 construct (or the CXCR2T1 chimeras T346 and T349, where indicated) were further mutated at the codons for amino acids 346, 348, and 349. Primers for T346P were 5⬘-GGCACGTCATCGTGTTCCCTCCTACACTTCT- IL-8 AND PiT-1 IN CHONDROCYTE PHOSPHATE UPTAKE 147 Figure 1. Up-regulated expression of phosphate transporter/retrovirus receptor 1 (PiT-1) in osteoarthritic (OA) human knee articular cartilage. Immunohistochemical analysis of frozen sections of normal and OA cartilage was performed using an antibody to a PiT-1–specific polypeptide, as described in Materials and Methods. At an original magnification of 25⫻, all 3 cartilage zones are visible; the superficial and middle zones are visible at 63⫻ original magnification. Minimal PiT-1 expression was detected in normal donor cartilage, including the regions of the middle zone indicated by boxes and magnified to 116⫻. In contrast, in the middle zone of OA cartilage, there is abundant expression of PiT-1. The cells in which PiT-1 expression was robust in OA cartilage included grossly enlarged chondrocytes (⬎5 times the approximate cell volume of chondrocytes seen in normal cartilage in the top panels), consistent with chondrocyte hypertrophy (examples indicated by arrows). Background staining using rabbit preimmune serum is shown at 25⫻ original magnification for both normal and OA cartilage. As a control for specificity of PiT-1 detection, OA cartilage was stained with anti–PiT-1 that had been preincubated with the immunogenic peptide for PiT-1 antibody generation as described in Materials and Methods, with results shown at 116⫻. Results shown are from 2 normal and 2 OA donors, representative of 5 normal donors and 6 OA donors analyzed. TCG-3⬘ (sense) and 5⬘-CGAAGAAGTGTAGGAGGGAACACGATGACGTGCC-3⬘ (antisense); primers for Y348F were 5⬘-GTCATCGTGTTACCTCCTTCACTTCTTCGTCTGTC-3⬘ (sense) and 5⬘-GACAGACGAAGAAGTGAAGGAGGTAACACGATGAC-3⬘ (antisense); primers for T349V were 5⬘-CGTGTTACCTCCTACGTTTCTTCGTCT-3⬘ (sense) and 5⬘-GACATTGACAGACGAAGAAACGTAGC-3⬘ (antisense); primers for TT346/349PV were 5⬘-CATCGTGTTCCCTCCTACGTTTCTTCG-3⬘ (sense) and 5⬘-CATTGACAGACGAAGAAACGTAGGAC-3⬘ (anti- 148 CECIL ET AL sense). All constructs were sequenced to verify that no extraneous mutations were introduced during mutagenesis. Statistical analysis. Results are presented as the mean ⫾ SD. Single-factor analysis of variance was used to determine statistically significant differences between samples. RESULTS PiT-1 expression in OA cartilage in situ and PiT-1 regulation of chondrocyte differentiation in vitro. PiT-1 expression was detectable in normal human knee articular cartilage, but was more robust in chondrocytes in the superficial, middle, and deep zones of OA cartilage (Figure 1). In the middle zone of OA knee cartilage, markedly enlarged chondrocytes, consistent with hypertrophic cells, exhibited abundant PiT-1 expression (Figure 1). Therefore, we assessed for direct effects of PiT-1 on up-regulated TGase activity and type X collagen expression, features characteristic of chondrocyte hypertrophy in vitro. To do so, normal bovine knee chondrocytes were transfected with PiT-1. We observed no change in TGase activity in chondrocytes that had been simply transfected with PiT-1 (Figure 2A). In chondrocytes in medium supplemented with 2.5 mM sodium Pi, however, PiT-1 transfection induced an ⬃2-fold increase in TGase activity in comparison with transfection of vector, an effect attenuated by addition of the competitive sodium/phosphate cotransport inhibitor phosphonoformic acid (PFA) (25,35) (Figure 2A). Similarly, bovine chondrocytes transfected with PiT-1 and supplemented with Pi exhibited increased type X collagen expression that was inhibited by PFA (Figure 2B). Induction by IL-8 of the PiT-1 expression and Pi uptake involved in promotion of chondrocyte hypertrophy. We observed that IL-8 (10 ng/ml), but not GRO␣ or IL-1, induced PiT-1 expression and increased Pi uptake in normal bovine knee articular chondrocytes (Figure 3). The capacity of IL-8 to induce TGase activity and type X collagen expression was significantly inhibited by PFA (Figures 3C and D), which prevented the induction of Pi uptake by IL-8 (results not shown). These changes were specific for IL-8: PFA did not significantly inhibit the capacity of GRO␣ to induce TGase activity or type X collagen expression (Figures 3C and D). Role of Pyk-2 in PiT-1 expression, Pi uptake, and chondrocyte hypertrophy in response to IL-8. We next tested for potential effects of IL-8 through the focal adhesion kinase family member Pyk-2, since Pyk-2 transduces signals in leukocytes that regulate adhesion and Figure 2. Effects of phosphate transporter/retrovirus receptor 1 (PiT-1) and supplemental inorganic phosphate (Pi) on transglutaminase (TG) activity and type X collagen expression. Bovine chondrocytes were transfected with PiT-1 and vector (Vec) control as described in Materials and Methods. After transfection, 1 ⫻ 105 cells were transferred to a 96-well polyHEME-coated plate and incubated with 25 g/ml ascorbic acid with or without supplementation of the medium with 2.5 mM sodium phosphate. Where indicated, the competitive inhibitor of sodium/phosphate cotransport, phosphonoformic acid (PFA), also was present. A, TG activity. Values are the mean and SD from experiments performed in triplicate with samples from 5 different donors. Basal TG activity in bovine chondrocytes transfected with the empty vector was 422 ⫹ 106 units/g protein; after transfection with PiT-1 and supplementation of the medium with Pi, this increased to 1,041 ⫾ 250 units/g protein. ⴱ ⫽ P ⬍ 0.05 versus vector control treatment, by analysis of variance. B, Results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting analysis for type X collagen, performed on cell lysates after 5 days of culture as described in Materials and Methods. Results shown are representative of findings in 3 different donors. migration in response to several chemokines (37). We observed that IL-8 stimulated rapid phosphorylation of Pyk-2 in bovine chondrocytes (results not shown). To assess the functional significance of Pyk-2 in PiT-1 expression and sodium-dependent Pi uptake in response to IL-8, we used the readily transfected normal human knee immortalized articular chondrocyte cell line CH-8 (31), which demonstrated expression of CXCR1, and to a lesser degree CXCR2, by FACS (results not shown). We transfected the CH-8 cells with an inhibitory con- IL-8 AND PiT-1 IN CHONDROCYTE PHOSPHATE UPTAKE 149 Figure 3. Differential induction of PiT-1 expression, sodium-dependent Pi uptake, TG activity, and type X collagen expression in response to interleukin-8 (IL-8) and growth-related oncogene ␣ (GRO␣). A, Human articular chondrocytes from normal knees (5 ⫻ 105 cells/well in a 96-well polyHEME plate) were incubated with 10 ng/ml IL-1, IL-8, or GRO␣. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis for PiT-1 (using the antipeptide antibody to human PiT-1, generated as described in Materials and Methods) were performed on cell lysates as above, 6 hours after cytokine treatment. Tubulin was used as a loading control. Results shown are representative of findings in 3 different donors. B, Bovine chondrocytes (2 ⫻ 105 cells/well in a 96-well polyHEME-coated plate) were treated as described in A. Sodium-dependent Pi uptake was measured in phosphate-starved cells as described in Materials and Methods, and expressed as the percent increase in cellular 33P cpm per microgram cell DNA relative to unstimulated cells. Values are the mean and SD from 4 donors. Basal 33P uptake in unstimulated bovine chondrocytes in these studies was 875 ⫾ 75 cpm. ⴱⴝP ⬍ 0.05 versus IL-1 and GRO␣ treatment, by analysis of variance. C, Bovine chondrocytes (1 ⫻ 105 cells/well in a 96-well plate) were stimulated with 10 ng/ml IL-8 or GRO␣ in the presence or absence of 300 mM PFA. TG activity was measured at 48 hours as described above. Values are the mean and SD. Basal TG activity in untransfected bovine chondrocytes was 66 ⫾ 22 units/g protein. ⴱ ⫽ P ⬍ 0.05 versus findings in the absence of PFA, by analysis of variance. D, Bovine chondrocytes (1 ⫻ 105 cells/well in a 96-well polyHEME-coated plate) were stimulated with 10 ng/ml IL-8 or GRO␣ in the presence or absence of 300 mM PFA. SDS-PAGE and Western blotting analysis for type X collagen were performed on cell lysates at 5 days. Tubulin was used as a loading control. Results shown are representative of findings in 3 different donors. See Figure 2 for other definitions. struct for Pyk-2 (Pyk-2–Y402F) (34), which inhibited PiT-1 expression in response to IL-8 and suppressed Pi uptake in response to IL-8 by 82% in CH-8 cells (Figure 4). Pyk-2–Y402F also markedly suppressed IL-8– induced TGase activity and type X collagen expression (Figure 4). Up-regulation of CXCR1, but not CXCR2, transduces Pi uptake in response to IL-8. Given the differential effects of the CXCR1/CXCR2 ligand IL-8 and the CXCR2 ligand GRO␣ on Pi uptake, we tested whether IL-8 induced Pi uptake selectively via CXCR1. To do so, we performed “gain-of-function” experiments, transiently transfecting CH-8 cells and confirming that transfection up-regulated the wild-type chemokine receptors (results not shown). Wild-type CXCR1 or CXCR2, chimeras of CXCR1 and CXCR2, and certain site mutants of CXCR1 were expressed. The mutants were selected by examining the amino acid sequences of 150 Figure 4. Role of Pyk-2 in interleukin-8 (IL-8)–induced PiT-1 expression, Pi uptake, TG activity, and type X collagen expression in human chondrocytic CH-8 cells. Immortalized human normal knee chondrocytic CH-8 cells were transfected with wild-type Pyk-2 (Pyk-2–WT), the inhibitory Pyk-2 construct Pyk-2–Y402F, or vector control as described in Materials and Methods. The transfected cells were then aliquoted for specific assays after treatment with IL-8 (10 ng/ml). A, Total RNA was collected at 2 hours and reverse transcriptase– polymerase chain reaction (RT-PCR) analysis for PiT-1 expression was performed. Results shown are representative of 3 different experiments. B, Pi uptake in CH-8 cells transfected with Pyk-2–WT, Pyk-2– Y402F, or vector control after 6 hours of IL-8 treatment. Values are the mean and SD from 3 independent experiments. Basal 33P uptake in unstimulated CH-8 cells in these studies was 584 ⫾ 46 cpm. ⴱ ⫽ P ⬍ 0.05 versus vector control and Pyk-2–WT treatment, by analysis of variance. C, TG activity in CH-8 cells transfected with Pyk-2–Y402F or vector control, after 48 hours of treatment with IL-8. Values are the mean and SD from 3 experiments performed in triplicate. Basal TG activity in CH-8 cells transfected with vector control was 462 ⫾ 154 units/g protein; after IL-8 treatment, this increased to 1,287 ⫾ 269 units/g protein. ⴱ ⫽ P ⬍ 0.05 versus vector control treatment, by analysis of variance. D, Results of sodium dodecyl sulfate– polyacrylamide gel electrophoresis and Western blotting analysis for type X collagen, performed on cell lysates from CH-8 cells transfected with Pyk-2–WT, Pyk-2–Y402F, or vector control, after 5 days of treatment with IL-8. Results shown are representative of 3 different experiments. See Figure 2 for other definitions. CXCR1 and CXCR2 for differences in their cytosolic tail regions potentially involved in differential effects on PiT-1 expression and Pi uptake. CXCR1 and CXCR2 have an overall identity of 76% (11) (Figure 5A), with sequence divergences identified by BLAST analysis at the ligand-binding N-terminal extracellular domain (25% identical) and the fourth transmembrane and C-terminal cytoplasmic domains (each 53% identical). We focused our attention on the C-terminal CXCR1 cytosolic tail domain. Figure CECIL ET AL 5B shows the sequence alignment of the last 42 amino acids of the C-terminal cytoplasmic domain in each receptor. The threonines and tyrosine in the amino acids 346–349 domain of CXCR1 were divergent from the same region in CXCR2 (TSYT versus PSFV, respectively). Using site-directed mutagenesis on wild-type CXCR1, we introduced a proline at position 346 for the threonine, a phenylalanine at position 348 for the tyrosine, and a valine at position 349 for the threonine and also constructed a double mutation replacing both threonines in the 346–349 motif with a proline and a valine, respectively. First, we observed that direct expression of wildtype CXCR1, but not wild-type CXCR2, via transfection significantly increased sodium-dependent Pi uptake (by ⬃84%) in response to IL-8 in CH-8 cells (Figure 6A). Second, transfection of the chimera of CXCR1 with the cytosolic tail of CXCR2 (generated as described in Materials Methods, and termed CXCR1T2) was associated with loss of Pi uptake in response to IL-8 (Figure 6A). Conversely, transfection of the CXCR2T1 chimera into CH-8 cells was associated with a 25% increase in Figure 5. Regions of sequence identity and divergence in CXCR1 and CXCR2 (A) and amino acid sequence alignment of the last 42 amino acids of the C-terminal cytoplasmic domain in CXCR1 and CXCR2 (B). This graphic representation summarizes the results of BLAST analysis comparing CXCR1 and CXCR2 cytosolic tail domains. Conservative substitutions are indicated in italics; nonconservative amino acid substitutions are underlined. IL-8 AND PiT-1 IN CHONDROCYTE PHOSPHATE UPTAKE 151 decreased ⬃50% relative to wild-type CXCR1 (Figure 6B). In contrast, mutation of either or both of the threonines in the 346–349 domain of wild-type CXCR1 (and of the CXCR2T1 chimera [data not shown]) attenuated Pi uptake in response to IL-8 (Figure 6B). DISCUSSION Figure 6. Evidence that the CXCR1 cytoplasmic tail is critical for mediating interleukin-8 (IL-8)–induced uptake of Pi. CH-8 cells were transfected with constructs as indicated, and after transfection, the cells were stimulated with 10 ng/ml of IL-8. Pi uptake was assessed at 6 hours after addition of IL-8. Values are the mean and SD from 6 independent experiments. ⴱ ⫽ P ⬍ 0.05 for comparison of CXCR1/T2, or all CXCR1 site mutants, with CXCR1, by analysis of variance. # ⫽ P ⬍ 0.05 versus CXCR2, by analysis of variance. See Figure 2 for other definitions. IL-8–induced Pi uptake compared with the wild-type CXCR2 (Figure 6A). In addition, transfection of CH-8 cells with the CXCR2T1 chimera, but not with CXCR2, led to a significant GRO␣-induced increase in sodiumdependent Pi uptake (data not shown). We then performed structure/function studies of transfected site mutants in the CXCR1 TSYT346–349 motif. Induction of sodium-dependent Pi uptake was detected in response to IL-8 in association with mutation of the Y348 residue in CXCR1, although Pi uptake was This study demonstrated markedly up-regulated expression of the type III sodium-dependent phosphate cotransporter PiT-1 in chondrocytes throughout OA cartilage, including hypertrophic chondrocytes. IL-8 induced both PiT-1 expression and sodium-dependent Pi uptake in normal chondrocytes. Since IL-8–induced chondrocyte hypertrophic differentiation was suppressed by treatment with the sodium phosphate cotransporter inhibitor PFA, our results suggested a critical role of sodium-dependent Pi uptake in chondrocyte maturation triggered by IL-8. PFA is a competitive inhibitor of multiple sodium phosphate cotransporters, and expression of a second type III sodium-dependent phosphate cotransporter, PiT-2 (Ram-1), occurs in chondrocytic cells (35). Hence, it remains possible that IL-8 regulates more than one type III sodium-dependent phosphate cotransporter involved in articular chondrocyte hypertrophy. We previously demonstrated that IL-8, as well as GRO␣, induces the Pi-generating ectoenzyme alkaline phosphatase in cultured articular chondrocytes (2). Exogenous Pi, generated in large part by alkaline phosphatase (6), has been observed to play a major role in the maturation of growth plate chondrocytes in vitro (17,18). PiT-1 expression and Pi uptake also were recently implicated in chondrogenic commitment of CFK2 cells (35). However, calcium sensing played a larger role than did Pi uptake in the maturation to hypertrophy of CFK2 cells committed to chondrocytic differentiation (35). Thus, the functional significance of PiT-1 expression and Pi uptake in chondrocytic cells may depend on differentiation status. Normal articular chondrocytes express both CXCR1 and CXCR2 in situ and in vitro (12,13). Furthermore, IL-8 and GRO␣ both induce hypertrophic differentiation of articular chondrocytes in vitro (2). It is noteworthy that both IL-8 and GRO␣ are expressed by articular chondrocytes in vivo (3,38). However, in this study it was demonstrated that the dual CXCR1/CXCR2 ligand IL-8, but not the CXCR2 ligand GRO␣, induced PiT-1 expression and Pi uptake in chondrocytes. Furthermore, forced expression of CXCR1, but not 152 CXCR2, directly promoted increased Pi uptake in chondrocytic CH-8 cells. The demonstration that IL-8–induced, but not GRO␣-induced, chondrocyte hypertrophy was dependent on Pi uptake was consistent with the notion that differential CXCR1 and CXCR2 signaling results in differential functional impact. However, certain limitations of this study must be considered when interpreting the results with regard to selective CXCR1 effects. First, we used SV40-immortalized chondrocytic CH-8 cells rather than untransformed articular chondrocytes in transfection-based CXCR1 “gain-of-function” experiments, to allow adequate efficiency to examine CXCR1 structure/function. Second, CH-8 cells had higher basal expression of CXCR1 than of CXCR2. The concurrent basal expression of both CXCR1 and CXCR2 by chondrocytic cells does not exclude the possibility of crosstalk between CXCR1 and CXCR2. Secondary events might include priming of CXCR1 by CXCR2 activation (39), faster phosphorylation and down-regulation of CXCR2 (40,41), or increased affinity of N-terminally truncated IL-8 for the CXCR1 following cleavage by proteases, as described for neutrophils mediated by gelatinase B (42). Up-regulation of CXCR1, but not CXCR2, was previously observed in OA cartilage chondrocytes in situ (12), which highlights the significance of effects mediated selectively by IL-8 through CXCR1 in chondrocytes. The cytosolic tail domains of both CXCR1 and CXCR2 are rich in serine residues, and phosphorylation of certain serine residues in both of these cytosolic tails has been shown to impact receptor functions including cell migration, phospholipase D activation, exocytosis, and receptor desensitization (40,43–45). However, the role of the TSYT346–349 motif unique to wild-type CXCR1 has not previously been studied in leukocytes. Our results revealed a major role of the TSYT346–349 motif of CXCR1 in Pi uptake in response to IL-8. A BLAST search for the TSYT346–349 motif in CXCR1 has shown that it is unique among all chemokine receptors (Liu-Bryan R, et al: unpublished observations). We do not know if the threonines and other individual amino acids in the TSYT346–349 motif in CXCR1 can be phosphorylated and, if so, affect potential protein–protein interactions and signaling. However, analysis with a phosphorylation prediction algorithm (NetPhos2.0; Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark), has indicated that T346 has a high probability of phosphorylation, potentially by glycogen synthase kinase 3 (Liu-Bryan R, et al: unpublished observations). Al- CECIL ET AL though the TSYT346–349 motif of CXCR1 was essential for IL-8–induced Pi uptake, our results also suggested a role of other CXCR1 domains since IL-8–induced Pi uptake was substantially lower in cells transfected with the chimera of CXCR2 with the cytosolic tail of CXCR1 (CXCR2T1), in comparison with wild-type CXCR1. Pyk-2 mediates chemokine-induced migration and adhesion responses in leukocytes (37), as well as MMP expression induced by fibronectin fragments and certain other stimuli in chondrocytes (34,46). However, as illustrated in studies using monocyte chemoattractant protein 1 (CCL2), Pyk-2 does not mediate all signaling or responses of cells to chemokines (47). In the present study, we observed that Pyk-2 was centrally involved in IL-8–induced PiT-1 expression and Pi uptake in chondrocytic cells, but GRO␣ also induced tyrosine phosphorylation of Pyk-2 in chondrocytes (Liu-Bryan R, et al: unpublished observations). Potential clues to alternative early divergent CXCR1 and CXCR2 signaling responses in chondrocytes might be gleaned from previous studies with neutrophils, in which IL-8–induced cell migration, phospholipase D activation, and superoxide anion generation were mediated by CXCR1 (14–16). Such effects appear to be cell type–specific, since CXCR2 was able to independently mediate cell migration in response to IL-8 in T lymphocyte–derived Jurkat cells (48). In addition, CXCR2 serves as the primary mediator of neutrophil migration in response to GRO␣ and several other chemokine CXCR2–selective ligands (16). Thus, at least some differential signaling responses through CXCR1 and CXCR2 may ultimately be found to be chondrocyte-specific. Our results augment a growing body of evidence that IL-8 functions beyond recruitment and activation of leukocytes (2,49). Timing of IL-8 exposure to chondrocytes and differential levels of CXCR1 and CXCR2 expression have the potential to modulate chondrocyte hypertrophic differentiation in vivo. The chondrocyte growth factors transforming growth factor ␤ and insulinlike growth factor 1 induce PiT-1 expression in skeletal cells (28,50). Hence, cytokines other than IL-8 might regulate articular chondrocyte differentiation and function in OA partly by affecting PiT-1 expression and sodium-dependent Pi uptake. Our identification of PiT-1 expression and sodium-dependent Pi uptake as essential mediators of the capacity of IL-8 to stimulate hypertrophy in cultured articular chondrocytes, and of the specific role of the CXCR1 TSYT346–349 motif in this function, is noteworthy. These results reveal a novel mechanism by which chronic, indolent inflammation can IL-8 AND PiT-1 IN CHONDROCYTE PHOSPHATE UPTAKE 153 alter the differentiation and function of resident cells of mesenchymal origin in OA. 15. 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