CITED2-mediated regulation of MMP-1 and MMP-13 in human chondrocytes under flow shear.

CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2) is a member of the Cited family of nuclear regulators, previously known as mrg1 (melanocyte-specific gene-related gene 1). CITED2 is inducible by varying stimuli including lipopolysaccharide, hypoxia, and cytokines such as interleukin 9 and interferon gamma. Using the immortalized human chondrocyte cell line, C-28/I2, we investigated whether CITED2 could be responsive to mechanical stimuli, and if so, whether CITED2 could mediate shear-driven regulation of matrix metalloproteinase (MMP) genes. The C-28/I2 cells were cultured under flow shear at 1-20 dyn/cm2, and the role of CIT-ED2 in regulation of MMPs was examined using the plasmids encoding sense and antisense CITED2 DNA sequences. The results showed that flow shear at 5 dyn/cm2 increased CITED2 mRNA and protein levels and down-regulated MMP-1 and MMP-13 mRNA and protein levels as well as enzyme activities. Consistent with the coordinated expression patterns of CITED2 and MMPs, overexpression of CITED2 repressed MMP-1 and MMP-13 mRNA levels and activities, whereas antisense CITED2 plasmids prevented the shear-induced down-regulation of MMP expression. Interleukin-1beta induced the formation of p300-Ets-1 complexes without affecting expression of CITED2. Transforming growth factor-beta as well as flow shear at 5 dyn/cm2 stimulated not only the expression of CITED2 but also the association of CIT-ED2 with p300 by dissociating Ets-1 from p300. These results indicate that CITED2 plays a major role in shear-induced down-regulation of MMP-1 and MMP-13 via a transforming growth factor-beta-dependent pathway.

Physical stimuli at appropriate intensities are essential for growth and maintenance of bone and joint tissues (1)(2)(3). In vivo studies demonstrate that mechanical loading facilitates the strengthening of bone, and flow-induced shear in articular cartilage stimulates a repair response (4,5). Chondrocytes in cartilage experience a variety of stresses, strains, and pressure that result from normal activities of daily living. Determining how shear stress alters chondrocyte metabolism is fundamental to understanding how to limit matrix destruction and stimulate cartilage repair and regeneration (31). Matrix metallo-proteinases (MMPs) 1 are a family of collagen-degrading proteinases whose expression and activities are altered by mechanical stimuli in various cell types (6 -8). In inflammatory joint diseases such as rheumatoid arthritis and osteoarthritis, MMPs are considered pivotal proteinases of cartilage degradation. Because IL-1 and tumor necrosis factor-␣ are known to stimulate the expression and activities of MMPs, cytokine antagonists and receptor-blocking antibodies have been studied as potential agents for blocking cartilage destruction in joint diseases (9 -11). Our recent study using a human synovial cell line showed that gentle mechanical shear with intensity at a few dyn/cm 2 had anti-inflammatory effects and reduced the expression and activities of many MMPs including MMP-1 and MMP-13 (7). The molecular mechanism underlying anti-inflammatory responses to flow shear, especially down-regulation of MMPs, has not been identified.
Many MMPs possess transcription-factor binding motifs such as AP1, AP2, NFB, PEA3, Sp1, and STAT in the 5Јflanking regulatory DNA sequences (12)(13)(14). Transcription factors such as Ets-1, NFB, and Stat2 not only bind DNA directly but also interact with CREB-binding protein (CBP) and p300 (15). CBP and p300 are transcriptional co-activators, and they play a critical role in many cellular functions through interactions with TFIIB and RNA polymerase II as well as regulatory transcription factors (16). Recently, a new family of transcriptional co-regulators, the CITED (CBP/p300-interacting transactivator with ED-rich tail) family, was discovered. These factors interact with the first cysteine-histidine-rich (CH1) region of CBP/p300 (17) and appear to be key transcriptional modulators in embryogenesis, inflammation, and stress responses (18 -21). Recent studies have shown that CITED2 is involved in regulation of stress-responsive genes such as HIF1 and LIM (21). To our knowledge, however, the role of CITED in mechanical stress and regulation of MMPs has not been investigated.
MMP-1 and MMP-13 are collagenases whose expression is elevated in inflamed joints (22) and are associated with regions of collagen destruction in osteoarthritic chondrocytes (21,23). Thus, we investigated whether CITED2 could be induced by flow shear in chondrocytes and, if so, whether CITED2 could mediate the down-regulation of MMP expression and activities in response to flow shear. As a reproducible model, we used the C-28/I2 chondrocyte cell line and analyzed MMP-1 and MMP-13 mRNA levels and enzyme activities after application of flow shear with intensity up to 20 dyn/cm 2 . Our results using the sense and antisense CITED2 plasmids, overexpressed in the C-28/I2 cells prior to application of flow shear, support the critical role of CITED2 in flow shear-induced down-regulation of MMP-1 and MMP-13. Ets-1 is responsive to IL-1 and known to activate the transcription of many MMPs (24,25), and TGF-␤ leads to suppression of Ets-1 through its dissociation from CBP/p300 (26). In this report, we have identified a mechanism whereby IL-1␤ and inflammatory flow shear at 20 dyn/ cm 2 facilitate association of Ets-1 with p300 and TGF-␤ and anti-inflammatory flow shear at 5 dyn/cm 2 stimulate formation of p300-CITED2 complexes.

EXPERIMENTAL PROCEDURES
Cell Culture-The immortalized human chondrocyte cell line, C-28/ I2, was used for this study (27,28). The use of human cell cultures was approved by the Indiana University-Purdue University Indianapolis Institutional Committee. Cells were plated on a glass slide coated with 2 g/ml type I collagen and grown to reach ϳ70% confluency in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. After a mild starvation in medium containing 1% serum for 18 h, the cells were exposed to 1-h flow shear at intensity of Ͻ20 dyn/cm 2 in a Streamer Gold flow device (Flexcell International). In some experiments, the cells were incubated with recombinant human IL-1␤ (Calbiochem) or TGF-␤ (Calbiochem) at concentrations of 5-100 ng/ml for 0 -6 h.
Transfection of Sense and Antisense CITED2 Plasmids-Expression vectors containing the sense or antisense CITED2 DNA sequences were constructed in pcDNA3.1 to examine the function of CITED2 (Fig. 1). Sequences from the coding region of human CITED2 DNA was amplified by PCR using the primers containing EcoRI and BamHI sites, and the amplified fragments were subcloned into the pcDNA3.1 vector. The sense CITED2 plasmid (pcDNA3.1-CITED2S) was used to overexpress CITED2, and the antisense CITED2 plasmid (pcDNA3.1-CITED2AS) was designed to block translation of CITED2 mRNA. The plasmids were transfected in chondrocytes at a concentration of 0.1 or 1 g of DNA/2 ϫ 10 5 cells using the Effectene transfection reagent (Qiagen). In a parallel experiment using pIRES-EGFP plasmids (5.3 kb), transfection efficiency was estimated as 14.1 ϩ 2.2% (0.1 g of DNA) and 46.7 ϩ 2.4% (1.0 g of DNA). The CITED2 protein level was determined by immunoblotting using the antibody specific for CITED2.
Reverse Transcription (RT)-PCR-RT-PCR was conducted to determine the mRNA levels of CITED2, MMP-1, and MMP-13. Total RNA was isolated using the RNeasy mini kit (Qiagen) and reverse-transcribed with random hexamers. Semi-quantitative PCR was performed by hot-start at 94°C for 5 min using the primers listed in Table I. We employed 32 PCR cycles with a temperature profile at 94°C for denaturation (1 min), 55-65°C for annealing (1 min), and 72°C for extension (45 s to 1 min, 30 s). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as RT-PCR control. To further quantify the effects of shear and/or IL-1␤ on MMP-1 mRNA and MMP-13 mRNA, real-time PCR was conducted using ABI Prism 7700 sequence detection system (PE Applied Biosystems) as described previously (30). The primers were 5Ј-CTCAATTTCACTTCTGTTTTCTG-3Ј and 5Ј-CATCTCTGTCGGCA-AATTCGT-3Ј (MMP-1) and 5Ј-AAGGAGCATGGCGACTTCT-3Ј and 5Ј-TGGCCCAGGAGGAAAAGC-3Ј (MMP-13). The fluorescent probes were 5Ј-FAM-CACAACTGCCAAATGGGCTTGAAGC-TAMRA-3Ј (MMP-1) and 5Ј-FAM-CCTCTGGCCTGCTGGCTC-TAMRA-3Ј (MMP-13). A threshold cycle was determined to estimate differences in the starting cDNA copy number with and without IL-1␤ and/or the shear treatment. GAPDH was used as an internal control with the primers and the probe in the TaqMan assay control kit (PE Applied Biosystems).
Immunoblot/Immunoprecipitation Assay-An immunoblot assay was performed by using antibodies specific for MMP-1 (Calbiochem), MMP-13 (Calbiochem), and CITED2. The primary antibodies for MMP-1 and MMP-13 recognized both the latent and active forms.
Approximately 10 g of the extracted proteins were separated by electrophoresis (SDS-PAGE) in a 10% polyacrylamide gel, and the separated proteins were transferred to nitrocellulose membranes (Hybond, Amersham Biosciences). Membranes were incubated with primary antibodies followed by incubation with secondary antibodies conjugated to horseradish peroxidase (ECL Western blotting analysis system, Amersham Biosciences). The expected molecular sizes were 42/46 kDa (MMP-1), 48/60 kDa (MMP-13), and 28 kDa (CITED2). A mouse antibody specific for ␤-actin (42 kDa, Sigma) was used as loading control.
For immunoprecipitation, 100 g of nuclear extracts were mixed with 8 g of p300-specific antibody (Upstate Biotechnology) and incubated with gentle rocking at 4°C overnight. The immunocomplexes were captured by mixing with protein G-agarose beads for 2 h at 4°C. The collected agarose beads were washed four times with an ice-cold cell lysis buffer, and the precipitated proteins were eluted by boiling the beads for 5 min in the SDS sample buffer. Fractions of the supernatant were analyzed by SDS-PAGE and immunoblotting using antibodies against p300, CITED2, and Ets-1 (Santa Cruz Biotechnology). The expected molecular sizes of p300 and Ets-1 were 300 and 54 kDa, respectively.
MMP Activity Assay-MMP activities were determined using fluorophore-labeled substrates specific for MMP-1 or MMP-13 and collagenases or gelatinases (Molecular Probes) (30). The proteins isolated from the culture medium were incubated with the fluorescent substrates in a reaction buffer consisting of 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl 2 , and 0.2 mM sodium azide at room temperature for 2 h. Fluorescent intensity, a measure of MMP activity, was determined by a FluoroMax-2 spectrofluorometer (Instruments S. A., Inc.). An absorption/emission wavelength was set to 382/441 nm (MMP-1), 325/393 nm (MMP-13), and 495/515 nm (collagenases and gelatinases). The culture medium itself exhibited basal MMP activity that was ϳ5% of that in the medium incubated with control cells.
Data Analysis-All of the experiments were performed three times. To examine statistically significant changes in the levels of mRNAs, proteins, and MMP activities, a Student's t test was conducted using StatView software (version 5.0, SAS Institute Inc.) and p values Ͻ0.05 were considered statistically significant. conditions of culture. The levels of mRNA were reflected in the protein levels detected by immunoblotting and the respective enzyme activities. In response to flow shear at 0 (control), 1, 2, 5, 10, and 20 dyn/cm 2 for 1 h, MMP-1 and MMP-13 mRNA, protein levels, and enzyme activities were modulated in a stress intensity-dependent manner (Fig. 2). Low intensities of shear at 1 and 2 dyn/cm 2 had no effect, whereas shear at 5 dyn/cm 2 reduced significantly (p Ͻ 0.05) the MMP-1 and 13 mRNA levels ( Fig. 2A), protein levels (Fig. 2B), and enzyme activities (Fig. 2C). The antibody against MMP-1 detected the active form (46 kDa), and the antibody against MMP-13 identified both the active form (48 kDa) and the proenzyme (60 kDa). However, increasing the shear to 10 dyn/cm 2 resulted in less inhibition, whereas a further elevation to 20 dyn/cm 2 increased protein and activity levels of these collagenases but did not change the mRNA levels relative to controls. MMP-1 responses to shear at both 5 and 20 dyn/cm 2 were altered to a greater extent than MMP-13. A positive correlation was observed in Fig. 2 between the mRNA level and the protein level with r (correlation coefficient) ϭ 0.75 and between the protein level and the activity level with r ϭ 0.89. We determined not only the protein level but also the activity level, because both MMP-1 and MMP-13 need to be post-translationally modified for activation.

MMP
Increased Expression of CITED2 in Response to Flow Shear at 5 dyn/cm 2 -CITED2 is a transcriptional activator known to be inducible by many biological stresses, but no study on the responses to mechanical stress has been conducted previously. Thus, we wished to determine whether CITED2 could be expressed in chondrocytes in response to flow shear. The C-28/I2 cells expressed detectable levels of CITED2 mRNA and proteins as shown in Fig. 3. Application of flow shear at 5 dyn/cm 2 for 1 h increased CITED2 mRNA and protein levels by 1.9-and 2.0-fold, respectively. However, flow shear at 10 or 20 dyn/cm 2 did not alter either mRNA or protein level relative to controls (Fig. 3). These data indicate that the induction of CITED2 expression occurs at the same level of fluid sheer, 5 dyn/cm 2 , that causes down-regulation of MMP expression.
Suppression of the Levels of MMP-1 and MMP-13 mRNA, Protein, and Enzyme Activities by Overexpression of CIT-ED2-To examine the role of CITED2 in regulation of MMP-1 and MMP-13, we overexpressed CITED2 in C-28/I2 cells. Transfection of pcDNA3.1-CITED2S elevated the mRNA protein levels of CITED2, and the increases were dependent on the dosage of plasmids used for transfection. A 10-fold increase in the amount of plasmid DNA raised transfection efficiency by 3.3-fold (Fig. 4, A and B). The MMP-1 and MMP-13 mRNA levels and enzyme activities were reduced by overexpression of CITED2 (Fig. 4, A and C). Compared with the cells transfected with the empty pcDNA3.1 vector, transfection of the sense CITED2 plasmids at 0.1 and 1 g/2 ϫ 10 5 cells reduced MMP-1 enzyme activity by 43 and 66% and MMP-13 activity by 23 and 43%, respectively.
Loss of Shear-induced Repression of MMP-1 Expression after Overexpression of Antisense CITED2-To determine whether flow shear-induced CITED2 was indeed capable of suppressing MMP-1 expression, we transfected antisense CITED2 in the C-28/I2 cells for 72 h followed by application of flow shear at 5 dyn/cm 2 for 1 h. The antisense plasmid reduced the level of CITED2 protein by 81% in control cells and 80% in sheartreated cells at 5 dyn/cm 2 (Fig. 5A). In the cells transfected with the empty vector alone, the expression of CITED2 was not altered either in the presence or absence of flow shear (Fig. 5A). However, the level of MMP-1 mRNA was increased by overexpression of antisense CITED2 in either the absence or presence of flow shear (Fig. 5B).
CITED2 Expression in C-28/I2 Chondrocytes Was Unaltered by IL-1␤ but Activated by TGF-␤-We wished to determine whether the anti-catabolic effects of CITED2 were via modulation of IL-1␤ expression or TGF-␤ expression in C-28/I2 chondrocytes. After a 1-h incubation with IL-1␤ at a concentration of 5, 25, 50, and 100 ng/ml, the level of CITED2 mRNA was unchanged (Fig. 7A). Furthermore, the level of CITED2 mRNA was not affected by incubation with 25 ng/ml IL-1␤ for up to 4 h (Fig. 7B). On the other hand, a 1-h incubation with TGF-␤ at a concentration of 5, 25, 50, and 100 ng/ml elevated the mRNA level of CITED2 (Fig. 7C). The elevated level of CITED2 mRNA after a 30-min incubation with 25 ng/ml TGF-␤ was maintained at least for up to 4 h (Fig. 7D). The mRNA level of MMP-1 was undetectable in the presence of TGF-␤ (data not shown).

Formation of p300-Ets-1 Complexes by IL-1␤ and Inflammatory Shear at 20 dyn/cm 2 and Formation of p300-CITED2 Complexes by TGF-␤ and Anti-inflammatory Shear at 5 dyn/cm 2 -
Because CITED2 and Ets-1 are known to interact with p300, we determined whether IL-1␤ or TGF-␤, which have opposite effects on MMP expression, could modulate the interactions with this cofactor. The C-28/I2 cells were incubated with 25 ng/ml IL-1 ␤ or 25 ng/ml TGF-␤ for 1 h. Nuclear extracts were prepared and incubated with the antibody specific for p300 to immunoprecipitate p300 protein complexes. Western blotting identified equal amounts of p300 in control and treated extracts (Fig. 8). In control cells, no p300-Ets-1 or p300-CITED2 complex was detectable. In contrast, p300-Ets-1 complexes were detected in the extracts from IL-1␤-treated cells and cells under flow shear at 20 dyn/cm 2 and p300-CITED2 complexes were identified in the TGF-␤-treated cells and cells under flow shear at 5 dyn/cm 2 . These results suggest that CITED2 medi-ates TGF-␤-dependent effects on gene transcription via protein-protein interactions with p300. DISCUSSION In this study, we provide evidence that CITED2 is a shearresponsive transcriptional mediator in chondrocytes and that it plays a critical role in reducing expression and activity of MMP-1 and MMP-13 at intensities of flow shear that are physiologically relevant (31). We used the immortalized human chondrocyte cell line, C-28/I2, which has served as a reproducible model for examining intracellular signaling and gene transcriptional mechanisms relevant to chondrocyte functions (28,(32)(33)(34). Our results clearly show that CITED2 mRNA and protein levels are elevated most effectively by shear stress at 5 dyn/cm 2 and that this shear intensity down-regulates expression and activities of MMP-1 and MMP-13. A positive correlation between the MMP protein level and the activity level indicates that expression of tissue inhibitors of metalloprotein- The observed anti-inflammatory effects of mechanical stimuli are consistent with previous studies in various cell types, although excessive mechanical loads elevate expression of MMPs in chondrocytes (35). In peridontal ligament cells, 6 -10% of mechanical strain was shown to reduce IL-1␤-induced expression of proinflammatory cytokines such as IL-6 and IL-8 (8). Expression of MMP-1 was decreased by 1-4% of strain in vascular smooth muscle cells and by 6% of strain in fibrochondrocytes (36,37). Using synovial cells, we have shown previously that mechanical stimuli such as small strain at ϳ1% and flow shear at ϳ2 dyn/cm 2 are effective in reducing expression and activities of MMPs induced by IL-1␤ and tumor necrosis factor-␣ (30).
Elucidation of the molecular mechanism underlying the CIT-ED2-mediated down-regulation of MMP-1 and MMP-13 re- quires investigation of the interactions of CITED2 with p300/ CBP and many transcription factors that control MMP promoter activities by interacting with p300/CBP, such as c-Fos, Ets-1, NFB, and SMAD family members (16,25,26). In our previous studies using human synovial cells, the mRNA expression of c-Fos and Ets-1 was responsive to mechanical stimuli and the response was dependent on shear intensity (7). In computational simulations where the expression of MMPs in response to mechanical stimuli was modeled using multiple transcription-factor binding motifs, the PEA3 binding motif, which is recognized by Ets-1, was identified as a stimulatory factor (12). In the same model, the AP-2 binding motif was predicted as an inhibitory factor that is known to be activated by CITED2 (19). Taken together, these experimental and computational results suggest that CITED2 could orchestrate the expression of MMPs by regulating interactions between p300 and other transcription factors that either down-regulate (AP-2) or up-regulate (Ets-1 and NFB) MMP gene expression. The multifunctional p300/CBP has been shown to play a major role in SMAD-dependent TGF-␤ stimulation of the type I collagen promoter (38). Our studies suggest that CITED2 provides an alternative mechanism of TGF-␤-induced metabolic responses.
The observed link of the CITED2-mediated reduction in MMP expression to the TGF-␤ pathway is consistent with the known role of TGF-␤ in down-regulation of MMP-1 expression (28). TGF-␤ leads to dissociation of Ets-1 from the p300/CBP complexes (26), and TGF-␤-null mice develop uncontrolled inflammation and joint destruction (18). The observation that the anti-catabolic effects of CITED2 were not directly linked to the IL-1␤ signaling pathway was unexpected, because a previous study in mouse D10 T cells reported that CITED2 was inducible by IL-1␣. Differential responses to IL-1 in T cells and chondrocytes may result from cell-type dependent signaling pathways. We used IL1-␤ (29), because this is the dominant IL-1 isoform in human tissues and it plays a central role in the inflammation and connective tissue destruction observed in rheumatoid arthritis and osteoarthritis. The observed quick increase in MMP-1 expression in chondrocytes after 1 h of the IL-1␤ treatment is consistent with the responses to IL-1 in synoviocytes (30). IL-1␤ suppresses the expression of the type II collagen gene in C-28/I2 chondrocytes (39). However, because CITED2 down-regulates and IL-1␤ up-regulates MMP expression in chondrocytes, CITED2 could be interfering with the actions of IL-1-induced transcription factors on MMP promoters. Consistent with our observation in C-28/I2 chondrocytes, Xu et al. (40) reported that cyclic tensile strain, which generated an anti-inflammatory signal in chondrocytes, was not mediated through IL-1 receptors but instead was an antagonist of IL-1 actions (40).
In conclusion, we have identified CITED2 as the critical repressor of MMP-1 and MMP-13 gene expression and associated proteolytic activities using flow shear as a probe. Further elucidation of CITED2-mediated mechanotransduction of chondrocytes would contribute to controlling catabolic responses in rheumatoid arthritis and osteoarthritis.