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Transcriptional regulation of the HOX4C gene by basic fibroblast growth factor on rheumatoid synovial fibroblasts.

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Vol. 40, No. 9, September 1997, pp 1628-1635
0 1997. American College of Rheumatology
Objective. To examine the expression of genes of
the HOX D cluster in the synovial tissue of patients with
rheumatoid arthritis (RA), and to determine whether
basic fibroblast growth factor (bFGF) influences the
expression and transcriptional regulation of the gene.
Methods. The expression of genes of the HOX D
cluster, including HOX4C, HOX4D, HOX4H, and
HOX41, was determined in the synovium of 4 patients
with RA and 4 with osteoarthritis (OA) by in situ reverse
transcription (RT) and RT-polymerase chain reaction
(RT-PCR). The induction of HOX4C messenger RNA
(mRNA) by bFGF was determined by RT-PCR. The
binding activity of a transcriptional regulator of the
HOX4C gene, C2, was analyzed by the mobility shift
assay. NIH-3T3 cells transfected with a construct containing C2 binding sequence were incubated with bFGF,
and the activity of the reporter was measured by luciferase assay.
Results. Using an in situ RT assay, specific expression of HOX4C mRNA was detected in 3 of 4 RA
synovial samples, whereas none of the OA synovia
expressed HOX4C. HOX4D, HOX4H, and HOX41 genes
were expressed in all synovial samples from RA and OA
Supported by grants from the Ministry of Education, Science
and Culture of Japan, the Ministry of Health and Welfare of Japan,
and the Kanagawa Foundation for Medical Science.
Chengsen Xue, MD: St. Marianna University School of
Medicine, Kawasaki, Japan, and Peking Union Medical College Hospital, Beijing, China; Tomoko Hasunuma, MD, PhD, Hiroshi Asahara,
MD; PhD, Weihong Yin, MS, Toshiro Maeda, MD, PhD, Koushi
Fujisawa, MS, Takayuki Sumida, MD, PhD, Kusuki Nishioka, MD,
PhD: St. Marianna University School of Medicine, Kawasaki, Japan;
Yi Dong, MD: Peking Union Medical College Hospital, Beijing,
Address reprint requests to Kusuki Nishioka, MD, PhD,
Rheumatology, Immunology and Genetic Program, Institute of Medical Science, St. Marianna University School of Medicine, 2-16-1
Sugao, Miyamae-ku, Kawasaki 216, Japan.
Submitted for publication November 22, 1996; accepted in
revised form April 30, 1997.
patients. The presence of HOX4C mRNA was also
confirmed by RT-PCR and Southern blotting. Treatment with bFGF increased the expression of HOX4C
mRNA in RA fibroblasts. The mobility shift assay and
luciferase assay showed that bFGF enhanced C2 binding activity and significantly increased the transcriptional activity on RA fibroblasts.
Conclusion. Our findings suggest that HOX4C is
involved in synovial hyperplasia, and that the transcriptional regulation of HOX4C genes by bFGF may play a
crucial role in the pathogenesis of RA.
Rheumatoid arthritis (RA) is characterized by
hyperplasia of synovial tissue, which contains morphologically transformed mesenchyme-like cells (1,2).
Transformed growth of synoviocytes could play an
important role in the pathogenesis of the disease.
Fibroblast transformation is regulated by a variety of
transcriptional factors, such as AP-1, nuclear factor
KB,and homeobox (HOX) gene products (3-6). HOX
genes are a family of transcriptional regulators that
encode a 61-amino acid domain with binding activity to
DNA. These HOX genes control cell growth and pattern
formation during embryogenesis (7,s).
Basic fibroblast growth factor (bFGF) is present
in abundance in rheumatoid synovium (9-13) and
strongly expressed in rheumatoid pannus (11).In the rat
model of experimental adjuvant arthritis, there is a close
association between increased bFGF expression and
destruction of arthritic joints (14). As a potent angiogenic and mitogenic polypeptide, bFGF has been implicated in tissue differentiation, angiogenesis (15,16), and
synovial proliferation (17,18). Thus, bFGF is involved in
synovial hyperplasia, neovascularization, and joint destruction in RA.
FGFs also play a critical role in the induction of
the limb bud during embryogenesis. A bead soaked in
FGF1, FGF2, o r FGF4 implanted in the presumptive
flank of a chick embryo is capable of inducing the
formation of a complete, morphologically normal limb
(19). It is thought that an endogenous local source of
FGF from the mesoderm of the flank may initiate
mesenchymal proliferation in normal development (20).
The initiation of the limb bud has also been linked to the
HOX D cluster (21,22). It has been shown that some
HOX genes, such as XIHbox 6 and HOXD1, are
activated by bFGF in Xenopus (23-25).
Based on the above findings, we postulated that
bFGF, through activation of a HOX gene, is involved in
the proliferation and transformation of synovial fibroblasts in the rheumatoid joint. To investigatethis hypothesis, we examined the expression of HOX D genes in
rheumatoid fibroblasts and analyzed whether the effect
of bFGF on the proliferation of synovial fibroblasts is
mediated through the transcriptional regulation of HOX
genes. Our results show that HOX4C is specifically
expressed on rheumatoid synovial fibroblasts, and that
its expression and transcriptional activity are regulated
by bFGF through the C2 regulatory region, a highly
conserved element on the upstream of the gene (26).
Tissue and cell preparation. Synovial tissue samples
were obtained, during arthroplasty, from the knee joints of 4
patients with RA and 4 patients with osteoarthritis (OA). The
diagnosis of RA was based on ACR criteria (27) and that of
OA on clinical and radiologic criteria. The experimental
protocol was approved by the Ethics Review Committee for
Human Experimentation at our institution, and written consent was obtained from all subjects. Separation of synoviocytes
was performed according to the method described by Goto et
a1 (28). Briefly, following removal of excess adipose tissue from
the sample, the specimen was minced into small pieces and
digested with 1.0 mg/ml of collagenase (Sigma, St. Louis, MO)
in Eagle’s medium (Gibco, Grand Island, NY) at 37°C for 2
hours. After digestion, the dissociated cells were collected by
centrifugation at 500g for 5 minutes, and resuspended in
Ham’s F-12 medium (Gibco) supplemented with 10% fetal calf
serum (FCS; Bioserum, Melbourne, Victoria, Australia), 100
unitsiml penicillin, 100 mgiml streptomycin, and 5 X 1OP5M
2-mercaptoethanol. The cell suspension was then poured into
90 mm-diameter dishes (Sumitomo Medical Co., Tokyo, Japan). Human bFGF (TaKaRa Biochemical, Kyoto, Japan) was
used for bFGF treatment.
In situ reverse transcription assay. An in situ reverse
transcription (RT) assay was performed using the Digoxigenin
Detection System Kit (Kreatech Biotechnology BV, Amsterdam, The Netherlands) (29). The primers used in this experiment were as follows: BACl 5’-AAGGCCAACCGCGAGA
AAC-3’ for p-actin, HHox4Cl 5‘-ACTTCCTCCTCCTCTTC
CTTTGGTCT-3‘ for HOX4C, and HHox4D1 5’-GCTTCA
Primers for HOX4H and HOX4I were synthesized
according to the method described by Davis and Capecchi (30).
The antisense primers and sense primers (for control) corresponding to individual HOX genes (HOX 4C, 4D, 4H, and 41)
were used to examine the expression of the genes. Briefly, fresh
tissue samples were immediately embedded in Tissue-Tek
embedding medium (Miles, Elkhart, IN), snap frozen, and cut
into 5 wm-thick sections. The sections were fixed with 10%
paraformaldehyde for 1 hour, and the cells were treated with
0.5% Nonidet P40 (NP40) for 1 hour to ensure permeability.
We then added 10 PI of a reaction mixture containing 5 pmoles
primer, 10 mmoles dithiothreitol (DTT), 1.0 mM dNTP, 10
unitsiml RT, 2 unitsiml RNase inhibitor, and 0.1 nmole
digoxigenin-11-dUTP. The slides were covered with coverslips
and incubated at 42°C for 1 hour. The coverslips were removed
at a later stage by washing with 2X standard saline-sodium
citrate buffer, pH 7.0. The slides were incubated with freshly
prepared dye solution and later rinsed in phosphate buffered
saline. All sections were evaluated by light microscopy.
RNA preparation and RT-polymerase chain reaction
(RT-PCR). Cells ( 5 X lo5) were cultured to near-confluence.
RNA was extracted from the fresh synovial tissue samples and
cultured cells using the acid guanidinium thiocyanate-phenolchloroform method (31). RNA was analyzed by RT-PCR with
Moloney murine leukemia virus reverse transcriptase (Gibco)
(32). Amplification of p-actin messenger RNA (mRNA) was
performed as a loading control. RT-PCR was modified in a
cycle-dependent manner to accurately control the PCR amplification of all genes for the plateau phenomenon. PCR was
performed on a thermocycler (Hybaid, Middlesex, UK) in a
50-pl reaction volume, including complementary DNA sample, 2.0 units Tuq DNA polymerase (Gibco), 50 pmoles of each
primer, 200 mmoles of each deoxynucleotide triphosphate, and
5 pCi (w3’P)-dCTP (NEN, Wilmington, DE). The annealing
temperature was set at 64°C for 1 minute. The PCR products
were then separated by 10% polyacrylamide gel electrophoresis and the gel was exposed to x-ray film.
Mobility shift assay. All animal experiments were
performed according to St. Marianna University School of
Medicine’s Guidelines for Laboratory Animal Experimentation. Cells (lo’) were collected from human embryonic cell
line NTI-5, mouse NIH-3T3 cells, and fibroblasts from RA and
OA synovia. The cells were lysed by mixing with 0.58% NP40,
and nuclear proteins were mixed with radiolabeled oligonucleotides in a buffer consisting of 50 mM NaCI, 10 mM
Tris HCI, 0.5 mM DTT, 0.05 mgiml poly(d1-dC), 12.5%
glycerol, and 0.05% NP40. Two complementary oligonucleotides corresponding to the sequence C2 located on HOX4C
were annealed, and then labeled with [y3”P]dCTP using T4
DNA kinase. Sample5 were incubated with 1 ng of 32Plabeled double-stranded (annealed) C2 oligonucleotides
complexes were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel. For the competition analysis, the
same amounts of the unlabeled C2 oligonucleotide were used
in 10-fold and 100-fold excess. Oct-1 oligonucleotide (5‘-GAT
was used as a nonspecific competitor (33).
Transfection. Mouse fibroblast NIH-3T3 cells were
maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS. The pGL2-Promoter vector (Promega,
Madison, WI), a luciferase reporter plasmid carrying the SV40
early promoter followed by the luciferase gene, was used.
The reporter construct pGLC was generated by cloning human
C2 fragment of HOX4C into the pGL2-Promoter plasmid.
Briefly, genomic DNA extracted from human peripheral blood
lymphocytes and C2 region was amplified with primers that
contained the digestion sites for Kpn I and Xho I. After
amplification by PCR, the products were digested with I@z I
and Xho I, then ligated into pGL2-Promoter vector. Transfection was performed using the Tfx-50 Reagent (Promega). In
transfection experiments, 5 mg of reporter plasmid and 11.25
ml of Tfx-50 was used per well of a 6-well plate (Corning,
Corning, NY). After transfection, NIH-3T3 cells were incubated with 10 ngiml of bFGF. The cells were harvested 48
hours after transfection, and lysed with Cell Lysis Buffer
(Promega). For normalizing efficiency, we cotransfected the
vector pSV2Apap, containing the human placental alkaline
phosphatase gene (34,35).
Statistical analysis. Data were expressed as the
mean I
SEM. Differences between groups were examined for
statistical significance using Student’s t-test. P values less than
5% were considered significant.
HOX D gene expression in rheumatoid synovium. We evaluated the mRNA of HOX D cluster genes
in synovial tissue samples obtained from patients with
RA and OA, by in situ R T assay. HOX4C was expressed
in freshly prepared synovia from 4 R A patients, whereas
none of the synovia from OA patients exhibited HOX4C
mRNA (Figure 1). Fewer than 10% of synovial lining
cells and sublining cells in RA synovium were positive
for HOX4C. Other genes of the HOX D cluster, such as
HOX4D, HOX4H, and HOX41, were expressed in
synovia from both RA and OA patients, and, in R A
synovium, the pattern of expression of all 4 genes was
similar. The presence of HOX mRNA was confirmed by
RT-PCR followed by Southern blotting (Figure 2).
These findings suggest that the HOX4C gene is specifically expressed in rheumatoid synovium.
Induction of HOX4C expression by bFGF. To
determine the potency of bFGF in inducing HOX4C
gene expression, we analyzed gene expression following
bFGF stimulation of synoviocytes from RA and 3 OA
patients, using RT-PCR. Treatment with bFGF upregulated HOX4C mRNA expression in rheumatoid
fibroblasts after stimulation for 48 hours (Figure 3, lanes
6 and S), but had no effect on the expression of 0-actin
Figure 1. HOX D gene expression in arthritic synovium. Snap-frozen
rheumatoid arthritis (A, C, E, G, and I) and osteoarthritis (B, D, F, H,
and J) synovial tissue sections showing HOX D messenger RNA
expression as determined by in situ reverse transcription. Synovial
lining cells and sublining cells are positive for HOX D gene expression
except for HOX4C in the osteoarthritis sample (D). A and B, Hematoxylin and eosin (H.E.) staining. C and D, HOX4C. E and F, HOX4D.
G and H, HOX4H. I and J, HOX4I. (Original magnification X 200.)
M 1 2 3 4 5 6 7 8
M 1 2 3 4 5 6 7 8
21 4
C >
1 2 3 4 5 6 7 8
- 220
Figure 2. HOX4C mcssengcr RNA (mRNA) expression in rheumatoid arthritis (RA) and osteoarthritis (OA) synovium. Complementary
DNA (cDNA) from freshly obtained synovial tissue samples was
examined by reverse transcription-polymerase chain reaction followed
by Southern blotting. Four RA samples (lanes 1-4) and 4 OA samples
(lanes 5-8) were examined. a, Amplified p-actin mRNA. b, Amplified
HOX4C mRNA. c, Southern blotting of HOX4C cDNA.
(Figure 3, lanes 2 and 4). To determine the specificity of
the bFGF effect, we compared the effect of bFGF on
fibroblasts from R A patients versus fibroblasts from
patients with traumatic injury (representing noninflammatory control synovial cells). HOX4C was not induced
on fibroblasts obtained from trauma synovium (Figure 3,
lanes 5 and 7).
Basic FGF-induced increase in DNA binding
activity of a potential regulator of HOX4C. To understand the mechanism through which bFGF acts on the
HOX gene, we analyzed the induction of DNA binding
activity in a potential regulator of HOX4C, C2, by
bFGF, using the mobility shift assay. Previous studies
have shown that cultured undifferentiated embryonic
cells, such as mouse F9 cell line and other human fetal
cells, have special C2 factor binding activity (26). We
used F9 cells and human NTI-5 embryonic cells as
positive controls. As expected, NTI-5 cells, and especially F9 cells, were able to bind to C2 (Figure 4A, lanes
1 and 2).
In the next step, rheumatoid synovial fibroblasts
were stimulated at a concentration of 10 ng/ml. Nuclear
extracts from fibroblasts were incubated with bFGF with
"P-labeled C2 oligonucleotide probe. Two-hour pretreatment of fibroblasts with bFGF increased C2 binding
activity (Figure 4A, lanes 3-5). The rise in binding
activity appeared within 30 minutes (Figure 4A, lane 4).
1 2
5 6
- -
+ +
- -
+ +
Figure 3. Induction of HOX4C messenger RNA (mRNA) expression by basic fibroblast growth factor (bFGF).
Fibroblasts wcrc obtained from patients with rheumatoid arthritis (R) or trauma (control; C). The expression of
HOX4C mRNA in fibroblasts incubated with or without 10 ng/ml of bFGF was determined by reverse
transcription-polymerase chain reaction, as described in Materials and Methods. At the same time, p-actin was
used as a loading control (lanes 1-4). Size markers (bp) are indicated on the left. The expression pattern of
HOX4C was confirmed in other experiments.
Incubation Time
m-5 ~9
- - -
f *
Figure 4. Increase in C2 binding activity of basic fibroblast growth factor (bFGF)-treated rheumatoid fibroblasts. A, Mobility shift analysis with
nuclear extracts from embryonic ccll line NTI-5 (lane 1),undifferentiated F9 embryonic cells (lane 2), and rheumatoid arthritis (RA; lanes 3-5) and
osteoarthritis ( O A ,lanes 6-8) fibroblasts, and a double-strand annealed oligonucleotide corresponding to C2. Nuclear extracts from fibroblasts were
prepared after 30 minutes or 2 hours of culture in the presence of 10 ngiml of bFGF, as indicated. Arrowheads show the position of the free (f) and
bound (b) DNA. Equal amounts of nuclear protein were used in all lanes. NTI-5 and F9 cells served as positive controls. B, To determine specificity,
cold competitors (C2 or Oct-1) were added in 10-fold and 100-fold excess per assay.
In contrast, bFGF had no effect on the binding activity
of C2 in OA synovial fibroblasts (Figure 4A, lanes 6-8).
The binding was specific for C2, since C2 binding activity
was inhibited completely by a 100-fold excess of unlabeled C2 oligonucleotide probe (Figure 4B). This was in
contrast to the effect of Oct-1, a ubiquitous transcription
factor, which did not influence C2 binding activity.
Considered together, these results suggest that the binding activity of the C2 sequence of the HOX4C gene is
specific to RA fibroblasts.
Analysis of transcriptional regulation of HOX4C
by bFGF. To further confirm the effect of bFGF on the
transcriptional activity of synovial fibroblasts, a transient
expression assay was performed. We generated construct pGLC derived from the pGL2-Promoter vector by
inserting a 197-basepair segment of DNA carrying the
binding sequence of C2 factor (Figure 5A).
The experiments were performed 3 times, and
the mean t SEM from each set of experiments was
calculated and compared. The results showed that the
transcriptional activity of pGLC with C2 segment
(101,137.7 t 1,307.9 units) was significantly higher than
that of pGL2-Promoter vector (51,997.0 t 3,157.1 units;
P < 0.01), which contains the SV40 promoter but not the
enhancer element. These results confirmed that the C2
binding sequence, 2,326 bp from the transcriptional start
site of HOX4C, most likely has an enhancing activity on
the transcriptional regulation of HOX4C. Incubation of
NIH-3T3 cells transfected by pGLC with 10 ng/ml of
bFGF significantly increased the reported activity
(101,137.7 t 1,307.9 units before incubation, 191,743.7 &
13,344.2 units after incubation; P < 0.01) (Figure 5B). In
contrast to the findings with pGLC, incubation of pGL2Promoter with the same concentration of bFGF did not
increase the transcriptional activity (51,997.0 5 3,157.1
units before incubation, 70,279.7 t 5,732.0 units after
incubation; P < 0.05). Thus, it seems that bFGF is
capable of inducing the transcriptional activity of
HOX4C in the presence of the C2 segment.
Homeobox genes, a family of transcriptional regulators, are expressed on a variety of tissues and cells,
such as pancreatic beta cells (36) and hematopoietic
cells (37). Our results extend the findings of these earlier
Figure 5. Induction of transcriptional activity in HOX4C by basic fibroblast growth factor
(bFGF). A, Constructs used in transfection assay. The pGL2-Promoter vector contains the
SV40 promoter (SV40) upstream of the luciferase gene (Luc). The reporter construct pGLC
was generated by inserting C2 fragment of human HOX4C in sense orientation and
upstream of the promoter-luciferase transcriptional unit. B, After transfection of the
pGL2-Promoter (pGL2) or pGLC, NIH-3T3 cells were incubated with or without 10 ngiml
of bFGF as indicated. Luciferase assays were performed using a Luciferase Assay System
Kit (Promega). Luciferase activity was normalized relative to human placental alkaline
phosphatase activity. Values are the mean 5 SEM from 1 of 3 similar experiments. ** = P <
0.01 versus pGL2; * = P < 0.05 versus pGL2.
studies by identifying the presence of a diverse set of
HOX genes in arthritic synovium. The abundance and
diversity of HOX genes in arthritic tissues probably
reflect the remarkable complexity of the machinery
controlling synoviocyte development and differentiation.
Our results also show that one particular HOX gene,
HOX4C, is specifically expressed on rheumatoid synovium, suggesting that it may play a crucial role in the
development of RA.
HOX4C (HOX4.4 in the mouse) is considered to
be the earliest HOX gene for limb formation (22). In
human embryonic carcinoma cells, HOX4C is not affected by retinoic acid stimulation, but other HOX
genes, i.e., HOX4D, HOX4F, HOX4H, and HOX41, are
suppressed (38). This evidence suggests that HOX4C
plays a distinct role in the initiation of limb formation.
Several lines of evidence suggest that FGF has
multiple effects on the expression of HOX genes
(23,39,40). For example, in the developing mouse limb,
Evx-1, a horneobox-containing gene, was recently identified as a downstream gene in the FGF signal transduction pathway in limb patterning (41). It was also found
that the expression of other HOX genes, such as XIHbox6 (23,39) and Nkx-1.1, can be activated by bFGF
(40). Since our results showed that HOX4C was specifically expressed on rheumatoid synovium,we believe it is
involved in the signaling cascade of bFGF. To address
this hypothesis, we investigated whether the expression
of HOX4C was increased after treatment with bFGF.
Among several highly conserved short sequences located
in the noncoding areas of HOX4C, C2 is involved in the
regulation of HOX4C (26,42). We also established that
bFGF enhanced the DNA binding activity of the C2
element of HOX4C in rheumatoid synovial fibroblasts,
but not in OA fibroblasts. The notion of up-regulation of
the HOX4C gene by bFGF was supported by the results
of the transient expression assay. Considering these
results together, it can be concluded that the HOX4C
gene on rheumatoid synovial fibroblasts is activated by
bFGF through the C2 regulatory region of HOX4C.
Our findings that HOX4C was expressed on
rheumatoid synoviocytes and involved in the signaling
cascade initiated by bFGF may also suggest a functional
role for HOX4C in synovial hyperplasia. A number of
HOX genes are expressed in certain neoplasms and
involved in abnormal cellular proliferation (43-45). For
example, Jurkat T cells transfected with the HOX HB24
gene form tumors in the nude mouse (46). It is possible
that HOX4C and bFGF acting in concert in arthritic
synovial hyperplasia may lead to a combined effect
similar to mesenchymal cell growth during embryonic
The finding that bFGF modulates the homeodomain transcriptional regulator may also enhance our
understanding of how bFGF contributes to joint destruction in RA. The secretion of tissue inhibitor of metalloproteinases (TIMP) and collagenase by human synovial
fibroblasts is modulated by retinoids (47,48). Basic FGF
appears to stimulate collagenase primarily via increasing
transcription of the collagenase gene (49). Moreover,
all-trans-retinoic acid interacts synergisticallywith bFGF
to stimulate the production of TIMP from fibroblasts
(50). Because HOX4C was modulated by bFGF, it is
reasonable to assume that HOX4C may be part of the
bFGF signaling cascade in fibroblasts. To confirm this
hypothesis, it is important to determine the response of
bFGF on HOX4C-transfected cells. Such studies are
currently in progress in our laboratory.
In conclusion, the present study demonstrates
that the expression and transcriptional activity of a HOX
gene, HOX4C, are regulated by bFGF in RA. Basic
FGF is thought to be a growth factor responsible, at least
in part, for promoting fibroblast proliferation in RA.
Therefore, the results reported here not only expand our
understanding of the action of bFGF in RA, but also
suggest that the homeodomain transcriptional factor is
involved in the hyperplasia of rheumatoid synovium.
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factors, growth, basic, hox4c, transcription, genes, regulation, synovial, rheumatoid, fibroblasts
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