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The Journal of Immunology, 2007, 178: 1774-1782.
Copyright © 2007 by The American Association of Immunologists, Inc.

Vascular Endothelial Growth Factor Expression in Arthritic Joint Is Regulated by SAF-1 Transcription Factor1

Bimal K. Ray*,{dagger}, Arvind Shakya* and Alpana Ray2,*,{dagger}

* Department of Veterinary Pathobiology and {dagger} Department of Orthopaedic Surgery, University of Missouri, Columbia, MO 65211


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of arthritis by promoting angiogenesis in the synovial joint and infiltration of inflammatory cells in the synovial joint. Although ample information has been obtained on the mechanism of VEGF regulation during cancer and hypoxic condition, less is known about the control of VEGF expression during arthritis. From the studies on the experimentally induced arthritis in a transgenic mouse model that overexpresses a transcription factor, serum amyloid A activating factor-1 (SAF-1), leading to markedly higher levels of angiogenesis, synovial inflammation, and inflammatory cell infiltration, we have identified a novel mechanism of VEGF regulation. We present molecular evidence that VEGF expression is increased in SAF-1-transgenic mice and that SAF-1 induces VEGF transcription by directly binding to its promoter. Deletion of SAF-1 binding elements from the VEGF promoter as well as knockdown of endogenous SAF-1 markedly inhibited IL-1beta- and TGF-beta-mediated induction of VEGF expression in chondrocyte cells. By chromatin immunoprecipitation assay, in vivo, markedly higher levels of SAF-1 interaction with the VEGF promoter was detected in the cartilage tissues of arthritic mice as well as human osteoarthritic patients. Together, these results provide a new insight into the molecular mechanism of VEGF expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vascular endothelial growth factor (VEGF)3 is a potent stimulator of angiogenesis that plays an essential role during embryonic growth and postnatal development. However, in adults, angiogenesis is mostly associated with pathological conditions such as cancer during which increased blood supply fuels unwanted growth factors and cytokines to the diseased tissues (1). Control of VEGF synthesis or action for the prevention of angiogenesis is therefore intensely investigated and factors that potentiate or inhibit VEGF synthesis are matters of significant interest. Angiogenesis at the osteochondral junction as well as within the osteoarthritic synovium also poses a critical problem in the pathology of osteoarthritis (OA) (2, 3). About one-third of OA patients develop severe synovial inflammation which is not confined to only patients with end-stage disease (2). Although undetectable in normal adult synovium, VEGF is seen to be present at a markedly higher concentration in OA synovium and chondrocytes especially in clusters of superficial chondrocytes; immunostaining of VEGF is shown to correlate well with the severity of morphological changes (4, 5). It is also expressed in the inflamed synovium by synovial fibroblasts and activated macrophages (6, 7). In addition, VEGF also contributes to inflammation via plasma extravasation (1). VEGF expression is stimulated by cytokines and growth factors including IL-1 and TGF-beta that are present in increased concentration in OA synovium (1, 6, 8). The mechanisms of VEGF regulation during the switch from quiescent to proliferating stage when new blood vessels are formed are still incompletely understood. One of the potent regulators of VEGF is hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) transcription factor (9, 10). However in HIF-1{alpha}null mice growth plates, VEGF expression in the hypertrophic chondrocytes was not affected (11) which suggested the existence of an HIF-1{alpha}-independent mode of VEGF regulation. Indeed, there are many nonhypoxic conditions during which VEGF expression is strongly increased in multiple cell types and some of these processes have been shown to be regulated by different transcription factors including NF-{kappa}B (12), Sp1 (13), AP-2 (14), and estrogen receptors (15). A serendipitous finding from a transgenic line of mice overexpressing a novel transcription factor SAF-1 suggested the possibility that this protein may be involved in regulating VEGF expression.

SAF-1 is an inflammatory-responsive Cys2His2-type zinc finger transcription factor (16). We have recently reported that serum amyloid A activating factor-1 (SAF-1) transgenic mice develop inflammation-induced arthritis with significantly higher levels of synovial inflammation, inflammatory cell infiltration, angiogenesis, and cartilage degradation (17). The incidence of increased cartilage degradation was expected as SAF-1 is a transcriptional regulator of several matrix metalloproteinases (18, 19, 20) but why SAF-1-transgenic mice experienced such higher levels of inflammatory cell infiltration and angiogenesis remained elusive. In this study, we demonstrate that SAF-1 directly regulates VEGF expression by a novel mechanism and while exogenous expression of SAF-1 potentiates, knockdown of endogenous SAF-1 abrogates VEGF expression in the chondrocyte cells. Additionally, in vivo interaction of SAF-1 with the VEGF promoter in cytokine-stimulated as well as arthritic cartilage tissues was detected by chromatin immunoprecipitation (ChIP) assay. Together, our data provide new insight into the molecular mechanism of VEGF expression. The physiological significance of the SAF-1-transgenic mouse model is that it provides a system to study, in the same setting, the concurrent effects of VEGF-mediated angiogenesis and matrix metalloproteinase-mediated matrix degradation which together play a critical role in the pathogenesis of arthritis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Transgenic mice

The generation of transgenic mice expressing SAF-1 under the control of the CMV promoter was described previously (17). SAF-1-transgenic and nontransgenic littermate B6C3F1 mice, 8 wk old, were inoculated in both hind footpads with a single injection of 106 Borrelia burgdorferi organisms as described (17). Control mice received a single injection of vehicle. Induction of arthritis was evident from the swelling detected 7 days after the bacterial challenge and was assessed by daily measurement of tibiotarsal joints with a metric caliper.

Immunohistochemical analysis

Animals were sacrificed by cervical dislocation 21 days following infection. The hind feet from control and treated mice were dissected and one set was frozen immediately and the other set was fixed in formaldehyde, decalcified, and embedded in paraffin. Human articular cartilage tissues were also similarly fixed and embedded in paraffin. Deparaffinized sections (5 µm) were stained with H&E. Immunostaining was performed as described (17) using anti-VEGF Ab (sc-152; Santa Cruz Biotechnology) at a 1/250 dilution. Preimmune rabbit IgG was used as a control. HRP-conjugated goat anti-rabbit IgG was used as the secondary Ab. Color was developed with diaminobenzidine, and slides were counterstained with hematoxylin solution. The animal studies have been reviewed and approved by the University of Missouri Animal Care and Use Review Committee. The ages of human subjects (including cadavers) ranged between 45 and 81 years, with body weight ranging between 150 and 260 pounds. Severity of OA in cartilage specimens was graded by the Outerbridge system (21) and verified by histological examination. An institutional review board-approved protocol was followed for human specimens.

Plasmids

The 1.2VEGF-CAT reporter plasmid was constructed by ligating a 1.2-kb promoter DNA fragment of the human VEGF gene (22) in pBLCAT3 vector (23). A deletion plasmid, 1.2{Delta}(–100/–30)VEGF-CAT, lacking 71 nt of the VEGF promoter from –100 to –30 that contains the SAF-1-binding regions was prepared by creating two overlapping oligonucleotides that lack this region of the VEGF promoter. These oligonucleotides were annealed to form a duplex which was extended by the Klenow fragment of DNA polymerase to a dsDNA. This DNA fragment was ligated to the VEGF backbone to generate the internally deleted 1.2{Delta}(–100/–30)VEGF fragment and after sequence verification, the mutated DNA was subcloned in the pBLCAT3 vector. Expression plasmids pcDSAF-1 (16) and pcDrevSAF-1 (18) were described previously.

Transient transfection assay

HTB-94 (SW-1353) chondrocyte cells, a primary grade II chondrosarcoma of the right humerus from a 72-year-old female Caucasian, were obtained from American Type Culture Collection. Isolation and propagation of primary cultures of chondrocytes from human OA patients’ cartilage was described earlier (18). HTB-94 cells were transiently transfected with 0.5 µg of reporter plasmid, 0.5 µg of pSV-beta-gal as a control for measuring transfection efficiency, an increasing concentration of pCDSAF-1 plasmid or empty vector or pcDrevSAF-1, and carrier DNA so that the total amount of DNA in each transfection assay remained constant. The DNAs for transfection were prepared using METAFECTENE PRO (Biontex Laboratories). After 24 h, cells were harvested and a chloramphenicol acetyl transferase (CAT) assay was performed using equivalent amount of cell extracts as described (16). For stimulation, cells were incubated with IL-1beta and TGF-beta, at concentrations as described in the figure legends, in serum-free medium for 24 h.

ChIP

ChIP assays were performed on cartilage tissues obtained from normal and arthritic mice, as well as normal cadaver and human OA patients following the method as described with minor modifications (24). Cartilage tissues (similar weight) were ground in liquid nitrogen and cross-linked with 1% formaldehyde for 10 min followed by addition of 0.125 M glycine for 5 min and washing in PBS buffer. DNA-protein complexes were immunoprecipitated using anti-SAF-1 Ab, washed, and the immunoprecipitated complexes were recovered by the addition of protein A-Sepharose. Immunoprecipitated DNA was de-cross-linked by adding NaCl to a 200 mM final concentration and any contaminating RNA was removed by RNase digestion. Purified DNA was used as template in PCR with TaqDNA polymerase and specific primers spanning the SAF-1 DNA-binding element of the VEGF promoter. PCR products were resolved in 1.5% agarose gels and visualized by ethidium bromide staining. Primers used for amplification of the mouse VEGF promoter were 5'-TAGCTTTCCATTTCGCGG-3' and 5'-CGGCTGCCCCAAGCCTC-3'. Primers used for amplification of the human VEGF promoter were 5'-GAGCTTCCCCTTCATTGCGG-3' and 5'-CGGCTGCCCCAAGCCTC-3'.

DNase I footprint assay

VEGF promoter DNA (–260 to +5) was radiolabeled at one end and incubated with 0.25, 0.75, and 1.5 µg of purified SAF-1 protein. Full-length SAF-1 protein was prepared from a SAF-1 plasmid clone in pRSETA vector (Invitrogen Life Technologies) that expressed a histidine-tagged SAF-1 protein. This protein was purified by affinity chromatography in a nickel-Sepharose column as described previously (25). In some experiments, radiolabeled VEGF DNA was incubated with 20 µg of protein of nuclear extracts from cartilage tissues of human normal cadaver and OA patients. For Ab interaction studies, anti-SAF-1 Ab, which had been prepared as described earlier (18), was added to the reaction mixtures during a preincubation period of 30 min on ice. DNase I-protected regions were identified by following methods as described earlier (26).

RT-PCR

Total RNA from the joint cartilage tissue of normal cadavers and OA patients were isolated following a procedure described earlier (27). RT-PCR was performed using an RT-PCR kit and following the manufacturer’s protocol (Invitrogen Life Technologies). One microgram of DNase-treated RNA was used in the reverse transcription reaction with random hexamer and oligo (dT)12–18 as extension primers. PCR was performed for 25 cycles using the following gene-specific primers: VEGF, 5'-GGATGTCTATCAGCGCAGCTAC-3' and 5'-TCACCGCCTCGGCTTGTCACATC-3'; SAF-1, 5'-AGCGCACGTGGTCCGACACGAGGAG-3' and 5'-ATTGGACAAACCTCACCAGTAC-3'; GAPDH, 5'-TGCACCACCAACTGCTTAG-3' and 5'-TAGAGGCAGGGATGATGTTC-3'.

Reaction products were resolved in a 1.5% agarose gel and the identity of amplified DNA was verified by DNA sequence analysis. For semiquantitative RT-PCR analysis of VEGF and SAF-1 transcripts, DNA-free RNA samples were reverse transcribed to cDNA as described above. The cDNA products were amplified by PCR in four replicate reactions containing 32P-labeled dNTPs. One of the replicate reactions of each sample was removed from the thermal cycler at the end of cycle 15, 20, 25, and 30. PCR products were fractionated in a 1.5% agarose gel and the bands were detected by autoradiography and the radioactivity in each band was measured. The data was normalized to GAPDH amplification and minus-reverse transcription control was used for each sample to ensure specificity of the RT-PCR amplification.

Western immunoblot analysis

Chondrocyte cells, grown in 60-mm dishes, were lysed in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM DTT, 1% Nonidet P-40, 0.1% SDS, 1 mM PMSF, and 0.5 mg/ml benzamidine buffer followed by sonication. The extracts (50 µg of protein) were fractionated in a 5%/11% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. To evaluate the relative amount of proteins in each lane, proteins were stained with Ponceau S solution (Sigma-Aldrich). Immunoblotting was performed using a 1/3000 dilution of anti-SAF-1 Ab, prepared as described earlier (18) or anti-GAPDH Ab (Santa Cruz Biotechnology). Bands were detected by using a chemiluminescence detection kit (Amersham Biosciences).

ELISA for VEGF

The VEGF level in the serum-free conditioned medium from chondrocyte cells was measured by using a solid-phase sandwich ELISA kit obtained from American Laboratory Products. Primary cultures of chondrocyte cells (18) or HTB-94 (SW-1353) chondrocyte cells were used. The cells were transfected with either control vector (pcDNA3) or a SAF-1 expression plasmid (pcDSAF-1). ELISA was performed following the manufacturer’s protocol. Absorbance of each sample was measured at 450 nm and quantitated by using a standard curve derived from serial dilutions of known concentration of human VEGF provided in the manufacturer’s kit. The assays were performed in duplicate and the experiment was repeated at least three times.

EMSA

Nuclear extracts from joint cartilage tissue from normal human cadavers and OA patients were prepared by grinding the tissues under liquid nitrogen in a mortar pestle and resuspending the ground tissue in buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 0.5 µg/ml each of leupeptin, pepstatin, and antipain, 0.1 µg/ml chymotrypsin, 0.3 trypsin inhibitor unit/ml aprotinin, and 0.5 mg/ml benzamidine). The mixture was vortexed and centrifuged at 3000 rpm in a microfuge for 3 min. The supernatant was processed to prepare nuclear extract as described earlier (28). Protein content was measured by the Bradford method (29). EMSA was performed with equal amounts of proteins using a method described previously (16). Radiolabeled VEGF DNA from –108 to –25 containing an SAF-binding element of the VEGF promoter was prepared by using [{alpha}-32P]dCTP as the substrate to label the dsDNA probe by filling in the overhangs at the termini with Klenow fragment of DNA polymerase. Some DNA-binding assay mixture contained 100-fold molar excess (100 nM) of competitor oligonucleotides for AP-1, AP-2, NF-{kappa}B, Sp1, and SAF. AP-1, AP-2, NF-{kappa}B, and Sp1 consensus oligonucleotides were obtained from Promega. SAF consensus oligonucleotide contained the following sequence 5'-CCCTTCCTCTCCACCCACAGCCCCCATGG-3'. For Ab interaction studies, anti-SAF-1 Ab (18) or a preimmune IgG preparation was added to the reaction mixtures during a preincubation period of 30 min on ice. The DNA-protein complexes were separated in a nondenaturing 6% polyacrylamide gel and detected by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
VEGF level is higher in arthritic SAF-1-transgenic mice

In a previous study, we noted that synovial inflammation and macrophage cell infiltration are much more rapid and of higher magnitude in SAF-1-transgenic mice when these mice are challenged with B. burgdorferi, an arthritis-promoting agent in humans (17). As VEGF is a potent stimulator of angiogenesis and a positive correlation between VEGF expression, synovial inflammation and severity of OA is seen in humans (2), we examined its level of expression in SAF-1-transgenic mice during B. burgdorferi infection. Massive infiltration of inflammatory cells in the joints indicated much higher level of inflammation in the SAF-1-transgenic mice (Fig. 1, compare between A and D, and B and E). Synovial vessel density is also higher in arthritic SAF-1-transgenic mice (Fig. 1, compare between G and H). Inflamed joint tissues of SAF-1-transgenic mice also showed much higher levels of VEGF protein as compared with the nontransgenic littermates (Fig. 1, C and F). These findings suggested a potential link between increased abundance of SAF-1 and VEGF expression in the joint tissue.


Figure 1
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  		FIGURE 1. Increased inflammatory cell infiltration and VEGF expression in the tibiotarsal joints of arthritic SAF-1-transgenic mice. The hind tibiotarsal joints of four different groups of mice, eight in each group, were examined. Joint tissue samples were prepared as described in Materials and Methods, stained with H & E, and photographed. A representative sample from B. burgdorferi-infected arthritic nontransgenic (A–C and G) and SAF-1-transgenic mice (D–F and H) is shown. Boxed regions in A and D are shown in B and E, respectively, for a close-up view. Higher magnifications of the boxed regions in B and E are shown in G and H, respectively. Increased accumulation of inflammatory cells, including neutrophil and macrophage infiltrates and increased synovial angiogenesis in A and D are indicated by arrows. Synovial vessel density, indicated by arrows, is significantly increased in arthritic SAF-1-transgenic mice (H) as compared with arthritic nontransgenic mice (G). Sections of tibiotarsal joints were analyzed by immunohistochemistry using anti-VEGF Ab (C and F). Magnification, A and D, x25; B and E, x300; C and F, x200; G and H, x800.

 
Abundance of VEGF and SAF-1 in human OA cartilage

Because angiogenesis is a potential problem in the pathogenesis of human OA (2, 3), we examined the levels of VEGF and SAF-1 in the OA-affected cartilage tissues of human OA patients. The protein levels of VEGF and SAF-1 in OA and normal cartilage tissues were examined by immunohistochemistry. As shown in Fig. 2, individual and clustered chondrocytes in the superficial and transitional zones of the OA cartilage were immunopositive with the Abs against both VEGF (Fig. 2B) and SAF-1 (Fig. 2D). In contrast, only a few chondrocyte cells of normal cartilage showed little to negligible staining with both of these two Abs (Fig. 2, A and C). The specificity of the VEGF and SAF-1 immunostaining was confirmed by the omission of primary Abs which showed no staining (data not shown). In addition, nonimmune rabbit IgG was also used as a negative control (data not shown). Fig. 2, E and F, shows higher magnification of VEGF and SAF-1-immunopositive clustered chondrocyte cells in Fig. 2, B and D, respectively. In these clusters some empty lacunae, indicative of OA pathogenesis, were visible. The expression of VEGF isoforms and SAF-1 was examined by RT-PCR using total RNA isolated from normal and OA cartilage tissues. OA cartilage tissues contained higher levels of VEGF mRNA, corresponding to VEGF121, VEGF165, and VEGF189 isoforms, as compared with the normal tissues (Fig. 2G). The SAF-1 expression level was also very high in OA cartilage tissue (Fig. 2G). GAPDH mRNA was used as a control to ensure the use of equal amount of RNA in the RT-PCR. These results were confirmed by semiquantitative RT-PCR analysis which indicated a >2-fold increase of VEGF mRNA level in osteoarthritic cartilage (Fig. 2H).


Figure 2
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  		FIGURE 2. Increased VEGF expression in human OA cartilage. Cartilage tissue samples, collected from the knee joints of normal human cadavers (A and C) and OA patients (B and D) undergoing total knee replacement surgery, were prepared as described in Materials and Methods and were analyzed by immunohistochemistry using anti-VEGF Ab (A and B) and anti-SAF-1 Ab (C and D). In A and C, insets show higher magnification of a chondrocyte. E and F represent high magnifications of inset regions in B and D, respectively. G, RT-PCR analysis of VEGF and SAF-1 mRNAs of cartilage tissues of normal and OA patients. RT-PCR products, generated as described in Materials and Methods, were fractionated in a 1.5% agarose gel. Two representative normal and three OA cartilage samples, from a total of 30 patients’ samples tested, are shown. Three VEGF-specific products, which correspond to three splice variants, VEGF121, VEGF165, and VEGF189, were detected. SAF-1-specific RT-PCR product is indicated. RT-PCR product for GAPDH is also indicated. H, Relative increase of SAF-1, VEGF121, VEGF165, and VEGF189 mRNA level in OA cartilage in comparison to that of normal cartilage was determined by measuring radioactivity in the PCR products from reactions terminated at 20 cycles when the amplification was at linear range. Results represent the mean ± SE of five separate experiments. *, p < 0.05 compared with normal tissue.

 
SAF-1 increases VEGF promoter activity and VEGF protein level in chondrocyte cells

Correlation between the VEGF and SAF-1 protein level in human OA patients and also the presence of increased VEGF in arthritic SAF-1-transgenic mice suggested that the SAF-1 transcription factor might be involved in increasing VEGF expression. To determine, HTB-94 chondrocyte cells were cotransfected with a reporter gene containing 1.2 kb of upstream promoter DNA from –1179 to +21 of the human VEGF gene and the pcDSAF-1 expression plasmid. As seen in Fig. 3A, ectopic expression of pcDSAF-1 expression plasmid, but not the empty vector pcDNA3, increased the expression of 1.2VEGF-CAT reporter in a dose-dependent manner. This data indicated that SAF-1 stimulates VEGF promoter activity. To test whether this activity of SAF-1 has any effect on cellular VEGF, we analyzed VEGF protein level in the culture medium of chondrocyte cells that are overexpressing SAF-1 by pcDSAF-1 expression plasmid. Cells were transfected in serum-free medium, and culture medium after 48 h of transfection was examined for VEGF protein level by ELISA. Indeed, SAF-1-overexpressing HTB-94 cells contained increased concentrations of VEGF protein which further demonstrated inducing effect of SAF-1 (Fig. 3B). Such an effect was specific because pcDNA3-transfected cells exhibited no stimulation. Similar results were obtained when we used the primary culture of human chondrocyte cells (data not shown). Results of these in vitro experiments suggested that SAF-1 can increase VEGF expression by directly interacting with the VEGF promoter.


Figure 3
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  		FIGURE 3. Induction of VEGF promoter activity and VEGF expression during ectopic expression of SAF-1. A, HTB-94 chondrocyte cells were cotransfected with 1.2VEGF-CAT reporter (0.5 µg) and increasing concentrations of either pcDSAF-1 or pcDNA3 plasmid (0.5, 1.0, and 1.5 µg), as indicated. CAT activity was determined 24 h after transfection using equivalent amount of cell extracts. Results are an average of three independent transfection experiments. B, ELISA analysis of VEGF expression in response to SAF-1. Chondrocyte cells were transfected with increasing concentrations of either pcDSAF-1 or pcDNA3 plasmid (0.5, 1.0, and 1.5 µg), as indicated. The cells were then grown for 24 h in a serum-free medium, and the VEGF level in the conditioned medium was determined as described in Materials and Methods.

 
Identification of the SAF-1 binding site in the VEGF promoter

To assess whether SAF-1 directly interacts with the VEGF promoter to regulate its expression, the SAF1-binding element of SAF-1 in the VEGF promoter was determined by a DNase I protection assay using affinity purified SAF-1 protein and an end-labeled VEGF promoter DNA (Fig. 4). This assay revealed two adjacently placed protected sites between –96 to –64 and from –47 to –30 at the proximal region of human VEGF promoter as the DNA-binding elements of SAF-1 (Fig. 4A, lanes 3–5). A similar type of protection was seen with OA-cartilage tissue-derived nuclear extract (Fig. 4A, lane 7). Almost no protection was obtained with normal cartilage nuclear extract (Fig. 4A, lane 6) and when OA cartilage nuclear extract was neutralized by SAF-1 Ab (Fig. 4A, lane 8). The DNA sequence of the SAF-1-protected regions in the VEGF promoter is shown in Fig. 4B.


Figure 4
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  		FIGURE 4. Identification of SAF-1 binding site in VEGF promoter. A, A single 5' end-labeled human VEGF promoter DNA was incubated without any protein (lane 2) or with increasing concentrations of a purified SAF-1 protein (0.25, 0.75, and 1.5 µg, respectively, in lanes 3–5) or nuclear extracts (20 µg of protein) from normal human (lane 6) and OA (lanes 7 and 8) cartilage tissues. In lane 8, anti-SAF-1 Ab was added to the DNA-binding reaction. DNA-protein complexes were incubated with DNase I, and the resultant DNA fragments were fractionated in a 12% sequencing gel containing 8 M urea. Lane 1, G-reaction, performed following Maxam and Gilbert method (39 ), was used as a marker. Two protected regions are indicated by I and II. B, DNA sequence of the VEGF promoter surrounding the protected regions I and II, indicated by shaded area, spans nucleotide positions –96 to –64 and –47 to –30, respectively.

 
Characterization of the VEGF promoter-binding factor(s) in chondrocyte nuclear extract

To provide further biochemical evidence of SAF-1 interaction to the VEGF promoter in chondrocyte cells, we performed EMSA (Fig. 5). The radioactive VEGF probe was interacted by nuclear proteins in the normal cartilage nuclear extract to form a few faint DNA-protein complexes (Fig. 5A, lane 1). On the contrary, three major inducible DNA-protein complexes, designated as I-III, were detected with nuclear extracts of OA cartilage tissues (Fig. 5A, lanes 2 and 3). Complex IV was present in both normal and OA cartilage nuclear extracts. Further characterization of these DNA-protein complexes was done by competition with molar excess of unlabeled consensus oligonucleotides of several transcription factors including SAF-1 (Fig. 5B, lanes 2–5). Only the SAF-binding oligonucleotide (16) was able to compete and reduce the levels of two inducible DNA-protein complexes (complexes I and II) formed with OA cartilage nuclear extract (Fig. 5B, lane 6). In correlation, complexes I and II were inhibited by anti-SAF-1 Ab but not by nonspecific IgG (Fig. 5B, lanes 7 and 8). As the inducible complex III remained unaffected by anti-SAF-1 Ab and competitor SAF oligonucleotide, we regarded the identity of this complex as undetermined. The above data indicated the presence of highly active SAF-1 in OA cartilage tissue that binds to the VEGF promoter.


Figure 5
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  		FIGURE 5. EMSA analysis of factors that bind to the SAF-1 element. A, Nuclear extracts were prepared from cartilage tissues from normal human cadavers (lane 1) and OA patients (lanes 2 and 3) and incubated with the 32P-labeled human VEGF promoter (–108/–25) as described in Materials and Methods. Four DNA-protein complexes are indicated by I–IV. B, In addition to the nuclear extract from an OA patient, a 100-fold molar excess of competitor oligonucleotides for AP-1, AP-2, NF-{kappa}B, Sp1, and SAF-1 binding (lanes 2–6), a preimmune normal IgG (lane 7) and anti-SAF-1 Ab (lane 8) were included as indicated. DNA-protein complexes were resolved in a 6% nondenaturing polyacrylamide gel. DNA-protein complexes are indicated.

 
In vivo interaction of SAF-1 with VEGF promoter under arthritic condition

To directly address whether SAF-1 interaction with VEGF promoter is active in the context of chromatin, we performed a ChIP assay in which DNA-binding proteins were covalently linked to genomic DNA by treatment with formaldehyde and thereby preserve the status of DNA-protein interaction occurring in vivo. Cross-linked chromatin from normal and arthritic joints was fragmented and immunoprecipitated with anti-SAF-1 Ab or nonspecific preimmune serum as negative control. DNA from the immunoprecipitate-specific chromatin fragment was isolated and amplified by PCR. Based upon the knowledge of the SAF-1 binding site in the VEGF promoter, we designed two sets of primers that span this region of VEGF promoter in humans and mice (Fig. 6A). Presence of a PCR-amplified product in the immunoprecipitated DNA of human OA cartilage tissues but not of normal articular cartilage revealed that the proximal promoter of VEGF was occupied by SAF-1 protein during arthritic condition (Fig. 6B, compare between lanes 3 and 6). Cross-linked chromatin from normal and arthritic mouse joints was also used for ChIP analysis, which showed prominent SAF-1 interaction with mouse VEGF promoter under arthritic condition (Fig. 6C, lanes 4–9). In contrast, very little to almost no SAF-1 interaction with the VEGF promoter was seen with immunoprecipitated DNA of normal mouse cartilage (Fig. 6C, lanes 1–3). These results are consistent with the findings of 1) increased abundance of SAF-1 in OA cartilage and 2) its ability to bind the VEGF promoter (Figs. 2, 4, and 5).


Figure 6
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  		FIGURE 6. In vivo interaction of SAF-1 with the VEGF promoter during arthritis. A, Schematic of VEGF promoter and DNA sequence with two potential binding sites of SAF-1 and the two primer sets (arrows) used in chromatin immunoprecipitation. Human and mouse VEGF promoter DNA sequences are aligned with the transcription start site and are indicated by the underlined letter A. B, ChIP assay was performed on cartilage tissues obtained from normal human cadavers (lanes 1–3) and OA patients (lanes 4–6). Tissues were fixed with formaldehyde and immunoprecipitated with SAF-1 Ab, followed by amplification of VEGF promoter using primers encompassing the SAF-1 element shown in A. Amplified products were resolved in 1.5% agarose gel. C, Tissues obtained from normal (lanes 1–3) and B. burgdorferi-infected arthritic (lanes 4–9) SAF-1-transgenic mice were treated as described above, amplified using mouse VEGF-specific primers as described in A and resolved in a 1.5% agarose gel.

 
Deletion of SAF-1 element of VEGF promoter and depletion of SAF-1 protein in chondrocyte cells markedly reduces VEGF expression

Having determined that SAF-1 interacts with the VEGF promoter both in vitro and in vivo and such interaction can increase VEGF expression and protein level, we sought to determine how crucial the SAF-1 element is in mediating VEGF expression in the chondrocyte cells. A VEGF promoter in which SAF-1 element is deleted was used for the analysis (Fig. 7A). Because the expression of VEGF in arthritic synovium is known to be induced by TGF-beta and IL-1beta (8, 30) and combination of these two inducers is shown to considerably stimulate VEGF gene expression (31), we compared the expression levels of wild-type and SAF-1 site-deleted VEGF reporters in response to these agents. Addition of both IL-1beta and TGF-beta increased transcription of wild-type 1.2VEGF-CAT reporter but these cytokines had very little stimulatory effect on a mutant reporter 1.2{Delta}SAF-1VEGF-CAT (Fig. 7B). Deletion of SAF-1-binding elements also led to a significant decrease in the basal promoter activity, i.e., the activity in the absence of IL-1beta and TGF-beta, when compared with the wild-type 1.2VEGF promoter, suggesting that this region is not only important for mediating cytokine response, it is also important for basal expression of the promoter in the chondrocyte cells.


Figure 7
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  		FIGURE 7. Deletion of the SAF-1 element and depletion of SAF-1 abrogates VEGF promoter activity in chondrocyte cells. A, Schematic depicting the wild-type VEGF promoter, 1.2VEGF-CAT, containing the human VEGF sequence from –1179 to +21 where two SAF-1 binding sites are present. Targeted deletion of sequences from –100 to –30 removed both the SAF-1 binding sites in 1.2{Delta}(–100/–30)VEGF-CAT B, HTB-94 chondrocyte cells were transfected with 0.5 µg of either 1.2VEGF-CAT or 1.2{Delta}(–100/–30)VEGF-CAT reporter plasmid and incubated with increasing concentrations of IL-1beta and TGF-beta, (100 U + 2 ng)/ml, (200 U + 4 ng)/ml, and (400 U + 4 ng)/ml, respectively, following the method as described in Materials and Methods. CAT activity was determined 24 h after transfection using equivalent amount of cell extracts. Results are an average of three independent transfection experiments. C, HTB-94 chondrocyte cells were cotransfected with 0.5 µg of 1.2VEGF-CAT reporter and increasing concentrations (0.5, 1.0, and 1.5 µg) of pcDrevSAF-1 and pcDNA3 plasmid. Following transfection, some cells were incubated with IL-1beta and TGF-beta (200 U + 4 ng)/ml, as indicated. Cells were harvested 24 h later and CAT activity was measured using equivalent amount of cell extracts. Results are an average of three independent transfection experiments. Inset, Western immunoblot analysis of extracts of chondrocyte cells grown as described above. The transferred proteins in nitrocellulose membrane were probed with anti-SAF-1 and anti-GAPDH Abs, respectively. D, Chondrocyte cells were transfected with increasing concentrations of either pcDrevSAF-1 (0.5, 1.0, and 1.5 µg) or pcDNA3 (1.5 µg), as indicated. The cells were then incubated for 24 h with IL-1beta and TGF-beta (200 U + 4 ng)/ml, as indicated, in a serum-free medium and the VEGF level in the conditioned medium was determined as described in Materials and Methods.

 
To independently confirm the role of SAF-1 in mediating cytokine response, we examined the impact of depleting endogenous SAF-1 during cytokine stimulation. HTB-94 cells were cotransfected with 1.2VEGF-CAT reporter and an expression plasmid containing SAF-1 cDNA in reverse orientation. Following transfection, cells were stimulated with IL-1beta and TGF-beta cytokines (Fig. 7C). The SAF-1 level was increased following cytokine treatment but the level declined progressively when the cells were transfected with pcDrevSAF-1 (inset of Fig. 7C). When the cells were transfected with empty vector, pcDNA3, the SAF-1 level was not depleted (data not shown). With the progressive depletion of SAF-1, VEGF promoter activity was inhibited in a dose-dependent manner (Fig. 7C). VEGF protein level in the chondrocyte cell culture medium was stimulated by cytokine treatment and progressively inhibited as the SAF-1 is depleted by pcDrevSAF-1 transfection (Fig. 7D). Together, these data indicated that SAF-1 involvement is necessary for induction of VEGF expression in chondrocyte cells.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study provides strong in vitro and in vivo evidence that SAF-1 is a transcriptional regulator of VEGF expression during arthritis. The following novel findings were obtained. 1) A higher level of VEGF immunoreactivity in the joints of arthritic SAF-1-transgenic mice than nontransgenic littermates; 2) a dose-dependent increase of the VEGF promoter by exogenously expressed SAF-1; 3) inhibition of cytokine-mediated VEGF induction during depletion of endogenous SAF-1; 4) reduction of VEGF promoter activity due to deletion of the SAF-1 element from the VEGF promoter; and 5) in vivo occupation of SAF-1 protein at the VEGF promoter in arthritic tissues and cytokine-induced cells.

Angiogenesis, inflammation, and macrophage infiltration are important interrelated processes for the mediation and progression of OA. Chronic inflammation at the joint can stimulate angiogenesis which perpetuates the inflammatory condition, leading to the classical symptoms of OA, such as progressive degeneration of articular cartilage, remodeling of the subchondral bone, and osteophyte formation. An association between macrophage infiltration and synovial angiogenesis in OA is well-established. Understanding the mechanism of increased expression of VEGF, one of the most important regulators of angiogenesis therefore is a critical step in solving OA-related pathology. The expression level of VEGF mRNA is tightly regulated by both transcriptional and posttranscriptional mechanisms (1, 32). A variety of growth factors and cytokines including TGF-{alpha} and TGF-beta (33), IL-1 (30), and transforming agents such as v-Ha-Ras and v-Raf (34) and hypoxia (9) are shown to induce VEGF expression in several cell lines. Accordingly, multiple routes of the VEGF induction process have been identified. However, the signaling cascades of VEGF induction during osteoarthritic condition is far from being resolved. Our studies provide, for the first time, a molecular mechanism in which VEGF overexpression during OA occurs by means of an inflammation responsive transcription factor, SAF-1.

We investigated the mechanisms by which SAF-1 regulates VEGF expression using transfection, DNA-binding, and ChIP assays. Exogenous SAF-1 expression markedly increased VEGF promoter activity in a dose-dependent manner. Exogenous SAF-1 expression also increased cellular VEGF protein level in transfected cells. Conversely, when we down-regulated endogenous SAF-1 by antisense SAF-1, the VEGF promoter activity was markedly inhibited. Antisense SAF-1 also inhibited IL-1beta- and TGF-beta-mediated induction of VEGF gene expression. The physical interaction between SAF-1 and VEGF promoter under arthritic conditions became evident from the ChIP assay. Such interaction in both human and mouse chromatin highlights the importance of SAF-1 in VEGF expression in diverse conditions. Deletion of the SAF-1 element not only inhibited IL-1beta- plus TGF-beta-mediated induction of VEGF, it also reduced the basal expression level of VEGF promoter. The latter observation suggests that the SAF-1-binding region of the VEGF promoter is also required for VEGF expression in normal physiological condition. From the present results, it is not clear whether under normal condition VEGF expression is regulated by SAF-1 as the results of ChIP and DNA-binding assays indicated very little SAF-1 and VEGF interaction under normal condition.

The SAF-1 DNA-binding element in the VEGF promoter is highly rich in G-C sequences which could act as the DNA-binding element of Sp1 transcription factor (35). The DNA-binding element of Sp1 is present in the regulatory regions of a wide range of vertebrate genes, and it is traditionally viewed as a constitutive transcription factor required for the maintenance of basal transcription. Earlier, we and others (36, 37) showed that SAF-1 and Sp1 can both bind to the same DNA element and synergize each other’s function (36). However, in this study, we did not detect any DNA-protein complex from the nuclear extracts of arthritic tissues that are composed of SAF-1 and Sp1. One of the interesting observations of the EMSA analysis (Fig. 5) is the apparent involvement of SAF-1 in the formation of multiple DNA-protein complexes including complexes I and II. These two complexes, but not complex III, were inhibited by both competitor SAF consensus oligonucleotide and anti-SAF-1 Ab. We believe that formation of these multiple complexes is the result of multimeric forms of SAF-1 or its association with other factors. Because recognition sequences of several known transcription factors showed no effect on these two complexes, we speculate that multimeric forms of SAF-1 are involved in forming DNA-protein complexes I and II.

In conclusion, we have uncovered a novel regulatory pathway of VEGF induction during arthritis. It is interesting in this context to mention that SAF-1 activity has been found to be high in metastatic prostate cancer tissues (B. K. Ray and A. Ray, unpublished observation) in the transgenic adenocarcinoma of mouse prostate cancer model (38). Because angiogenesis plays a critical role in malignant tumor formation and cancer metastasis, it is tempting to speculate that SAF-1 might be involved in increasing VEGF expression during the progression of cancer. Further studies will reveal whether such a mechanism of VEGF regulation is operative in tumorigenesis.


   Acknowledgments
 
We thank Jing Chen for assistance in transfection assays. We are grateful to Dr. B. Sonny Bal for kindly providing human cartilage tissue samples and Dr. Charles R. Brown for valuable help in generating B. burgdorferi-inflamed mice.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by U.S. Public Health Service Grant AR48762. Back

2 Address correspondence and reprint requests to Dr. Alpana Ray, Department of Veterinary Pathobiology, University of Missouri, 126A Connaway Hall, Columbia, MO 65211. E-mail address: rayal{at}missouri.edu Back

3 Abbreviations used in this paper: VEGF, vascular endothelial growth factor; OA, osteoarthritis; HIF, hypoxia-inducible factor; SAF-1, serum amyloid A activating factor-1; ChIP, chromatin immunoprecipitation; CAT, chloramphenicol acetyl transferase. Back

Received for publication August 16, 2006. Accepted for publication November 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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