探討SDF-1刺激對軟骨細胞分泌細胞間質水解酵素之作用; Stromal cell-derived factor-1 induces matrix metalloprotease-13 expression in human chondrocytes

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(1)中國醫藥大學臨床醫學研究所碩士學位論文探討 SDF-1 刺激對軟骨細胞分泌細胞間質水解酵素之作用 Stromal cell-derived factor-1 induces matrix metalloprotease-13 expression in human chondrocytes. 指導教授：許弘昌副教授. 共同指導教授：湯智昕助理教授. 研究生：邱詠証. 中華民國九十七年六月.

(2) Contents Contents ……………………………………………………...………….1 Abstract ……………………………………………………...………….3 Chinese Abstract………………………………………………………...4 Acknowledgement……………………………………………………….5 Figure contents…..………………………………………………………7 Abbreviation……..……………………………………………………...8 Introduction……………………………………………………………..9 Materials and methods………………………………………..…….…15 Results…………………………………………………………………..23 SDF-1α Increased the Expression of MMP-13 in Human Chondrocytes……….………………………………………………...23 SDF-1α/CXCR4 Interaction Was Responsible for the Expression of MMP-13 in Chondrocytes……………………..……………..………23 ERK Signaling Pathway Was Involved in SDF-1α-Mediated MMP-13 Up-Regulation…………………………………………………………24 SDF-1αIncreased the Binding of c-Fos and c-Jun to the AP-1 Element on the MMP-13 promoter………..………………………….………….25 Increase of MMP-13 Promoter Activity by SDF-1α…………….……..26 Discussion………………………………………………………………28 References………………………………………………………………32 Figure……………………………………………………………...……42 Fig. 1 Concentration- and time-dependent increase in MMP-13 expression by SDF-1α………………………………………...............42 Fig. 2 Involvement of CXCR4 receptor in SDF-1α-mediated MMP-13 expression in chondrocytes…………………………………...............44 Fig. 3 ERK is involved in the potentiation of MMP-13 expression by 1.

(3) SDF-1α……………………………………….....................................46 Fig. 4 c-Fos and c-Jun are involved in SDF-1α-induced MMP-13 expression………………………………….........................................48 Fig. 5 Time-dependent increase in the binding of c-Fos and c-Jun to the AP-1 site on MMP-13 promoter in chondrocytes...........................50 Fig. 6 Signaling pathways involved in the increase of MMP-13 promoter activity by SDF-1α………....................................................52 Clinical trial permission……..………………………………...………54 Publication……………………………………………………………...55. 2.

(4) Abstract The production of chemokine stromal cell-derived factor(SDF)-1 is significantly higher in synovial fluid of patients with osteoarthritis and rheumatoid arthritis. Matrix metalloproteinase (MMP)-13 may contribute to the breakdown of articular cartilage during arthritis. Here, we found that SDF-1α increased the secretion of MMP-13 in cultured human chondrocytes, as shown by reverse transcriptase-polymerase chain reaction, Western blot, and zymographic analysis. SDF-1α also increased the surface expression of CXCR4 receptor in human chondrocytes. CXCR4-neutralizing antibody, CXCR4-specific inhibitor[1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl) phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)], orsmall interfering RNA against CXCR4 inhibited the SDF-1α-induced increase of MMP-13 expression. The transcriptional regulation of MMP-13 by SDF-1α was mediated by phosphorylation of extracellular signal-regulated kinases (ERK) and activation of the activator protein (AP)-1 components of c-Fos and c-Jun. The binding of c-Fos and c-Jun to the activator protein(AP-1) element on the MMP-13 promoter and the increase in luciferase activity was enhanced by SDF-1α. Cotransfection with dominant-negative mutant of ERK2 or c-Fos and c-Jun antisense oligonucleotide inhibited the potentiating action of SDF-1α on MMP-13 promoter activity. Taken together, our results provide evidence that SDF-1α acts through CXCR4 to activate ERK and the downstream transcription factors (c-Fos and c-Jun), resulting in the activation of AP-1 on the MMP-13 promoter and contributing cartilage destruction during arthritis.. 3.

(5) 中文摘要 Stromal cell-derived factor-1(SDF-1)在退化性關節炎及風濕性關節炎病患的關節液中皆可以發現其含量顯著的提高。細胞間質水解酵素(Matrix metalloproteinases; MMP)則早已被證實會破壞關節軟骨導致關節炎的發生。但是到底經由何種訊息傳導路徑來調控人類軟骨細胞分泌MMP-13，則尚未有定論。在本論文中，我們經由RT-PCR， Western blot及Zymography發現SDF-1會誘導人類軟骨細胞分泌MMP13。SDF-1也會促使它在人類軟骨細胞上的接受器CXCR4提高表現量。相對的，CXCR4抗體(neutralizing antibody)，CXCR4化學抑制劑 (AMD3100)及CXCR4 si-RNA則會抑制SDF-1誘導人類軟骨細胞分泌 MMP-13的表現。此外，SDF-1誘導人類軟骨細胞分泌MMP-13的表現是經由磷酸化ERK訊息傳導路徑及增加轉錄因子c-Fos及c-Jun的表現來啟動MMP-13驅動子(promotor)的活力。轉錄因子c-Fos及c-Jun結合到 MMP-13驅動子(promotor)上AP-1序列從而增加MMP-13的表現可以由將人類軟骨細胞轉殖帶有MMP-13驅動子的質體(luciferase plasmid) 再給予SDF-1的刺激來證明。相對的，將人類軟骨細胞轉殖dominant -negative mutant of ERK(DN-ERK)或antisense c-Fos(AS c-Fos) 及antisense c-Jun(AS c-Jun)則會抑制SDF-1誘導人類軟骨細胞分泌 MMP-13的表現。此抑制效果也可由冷光偵測儀上的表現得知。綜合以上實驗結果，我們發現SDF-1可以經由人類軟骨細胞接受器CXCR4，接著磷酸化ERK訊息傳導路徑並活化轉錄因子c-Fos及c-Jun結合到 MMP-13驅動子(promotor)上AP-1序列，最後誘導人類軟骨細胞分泌 MMP-13破壞軟骨導致關節炎的產生。. 4.

(6) Acknowledgement 終於到了寫下碩士畢業感謝函的這一刻，回想初入學時對研究方法的一竅不通，到完成此論文，投稿至國外醫學期刊並榮獲刊登，其中辛苦實非筆墨能夠形容。我特別要感謝三位老師的提攜及指導，分別是我的指導教授許弘昌主任，共同指導教授湯智昕老師及臨醫所林清淵所長。沒有你們像燈塔般指引我研究方向，我注定要迷失在汪洋大海，遑論畢業了。身為公立醫學中心的臨床醫師，平時要應付醫療與行政上的煩瑣事務，已經令我有左支右絀分身乏術之感。但是現今醫療環境的激烈競爭，卻又驅使我不得不投入這場學位大戰中。所幸過去兩年來，我犧牲有限的與家人相處及運動休閒的時光，全心投入基礎研究的領域中。如今看來，實在是一項值得的投資。它帶給我的絕非只是一張碩士文憑，而是未來會影響我一生的真正的寶物!發現臨床工作無法解決的問題，卻在基礎研究中透露出解決問題的曙光。真是令人欣喜若狂。這讓我期許有朝一日能在某個骨科疾病的治療上為人類提供些微貢獻。回想初入學時，臨醫所標榜的，要指導我們成為醫師科學家。經過這些時間的薰陶,我不敢自居已經是一位醫師科學家，但毫無疑問的師父已經把我帶入門，其它就看日後的修行了。感謝我實驗室的研究夥伴們，多虧你們包容我的笨手笨腳及好心傳授我一些作實驗的小技巧。讓我在遇到瓶頸時能順利通過。感謝骨科部李土生主任本著栽培後進的好意，大力支持我在公餘之暇投入醫學研究。如今有初步的結果，我要特別感謝他在我無法兼顧公務時的寬容以對。最後我要感謝我生命中最重要的兩個女人，我的母親及太太幸宜。我的父親早逝，母親母代父職拉拔我讀完醫學院。但留給我感受最深的，卻是樂觀進取的人生觀。這讓我在人生旅途上受用無窮。畢竟人生不如意事十之八九!能常保樂觀才能披荊斬棘向前邁進。感謝我的太太對於我付出在家庭的時間如此的少，卻鮮有抱怨。尤其是我 5.

(7) 的第一個小孩弈升在我就讀碩一時出生。無法有較多的時間協助太太照顧他更讓我感到內疚。如今畢業在即，我深刻體會到，那是因為我周遭充滿了許多的愛與關懷才能帶領我走到這裡。絕非我一人之功. 感謝那些曾經給我協助，而我沒在這裡提到名字的親友及師長們。衷心感謝你們的協助，謹以此論文代表我最崇高的敬意獻給你們!. 6.

(8) Figure contents Fig 1.. Concentration- and time-dependent increase in MMP-13 expression by SDF-1α.. Fig 2. Involvement of CXCR4 receptor in SDF-1α-mediated MMP-13 expression in chondrocytes. Fig 3. ERK is involved in the potentiation of MMP-13 expression by SDF-1α. Fig 4. c-Fos and c-Jun are involved in SDF-1α-induced MMP-13 expression. Fig 5. Time-dependent increase in the binding of c-Fos and c-Jun to the AP-1 site on MMP-13 promoter in chondrocytes. Fig 6. Signaling pathways involved in the increase of MMP-13 promoter activity by SDF-1α.. 7.

(9) Abbreviation ChIP. Chromatin Immunoprecipitation. DAPA. DNA Affinity Protein-Binding Assay. DNA. Doxyribonucleic acid. ECM. Extracellular matrix. ERK. Extracellular signal regulated kinase. FACS. Flow cytometry analysis. MAPK. Mitogen-activated protein kinase. MMP-13. Matrix metalloproteinases. OA. Osteoarthritis. PBS. Phosphate buffer saline. PCR. Polymerase chain reaction. RA. Rheumatic arthritis. Si-RNA. Small interfering RNA. SDF-1. Stromal cell derived factor-1. SF. Synovium fluid. 8.

(10) Introduction Under normal physiological conditions, chondrocytes maintain an equilibrium between anabolic and catabolic activities that is necessary for preservation of the structural and functional integrity of the tissue. Chondrocytes express various proteolytic enzymes such as aggrecanases and matrix metalloproteinases(MMPs), which, under normal conditions, mediate a very low matrix turnover responsible for cartilage remodeling (Poole, 2001). However, in pathological conditions such as osteoarthritis (OA) or rheumatoid arthritis(RA), production of these enzymes by chondrocytes increases considerably, resulting in aberrant cartilage destruction(Pelletier et al., 2001; Aigner and McKenna, 2002). MMPs are a large family of structurally related calciumand zinc-dependent proteolytic enzymes involved in the degradation of many different components of the extracellular matrix (Nagase and Woessner, 1999; Vincenti, 2001). MMPs are expressed in a number of different cell types, and they play a key role in diverse cellular processes ranging from morphogenesis to tumor invasion and tissue remodeling (Sternlicht and Werb, 2001). Among the MMPs, MMP-13 (collagenase-3) is considered to be of particular interest because of its role in cartilage degradation. MMP-13 actively cartilage. MMP-13 has been previously shown to be overexpressed in OA (Reboul et al., 1996). Given their important role in cellular functions, the expression and activity of MMPs are tightly regulated at multiple levels of gene transcription, synthesis, and extracellular activity. Complete understanding of the various factors and pathways involved in regulation of MMP expression could be of interest with regard to potential therapies. Chemokines are a family of small, soluble peptides that regulate cell movement, morphology, and differentiation. They achieve their 9.

(11) regulation by signaling through a family of transmembrane G protein-coupled receptors. It has been reported that the concentration of an 8-kDa chemokine, stromal cell-derived factor (SDF)-1, is greatly elevated in the synovial fluid (SF) from patients with OA and RA (Kanbe et al., 2002). Such elevation of SDF-1α concentrations in SF is due, at least partially, to the stimulated synthesis of SDF-1α by synovial fibroblasts under OA and RA conditions (Kanbe et al., 2002). The source of SDF-1α in the joint is from synovium, as demonstrated by immunocytochemistry, protein chemistry, and reverse transcription-polymerase chain reaction (RT-PCR) analysis (Kanbe et al., 2002, 2004). In contrast, the SDF-1α receptor is expressed by chondrocytes in the superficial zone and in the deep zone in articular chondrocytes (Kanbe et al., 2002, 2004). Interaction of SDF-1α with its specific receptor CXCR4 on the surface of chondrocytes induces the release of MMP-3 from chondrocytes (Kanbe et al., 2002). Induction of the release of MMP-3 may contribute to the breakdown of articular cartilage during arthritis (Kanbe et al., 2002). STROMA-DERIVED FACTOR 1 (SDF-1, or CXCL12) was initially cloned by Tashiro et al. (Tashiro et al., 1993) and later identified as a growth factor for B cell progenitor cells, a chemotactic factor for T cells and monocytes, and in B cell lymphopoiesis and bone marrow myelopoiesis. CXCL12 is a 68-amino acid small (8kDa) cytokine that belongs to the CXC chemokine family. CXCL12 is expressed in two isoforms, SDF-1α and SDF-1β, from a single gene that encodes two splice variants. The two encoded proteins are almost identical, except for the last four amino acids of SDF-1β, which are absent in SDF-1α. Biological and functional differences between the CXCL12 isoforms have not been described. The CXCL12 gene is mapped in chromosome 10, whereas most of the other genes encoding CXC chemokines reside on 10.

(12) chromosome 4 (Shirozu et al., 1995). It was long thought that CXCL12 bound exclusively to CXCR4 and that CXCR4 was its sole receptor. However, CXCR7 was identified as another receptor for CXCL12 at the end of 2005 (Balabanian et al., 2005). The immunological activities of CXCL12/CXCR4 have been largely studied in the context of immune cell trafficking. Interestingly, both CXCL12 and CXCR4 knockout mice are embryonic lethal, with any surviving pups dying within an hour of birth, suggesting that the CXCL12/ CXCR4 pathway mediates multiple biological activities (Nagasawa et al., 1996, Tachibana et al., 1998). The lethal effect of CXCL12 and CXCR4 knockout is related to the pleiotropic activity of CXCL12 and CXCR4, which are critical for hematopoietic, neural, vascular, and craniofacial organogenesis (Ma et al., 1998, Nagasawa et al., 1996, Tachibana et al., 1998). CXCL12 was initially cloned from bone marrow stromal cells (Tashiro et al., 1993). Strikingly, CXCL12 is widely expressed in various organs including heart, liver, brain, kidney, skeletal muscle, and lymphoid organs. Vascular endothelial cells, stromal fibroblasts, and osteoblasts are the major cellular source for CXCL12 in these organs (Gupta et al., 1998, Katayama et al., 2006, Petit et al., 2002, Ponomaryov et al., 2000, Zou et al., 1998). Interestingly, high levels of functional CXCL12 were first reported in human ovarian cancer in 2001 (Kryczek et al., Scotton et al., 2001, Zou et al., 2001). Subsequent studies documented a strong correlation between CXCL12 expression and bone marrow and lymph node metastasis of breast (Muller et al., 2001) and prostate cancer (Taichman et al., 2002). Interest in the role of CXCL12/ CXCR4 in tumor pathology was provoked by these studies. In addition to ovarian cancer, CXCL12 expression is reported in breast cancer (Bachelder et al., 2002, Kang et al., 2005), glioblastoma (Barbero et al., 2003, Porcile et al., 11.

(13) 2005), pancreatic cancer (Koshiba et al., 2000, Marchesi et al., 2004), prostate cancer (Darash-Yahana et al., 2004, Sun et al., 2003), thyroid cancer (Hwang et al., 2003), and many other human tumors. The matrix metalloproteinase (MMP) family members are the major enzymes that degrade the components of the extracellular matrix (Vincenti MP, 2001, Nagase H and Woessner JF, 1999). At the time of writing this article, 20 members of this family have been identified (Stetler-Stevenson WG and Yu AE, 2001). All are active at neutral pH, require Ca2+ for activity and contain a central zinc atom as part of their structure. Most MMPs are secreted into the extracellular space in a latent proform, and require proteolytic cleavage for enzymatic activity. A few MMPs, however, are activated intracellularly by a furin-like mechanism and therefore, these enzymes are fully active when they reach the extracellular space (Nagase H and Woessner JF, 1999). Most cells in the body express MMPs, even though some enzymes are often associated with a particular cell type. For example, the principle substrate of MMP-2 (also known as gelatinase A) and MMP-9 (also known as gelatinase B) is the type IV collagen in basement membrane and thus, these enzymes are usually expressed by endothelial cells, although other cells (e.g. stromal fibroblasts, macrophages, tumor cells) also express them (Vincenti MP, 2001, Borden P and Heller RA, 1997). MMP-3 (also known as stromelysin) activates MMP-1 (also known as collagenase-1) and cleaves a broad range of matrix proteins (Vincenti MP et al., 1996); MMP-1, which is an interstitial collagenase, and MMP-3 are among the most ubiquitously expressed MMPs. In contrast, MMP-13 (also known as collagenase-3) has a more restricted pattern of expression within connective tissue, and is usually produced only by cartilage and bone during development, and by chondrocytes in osteoarthritis (OA) (Borden P et al., 1996, Mengshol JA, 2000). 12.

(14) Expression of MMPs is low in normal cells, and these low levels allow for healthy connective tissue remodeling. In pathologic conditions, however, the level of MMP expression increases considerably, resulting in aberrant connective tissue destruction. Excess MMP production is associated with the pathology of many diseases, including periodontitis, atherosclerosis, tumor invasion/metastasis and arthritic disease (Vincenti MP, 2001, Borden P and Heller RA, 1997). In rheumatoid arthritis (RA) and OA, connective tissue destruction is mediated primarily by chondrocytes, by synovial fibroblasts and on occasion, by osteoclasts (Mengshol JA et al., 2000, Goldring MB, 2000). The interstitial collagens (types I, II and III), are the principle targets of destruction, and the secreted collagenases (MMP-1 and MMP-13) have the major role in this process. These MMPs are induced in response to the cytokines and growth factors usually found in arthritic joints. MMP-9 is also an inducible MMP, but its role in connective tissue destruction in arthritis appears to be secondary, since it contributes to the degradation of collagen only after the chains of the triple helix have been cleaved by the interstitial collagenases (Nagase H and Woessner JF, 1999). In contrast, MMP-2 and MMP-14 (membrane type 1-MMP), are constitutively expressed, with minimal regulation, and they may have a relatively minor role in the pathophysiology of arthritis. Thus, the collagenases (MMP-1, MMP-8 [also known as neutrophil collagenase] and MMP-13) have the unique ability to cleave the triple helix of collagen, thereby allowing the chains to unwind; the chains then become susceptible to further degradation by other MMPs. Recently, MMP-8 (traditionally termed neutrophil collagenase) has been found in arthritic lesions, even in the absence of neutrophils, indicating that chondrocytes, and perhaps synovial cells, can produce this enzyme (Tetlow LC et al., 2001, Shlopov BV et al., 1997). MMP-13 may have a particular role in cartilage 13.

(15) degradation because it is expressed by chondrocytes, and because it hydrolyzes type-II collagen more efficiently than the other collagenases (Mitchell PG et al., 1996). However, MMP-1 is more abundant and it also degrades interstitial collagens effectively (Vincenti MP, 2001, Nagase H and Woessner JF, 1999). We will, therefore, focus this discussion on the mechanisms controlling transcription of MMP-1 and MMP-13 in arthritic disease, although the concepts may be applicable to other members of this gene family and to other pathologic conditions. During OA and RA, synovium may be involved in the induction of catabolic activities in the joint cartilage. Upon stimulation, chondrocytes in the joint cartilage release matrix degradation enzymes, such as MMP-3 and -13, which result in the destruction of cartilage (Pelletier et al., 2001). It has been reported that SDF-1α induced MMP-3 activity in human chondrocytes (Kanbe et al., 2002). However, the effect of SDF-1α on MMP-13 expression in human chondrocytes is mostly unknown. Here, we found that SDF-1α increased the expression of MMP-13. In addition, ERK, c-Fos/c-Jun, and AP-1 signaling pathways may be involved in the increase of MMP-13 expression by SDF-1α. The elevated level of SDF-1α in SF from patients with arthritis may contribute to release MMP-13 in cartilage during arthritic pathogenesis.. 14.

(16) Materials and methods Materials. Protein A/G beads. Santa Cruz Biotechnology. Anti-mouse IgG conjugated horseradish. Santa Cruz Biotechnology. peroxidase Anti-rabbit IgG conjugated horseradish. Santa Cruz Biotechnology. peroxidase Phosphorylated (p)-ERK. Santa Cruz Biotechnology. P-p38. Santa Cruz Biotechnology. P-c-Jun. Santa Cruz Biotechnology. NH2-terminal kinase (JNK). Santa Cruz Biotechnology. P-protein kinase B (Akt). Santa Cruz Biotechnology. Akt. Santa Cruz Biotechnology. ERK. Santa Cruz Biotechnology. p38. Santa Cruz Biotechnology. JNK. Santa Cruz Biotechnology. c-Fos. Santa Cruz Biotechnology. c-Jun. Santa Cruz Biotechnology. MMP-13. Santa Cruz Biotechnology. PD98059. Calbiochem. SB203580. Calbiochem. SP600125. Calbiochem. Ly294002. Calbiochem. Rabbit polyclonal antibody specific for. R&D Systems. CXCR4 Recombinant human SDF-1α. PeproTech 15.

(17) p38 dominant-negative mutant. Provided by Dr. J. Han. JNK dominant-negative mutant. Provided by Dr. M. Karin. ERK2 dominant-negative mutant. Provided by Dr. M. Cobb. pSV-β-galactosidase vector. Promega. Luciferase assay kit. Promega. All other chemicals. Sigma-Aldrich. Cell Cultures. Primary cultures of human chondrocytes were isolated from articular cartilage as described previously (Lee et al., 2002; Fong et al., 2007). Human articular chondrocytes were isolated from resected cartilage specimens obtained from undergoing primary total knee arthroplasty. Cartilage pieces were minced finely, and chondrocytes were isolated by sequential enzymatic digestion at 37°C with 0.1% hyaluronidase for 30 min and with 0.2% collagenase for 1 h. Isolated chondrocytes were filtered through 70-μm nylon filters. The cells were grown on plastic cell culture dishes in 95% air, 5% CO2 with Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA), which was supplemented with 20 mM HEPES and 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (pH adjusted to 7.6). The cells were used between the second and sixth passages.. Western Blot Analysis of the Cell Lysate and Supernatant. Proteins in the total cell lysate (30μg of protein) were separated by 12% SDS-polyacrylamide gel electrophoresis and electrotransferred to a. 16.

(18) polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Billerica, MA). Blot was blocked in a solution of 4% bovine serum albumin, and membrane-bound proteins were then probed overnight with primary antibodies against SDF-1α, CXCR4, MMP-13, p-ERK, p-p38, p-JNK, or p-Akt followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h. Antibody-bound protein bands were detected with enhanced chemiluminescence reagents (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK), and they were photographed with Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY). Quantitative data were obtained using a computing densitometer and ImageQuant software (GE Healthcare). Conditioned medium aliquots were concentrated 100-fold by acetone precipitation and resuspended in 2-fold concentrated reducing Laemmli buffer. Proteins were measured with the detergent-compatible protein assay from Bio-Rad Laboratories (Hercules, CA). Protein samples at 30μg were then separated on 10% SDS-polyacrylamide gel, and proteins were analyzed by Western blot analysis as described under Western Blot Analysis of the Cell Lysate and Supernatant.. Zymography Analysis. Conditioned media were collected, centrifuged, and concentrated 100-fold with a Centriprep concentrator (Millipore). Concentrated supernatants were mixed with sample buffer without reducing agent or heating. The sample was loaded into 1 mg/ml gelatin containing SDS-polyacrylamide gel, and then it underwent electrophoresis with constant voltage. Afterward, the gel was washed with 2.5% Triton X-100 to remove SDS, rinsed with 50 mM Tris-HCl, pH 7.5, and then incubated overnight at room temperature with developing buffer (50 mM Tris-HCl, 17.

(19) pH 7.5, 5 mM CaCl2, 1μM ZnCl2, 0.02% thimerosal, and 1% Triton X-100). The zymographic activities were revealed by staining with 1% Coomassie Blue. The sample was also loaded into SDS-polyacrylamide gel and stained with 1% Coomassie Blue as loading control (Chu et al., 2007). For examination of the downstream signaling pathways involved in SDF-1α treatment, chondrocytes were pretreated with various inhibitors (0.1% dimethyl sulfoxide as vehicle) for 30 min before SDF-1α administration.. mRNA Analysis by RT-PCR. Total RNA was extracted from chondrocytes using a TRIzol kit (MDBio Inc., Taipei, Taiwan). The reverse transcription reaction was performed using 2μg of total RNA that was reverse transcribed into cDNA using oligo(dT) primer and then amplified for 33 cycles using two oligonucleotide primers. The primers used are as follows: MMP-3, sense, AGAGGTGACTCCACTCACAT; antisense, GGTCTGTGAGTGAGTGATAG; MMP-13, sense, TGCTCGCATTCTCCTTCAGGA; antisense, ATGCATCCAGGGGTCCTGGC; CXCR4, sense, AATCTTCCTGCCCACCATCT; antisense, GACGCCAACATAGACCAC -CT; c-Fos, sense, GAATAACATGGCTGTGCAGCCAAATGCCGCAA; antisense, CGTCAGATCAAGGGAAGCCACAGACATCT; c-Jun, sense, GGAAACGACCTTCTATG -ACGATGCCCTCAA; antisense, GAACCCCTCCTGCTCATCTGTCACGTTCTT; and glyceraldehyde-3-phosphate dehydrogenase, sense, ACCACAGTCCATGCCATCAC; antisense, TCCACCACCCTGTTGCTGTA. Each PCR cycle was carried out for 30 18.

(20) s at 94°C, 30 s at 55°C, and 1 min at 68°C. PCR products were then separated electrophoretically in a 2% agarose DNA gel and stained with ethidium bromide.. Flow Cytometric Analysis. Human chondrocytes were plated in six-well dishes. The cells were then washed with phosphate-buffered saline (PBS) and detached with trypsin at 37°C. Cells were fixed for 10 min in PBS containing 1% paraformaldehyde. After rinsing in PBS, the cells were incubated with rabbit anti-human antibody against CXCR4 (1:100) for 1 h at 4°C. Cells were then washed again and incubated with fluorescein isothiocyanate-conjugated goat antirabbit secondary IgG (1:150; Leinco Technologies, Inc., St. Louis, MO) for 45 min and analyzed by flow cytometry using FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA) (Tang et al., 2005).. Oligonucleotide Transfection. Chondrocytes were cultured to confluence; the complete medium was replaced with Opti-MEM (Invitrogen) containing the antisense phosphorothioate oligonucleotides(5μg/ml) that had been preincubated with 10μg/ml Lipofectamine 2000 (Invitrogen) for 30 min. The cells were washed after 24 h of incubation at 37°C and washed before the addition of medium containing SDF-1α. All antisense ODNs were synthesized and highpressure liquid chromatography-purified by MDBio Inc. The sequences used are as follows: c-Fos antisense (AS)-ODN, GCGTTGAAGCCCGAGAA and missense (MS)-ODN, GCATTGACGCCAGAGCA; and c-Jun AS-ODN, 19.

(21) CGTTTCCATCTTTGCAGT and MS-ODN, ACTGCAAAGATGGAAACG (Naganuma et al., 2000; Zhang et al., 2002).. Generation of DNA Constructs Encoding a Small Interfering RNA against Human CXCR4. Oligonucleotides against human CXCR4 genes were generated and cloned into a pSilencer 3.1-H1 vector (Ambion, Austin, TX) as described previously (Lapteva et al., 2005). We used Lipofectamine 2000 reagent to transfect the chondrocytes with pSilencer 3.1-H1-siCXCR4 or pSilencer 3.1-H1- siCXCR4-mut. Twenty-four hours after transfection, cells were replated in Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% fetal calf serum.. MMP-13 Promoter Assay. We generated promoter constructs of human MMP-13 genes according to previous reports with some modifications (Penda´s et al., 1997; Chu et al., 2007). The primers used for PCR reactions for MMP-13 promoter construct were 5′primer, 5′-CTGAGAGCTCCAACAAGAGAT GCTCTCA-3′ (forward/SacI; nucleotides－186 to－166); and 3′primer, 5′-GGAAGCTTTCTAGATTGAATGGTGATGCCTGG- 3′(reverse/ HindIII; nucleotides＋10 to＋27). The pGL3-Basic vector containing a polyadenylation signal upstream from the luciferase gene was used to construct expression vectors by subcloning PCR-amplified DNA to MMP-13 promoter into the SacI/HindIII site of the pGL3-Basic vector. The PCR products were confirmed on the basis of their size as determined by electrophoresis and DNA sequencing. Human 20.

(22) chondrocytes were transiently transfected with MMP-13 promoter plasmid using Lipofectamine 2000 reagent. Luciferase activity was measured with the Luciferase reporter assay system (Promega) as described by the manufacturer, using a model TD-70/20 luminometer (Turner Designs, Sunnyvale, CA) (Tang et al., 2006).. DNA Affinity Protein-Binding Assay. Binding of transcription factors to the MMP-13 promoter DNA sequences was assayed as described previously (Penda´s et al., 1997). After treatment with SDF-1α, nuclear extracts were prepared. Biotin-labeled doublestranded oligonucleotides (2μg) synthesized based on the human MMP-13 promoter sequence were mixed at room temperature for 1 h with shaking with 200μg of nuclear extract proteins and 20 μl of streptavidin agarose beads in a 70% slurry. Beads were pelleted and washed three times with ice-cold PBS. The bound proteins were then separated by SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis with specific antibodies (Huang and Chen, 2005).. Chromatin Immunoprecipitation Assay. Chromatin immunoprecipitation (ChIP) analysis was performed as described previously (Tang et al., 2007). DNA immunoprecipitated by anti-c-Fos or antic- Jun antibody was purified. The DNA was then extracted with phenol-chloroform. The purified DNA pellet was subjected to PCR. PCR products were then resolved by 1.5% agarose gel electrophoresis and visualized by UV. The primers 5′-AACAAGAGAT GCTCTCA-3′ 21.

(23) and 5′-TGAATGGTGATGCCTGG-3′ were used to amplify across the human MMP-13 promoter region (－182 to＋27).. Statistics. The values given are means ± S.E.M. The significance of difference between the experimental groups and controls was assessed by Student’s t test. The difference is significant if p ﹤0.05.. 22.

(24) Results SDF-1α Increased the Expression of MMP-13 in Human Chondrocytes. It has been reported that SDF-1 is greatly elevated in the SF from patients with OA and RA. MMP-3 and -13 have been reported to participate actively in the destruction of cartilage (Pelletier et al., 2004). Therefore, we investigated the effect of SDF-1 on the MMP-13 expression in human chondrocytes. Human chondrocytes were incubated with SDF-1α(at various concentrations) for 24 h, and the cell lysates and culture medium were then collected. The results from RT-PCR, Western blot, and zymographic analysis indicated that SDF-1αsignificantly increased the expression of MMP-13 in both cell lysates and supernatant concentration- dependently (Fig. 1, A and B) (induction of MMP-3 expression was used as positive control; Fig. 1A). The induction of MMP-13 at concentration of 100 ng/ml occurred in a time-dependent manner (Fig. 1C).. SDF-1α/CXCR4 Interaction Was Responsible for the Expression of MMP-13 in Chondrocytes. Interaction of SDF-1 with its specific receptor CXCR4 on the surface of chondrocytes has been reported to induce the release of MMP-3 from chondrocytes (Kanbe et al., 2002); therefore, we then examined whether SDF-1/CXCR4 interaction is involved in the signal transduction pathway leading to MMP-13 expression caused by SDF-1α. Human chondrocytes were treated with SDF-1α for different times, and the cell lysates were collected. The results from RT-PCR, Western blot, and flow cytometry indicated that SDF-1α significantly increased both 23.

(25) mRNA or protein levels and the cell surface expression of CXCR4 time-dependently (Fig. 2, A and B). Pretreatment of chondrocytes for 30 min with CXCR4-specific chemical inhibitor AMD3100 (500 ng/ml), CXCR4- neutralizing antibody (12G5; 10μg/ml), but not mouse monoclonal immunoglobulin isotype control (isotype antibody; 10μg/ml) antagonized the SDF-1α-induced MMP-13 expression (Fig. 2D). Transient transfection of small interfering RNA against CXCR4 (siCXCR4), but not a mutant form of siCXCR4 (siCXCR4-mut), effectively inhibited the expression of MMP-13 caused by SDF-1α(Fig. 2D). These results suggest that induction of MMP-13 expression by SDF-1α might occur via the activation of CXCR4 receptor.. ERK Signaling Pathway Was Involved in SDF-1α-Mediated MMP-13 Up-Regulation. Because the SDF-1α/CXCR4 interaction has been shown to activate several signalingpathways, including phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase (MAPK), in various cell lines (Kijima et al., 2002; Barbero et al., 2003; Phillips et al., 2003), we performed Western blot analysis to elucidate the signal transduction pathways involved in the SDF-1α-induced up-regulation of MMP-13. SDF-1αactivated ERK1/2 in chondrocytes, as evidenced by the increase in phosphorylated p42 and p44 (p-ERK) (Fig. 3A). Other signaling pathways, including p38 MAPK, JNK, and Akt were not activated up to 4 h of treatment (Fig. 3A). SDF-1α-induced mRNA expression and gelatinase activity of MMP-13 were greatly reduced by treatment with ERK inhibitor PD98059 (30μM), but these processes were not affected by SB203580 (a p38 MAPK inhibitor; 10μM), SP600125 (a JNK inhibitor; 10μM), or Ly294002 (a phosphatidylinositol 3-kinase inhibitor; 10μM) 24.

(26) (Fig. 3B). To confirm that 10 _M SB203580 and 10μM SP600125 are effective on p38 and JNK activity, pretreatment of chondrocytes with 10 and 30μM SB203580 or 10 and 30μM SP600125 for 30 min completely inhibited 10 ng/ml TNF-α-induced p38 and JNK phosphorylation, respectively (Fig. 3C). In addition, transfection of cells with ERK2 but not p38, JNK, or Akt mutant also antagonized the potentiating effect of SDF-1α(Fig. 3D). Taken together, these data suggest that the activation of the ERK pathway is required for the SDF-1α-induced increase of MMP-13 in chondrocytes.. SDF-1αIncreased the Binding of c-Fos and c-Jun to the AP-1 Element on the MMP-13 Promoter. Because the promoter region of human MMP-13 contains an AP-1 binding site and phosphorylation of ERK can lead to AP-1 activation (Eferl and Wagner, 2003; Ala-aho and Kahari, 2005), we further examined the activation of AP-1 components c-Fos and c-Jun after treatment of SDF-1α. Time-dependent increase in the c-Fos and c-Jun mRNA expression in chondrocytes by SDF-1αwas observed (Fig.4A). SDF-1α-activated c-Fos and c-Jun were also evidenced by the accumulation of c-Fos and c-Jun in the nucleus (Fig. 4B). The SDF-1α-induced c-Fos and c-Jun activation was inhibited by PD98059 but not by SB203580, SP600125, and Ly294002 (Fig. 4C). SDF-1α-induced mRNA expression and gelatinase activity of MMP-13 were also inhibited by c-Fos and c-Jun AS-ODN but not by MS-ODN (Fig. 4E). It has been reported that human MMP-13 promoter contains an AP-1 binding site between -50 and -44 (Eferl and Wagner, 2003). We next investigated whether c-Fos and c-Jun bind to AP-1 element on the MMP-13 promoter after SDF-1α stimulation. DNA affinity 25.

(27) protein-binding assay experiments showed a time-dependent increase in the binding of c-Fos and c-Jun to the AP-1 element on the human MMP-13 promoter after treatment with SDF-1α(Fig. 5A). The in vivo recruitment of c-Fos and c-Jun to the MMP-13 promoter (-182 to +27) was assessed by ChIP assays. In vivo binding of c-Fos and c-Jun to the AP-1 element of MMP-13 promoter occurred as early as 30 min, and it was sustained to 240 min after SDF-1αstimulation(Fig. 5B). The binding of c-Fos and c-Jun to AP-1 element by SDF-1αwas attenuated by PD98059 or ERK mutant but not by SB203580, SP600125, and Ly294002 or p38, JNK, and Akt mutants (Fig. 5, C and D).. Increase of MMP-13 Promoter Activity by SDF-1α. To further study the pathways involved in the action of SDF-1αinduced MMP-13 expression, transient transfection was performed using the human MMP-13 promoter-luciferase construct, which contains the human MMP-13 gene between positions -186 and +27 fused to the luciferase reporter gene. Treatment with SDF-1αled to a 3.1-fold increase in MMP-13 promoter activity in chondrocytes. The increase of MMP-13 activity by SDF-1α was antagonized by 30μM PD98059 but not by 10μM SB203580, 10μM SP600125, and 10μM Ly294002 (Fig. 6A). Alternatively, a high concentration of SB203580 (30μM) or SP600125 (30μM) also did not affect SDF-1α-induced MMP-13 activity (Fig. 6A). In cotransfection experiments, the increase of MMP-13 promoter activity by SDF-1α was inhibited by the dominant-negative mutant of ERK2 or c-Fos and c-Jun AS-ODN but not by dominantnegative mutants of p38, JNK, and Akt (Fig. 6B). In addition, dominant-negative mutants of ERK2, p38, JNK, and Akt or c-Fos and c-Jun AS-ODN did not affect the basal luciferase activity (Fig. 6B). Taken together, these data suggest that the 26.

(28) activation of the ERK, c-Fos/c-Jun, and AP-1 pathway is required for the SDF-1α-induced increase of MMP-13 in human chondrocytes.. 27.

(29) Discussion SDF-1 is significantly higher in synovial fluid of patients with osteoarthritis and rheumatoid arthritis. MMPs have been demonstrated to contribute to the breakdown of articular cartilage during arthritis (Poole, 2001). In addition, SDF-1 also enhances MMP-3 production in human chondrocytes (Kanbe et al., 2002). Here, we found that MMP-13 is a target protein for the SDF-1 signaling pathway, which required an activation of CXCR4 receptor, ERK, c-Fos/c-Jun, and AP-1. The synovium of OA and RA patients produces many types of cytokines and chemokines, such as interleukin-1, TNF-α, macrophage inflammatory protein-1, and a variety of MMPs (Yoshihara et al., 2000). MMPs can induce the breakdown of cartilage. SDF-1 has the additional function to accumulate CD4+ memory T cells in the synovium. This indicates that SDF-1 is related to the immune system and the inflammation that attracts lymphocytes to develop RA (Nanki et al., 2000; Blades et al., 2002). It has been reported that SDF-1 is expressed in the synovium but not in cartilage, which can stimulate release of MMP-9 in chondrocytes (Kanbe et al., 2004). MMP-13 expression has been detected in several pathological conditions that are characterized by the destruction of normal collagen tissue architecture (Ala-aho and Kahari, 2005). However, the expression of MMP-13 by SDF-1α in chondrocytes is mostly unknown. Here, we found that SDF-1αincreased MMP-13 expression by using RT-PCR and zymographic analysis, which plays an important role during arthritis. Previous studies have shown that SDF-1α/CXCR4 interactions modulate cell migration, invasion, and MMP secretion in several cells (Bartolome et al., 2004, 2006; Fernandis et al., 2004; Ohira et al., 2006). In the present study, we used CXCR4-specific chemical inhibitor AMD3100 and CXCR4-neutralizing 28.

(30) antibody to determine the role of CXCR4, and we found that they inhibited SDF-1α- induced MMP-13 expression, indicating the possible involvement of CXCR4 in SDF-1α-induced MMP-13 expression in chondrocytes. This was further confirmed by the result that the small interfering RNA against CXCR4 inhibited the enhancement of MMP-13 production by SDF-1α, indicating the involvement of SDF-1/CXCR4 interaction in SDF-1α-mediated induction of MMP-13. A variety of growth factors stimulate the expression of MMP genes via signal transduction pathways that converge to activate AP-1 complex of transcription factors. MAPK pathways, including ERK, JNK, and p38, induce the expression of AP-1 transcription factors (Ala-aho and Kahari, 2005). We found that SDF-1α enhanced ERK1/2 phosphorylation without affecting phosphorylation of Akt and other MAPK pathways (e.g., p38 MAPK and JNK pathways) in human chondrocytes. Previous studies have revealed that SDF-1α treatment activates ERK1/2 in human lung cancer cells, astrocytes, and glioblastoma and basal cell carcinoma cells (Bajetto et al., 2001; Kijima et al., 2002; Barbero et al., 2003; Phillips et al., 2003; Chu et al., 2007). The SDF-1α- directed MMP-13 expression was effectively inhibited by ERK inhibitor but not by Akt and other MAPK pathway inhibitors. In addition, dominant-negative mutant of ERK but not p38, JNK, and Akt also inhibited the potentiating action of SDF-1α. This was further confirmed by the results that the dominant-negative mutant of ERK but not p38, JNK, and Akt inhibited the enhancement of MMP-13 promoter activity by SDF-1α. A similar signal pathway has also been reported in the invasion of basal cell carcinoma cells, which involved ERK-dependent MMP-13 expression (Chu et al., 2007). In addition, mechanical strain induced MMP-13 expression also through mitogen-activated protein kinase kinase-ERK signaling pathway to regulate mechanical adaptation (Yang et al., 2004). 29.

(31) Taken together, our results provide evidence that SDF-1αup-regulates MMP-13 in human chondrocytes via the ERK-dependent signaling pathway. Hormones and growth factors are known to regulate gene expression through AP-1 sites (Angel et al., 1988). It has been reported that SDF-1α induced MMP-13 secretion through AP-1-dependent pathway in human basal cell carcinoma (Chu et al., 2007). The AP-1 sequence binds to members of the Jun and Fos families of transcription factors. These nuclear proteins interact with the AP-1 site as Jun homodimers or Jun-Fos heterodimers formed by protein dimerization through their leucine zipper motifs. It has been observed that collagenase synthesis is induced in various tissues of transgenic animals overexpressing c-Fos or c-Jun, suggesting that an increase in c-Fos and c-Jun levels can stimulate collagenase expression (Wang et al., 1995). The results of this study show that SDF-1αinduced c-Fos and c-Jun expression and nuclear accumulation. Furthermore, SDF-1αincreased the binding of c-Fos and c-Jun to the AP-1 element on MMP-13 promoter, as shown by DNA affinity protein-binding assay and ChIP assay. Binding of c-Fos and c-Jun to the AP-1 element was attenuated by ERK inhibitor or ERK2 mutant but not by p38, JNK, and Akt inhibitor or p38, JNK, and Akt mutant. These results indicate that SDF-1αmight act through the ERK, c-Fos/c-Jun, and AP-1 pathway to induce MMP-13 activation in human chondrocytes. In conclusion, the signaling pathway involved in SDF-1α-induced MMP-13 expression in human chondrocytes has been explored. SDF-1αincreases MMP-13 expression and activity by binding to the CXCR4 receptor and activating ERK and the downstream transcription factors (c-Fos and c-Jun), resulting in the activation of AP-1 on the MMP-13 promoter and MMP-13, may contribute cartilage destruction 30.

(32) during arthritis.. 31.

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(43) Figure. 42.

(44) Fig. 1. Concentration- and time-dependent increase in MMP-13 expression by SDF-1α. Human chondrocytes were incubated with various concentrations of SDF-1α for 24 h. Then, the cell lysates were collected, and the mRNA levels of MMP-3 and MMP-13 were determined using RT-PCR. A, bottom, quantitative data are shown (n = 4). Cells were incubated with various concentrations of SDF-1α for 24 h (B) or with 100 ng/ml SDF-1αfor 2, 4, 6, 12, or 24 h (C). The cultured medium and cell lysates were then collected, and the mRNA level of MMP-13 in cell lysates was determined using RT-PCR. The protein level of MMP-13 in supernatant was determined using Western blot analysis, and the enzyme activity of MMP-13 in supernatant was determined using zymography. The quantitative data are shown at the bottom (n = 4). Data are expressed as means ± S.E. *, p ≦ 0.05 compared with control.. 43.

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(46) Fig. 2. Involvement of CXCR4 receptor in SDF-1α-mediated MMP-13 expression in chondrocytes. Chondrocytes were incubated with 100 ng/ml SDF-1α for indicated times. Then, cell lysates were collected, and the mRNA and protein level of CXCR4 was determined using RT-PCR and Western blot analysis, respectively. A, bottom, quantitative data are shown (n = 4). B, cells were incubated with 100 ng/ml SDF-1α for indicated times, and the cell surface expression of CXCR4 was determined using a flow cytometer. C, cells were transfected with siCXCR4-mut or siCXCR4 for 24 h, and then the mRNA and protein levels of CXCR4 were determined using RT-PCR and Western blot analysis, respectively. Chondrocytes were pretreated with 500 ng/ml AMD3100, 10μg/ml 12G5 antibody, and isotype antibody for 30 min or transfected with siCXCR4-mut and siCXCR4 for 24 h followed by stimulation with 100 ng/ml SDF-1α for 24 h. D, the mRNA level and enzyme activity of MMP-13 was determined by using RT-PCR and zymography analysis, respectively. The quantitative data are shown in the bottom panel (n = 4). Data are expressed as means ± S.E. *, p ≦ 0.05 compared with control. #, p ＜ 0.05 compared with SDF-1α-treated group.. 45.

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(48) Fig. 3. ERK is involved in the potentiation of MMP-13 expression by SDF-1α. A, chondrocytes were incubated with 100 ng/ml SDF-1α for the indicated times, and then p-ERK, p-p38, p-JNK, or p-Akt expression was determined by Western blot analysis. Chondrocytes were pretreated for 30 min with 10 and 30μM SB203580 or with 10 and 30μM SP600125 followed by stimulation with 10 ng/ml TNF-α for 15 min, and p-38 and p-JNK expression was determined by Western blot analysis. Note that 10 and 30μM SB203580 or 10 and 30μM SP600125 antagonized TNF-α-induced p38 or JNK phosphorylation, respectively. C, cells were pretreated for 30 min with 30μM PD98059, 10μM SB203580, 10μM SP600125, and 10μM Ly294002 (B), or they were transfected with dominant-negative (DN) mutant of ERK, p38, JNK, and Akt (D) for 24 h followed by stimulation with 100 ng/ml SDF-1αfor 24 h. The mRNA level and enzyme activity of MMP-13 was determined by using RT-PCR and zymography analysis, respectively. The quantitative data are shown at the bottom (n = 4). Data are expressed as means ± S.E. (percentage of vehicle control). *, p ≦ 0.05 compared with vehicle. #, p ＜ 0.05 compared with SDF-1α-treated group.. 47.

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(50) Fig. 4. c-Fos and c-Jun are involved in SDF-1α-induced MMP-13 expression. A, cells were treated with 100 ng/ml SDF-1α for the indicated times, and the mRNA levels of c-Fos and c-Jun were determined by using RT-PCR. Cells were treated with 100 ng/ml SDF-1α for the indicated times (B), or they were pretreated with 30μM PD98059, 10μM SB203580, 10μM SP600125, or 10μM Ly294002 for 30 min (C) before stimulation with SDF-1α for 240 min. The level of nuclear c-Fos and c-Jun was determined by immuno- blotting with c-Fos- and c-Jun-specific antibodies, respectively.D, cells were transfected with c-Fos or c-Jun AS-oligonucleotides or MS-oligonucleotides for 24 h, and then the protein level of c-For or c-Jun was determined by using Western blot analysis. Cells were transfected with c-Fos or c-Jun (AS) and (MS) for 24 h followed by stimulation with 100 ng/ml SDF-1α for 24 h, and then the mRNA level and enzyme activity of MMP-13 were determined by using RT-PCR and zymography analysis, respectively (E). The quantitative data are shown at the bottom (n = 4). Data are expressed as the means ± S.E. *, p ≦ 0.05 compared with vehicle control. #, p ＜ 0.05 compared with SDF-1α-treated group.. 49.

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(52) Fig. 5. Time-dependent increase in the binding of c-Fos and c-Jun to the AP-1 site on MMP-13 promoter in chondrocytes. A, top, schematic representing the consensus sequences of AP-1 site on the human MMP-13 promoter labeled with biotin. Chondrocytes were treated with 100 ng/ml SDF-1α for the indicated times, and nuclear extracts were prepared and incubated with biotinylated AP-1 probe. The complexes were precipitated by streptavidin-agarose beads as described under Materials and Methods, and c-Fos or c-Jun in the complexes was detected by Western blot. The equal amount of input nuclear protein was examined by the proliferating cell nuclear antigen protein level. B to D, cells were treated with 100 ng/ml SDF-1α for the indicated times, or they were pretreated for 30 min with 30μM PD98059, 10μM SB203580, 10μM SP600125, and 10μM Ly294002 or transfected with DN mutant of ERK, p38, JNK, and Akt for 24 h followed by stimulation with 100 ng/ml SDF-1α for 240 min. Then, ChIP assay was performed. Chromatin was immunoprecipitated with anti-c-Fos or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (input).. 51.

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(54) Fig. 6. Signaling pathways involved in the increase of MMP-13 promoter activity by SDF-1α. A, MMP-13 promoter activity was evaluated by transfection with the pMMP-13-Luc luciferase expression vector. Chondrocytes were pretreated with 30μM PD98059, 10 and 30μM SB203580, 10 and 30μMSP600125, or 10μMLy294002 for 30 min before incubation with 100 ng/ml SDF-1α for 24 h. B, cells were cotransfected with pMMP-13Luc and the DN mutant of ERK, p38, JNK, and Akt or c-Fos and c-Jun AS-oligonucleotides. Then, they were treated for 24 h with SDF-1α. Luciferase activity was measured, and the results were normalized to β-galactosidase activity. Data are expressed as means ± S.E. for three independent experiments performed in triplicate. *, p ≦ 0.05 compared with vehicle control; #, p ＜ 0.05 compared with SDF-1α-treated group.. 53.

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