Role of interleukin-8 in PiT-1 expression and CXCR1-mediated inorganic phosphate uptake in chondrocytes.

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: rterkeltaub@ucsd.edu.
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.
ACKNOWLEDGMENTS
We are grateful to Dr. Robert Crowl (Novartis, East
Hanover, NJ) for helpful comments, and to Jacqie Quach, Lilo
Creighton-Achermann, Dr. Martin Lotz (The Scripps Research Institute, La Jolla, CA), and Dr. Richard Coutts (Sharp
Hospital, San Diego, CA) for provision of and assistance with
human knee cartilage sections.
16.
17.
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