Stromal Cell-Derived Factor-1/CXCR4 Enhanced Motility of Human Osteosarcoma Cells Involves MEK1/2, ERK and NF-kB-Dependent Pathways CHUN-YIN HUANG, 1,2 CHUN-YI LEE, 1 MENG-YI CHEN, 1 WEI-HUNG YANG, 1,3,4 YING-HAO CHEN, 5 CHIA-HAO CHANG, 5 HORNG-CHAUNG HSU, 2,5 YI-CHIN FONG, 4,5 * AND CHIH-HSIN TANG 6,7 ** 1 Department of Orthopaedic Surgery, China Medical University Beigang Hospital, Yun-Lin County, Taiwan 2 Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan 3 Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 4 School of Chinese Medicine, China Medical University, Taichung, Taiwan 5 Department of Orthopaedic Surgery, China Medical University Hospital, Taichung, Taiwan 6 Department of Pharmacology, China Medical University, Taichung, Taiwan 7 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal-derived factor-1a (SDF-1a) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF-1 and CXCR4 (SDF-1 receptor). Treatment of osteosarcoma cells with SDF-1a increased the migration and cell surface expression of avb3 integrin. CXCR4-neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF-1a-induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF-1a-mediated migration and integrin expression. Stimulation of cells with SDF-1a increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kB inhibitor (PDTC) or IkB protease inhibitor (TPCK) also inhibited SDF-1a-mediated cell migration and integrin up-regulation. Stimulation of cells with SDF-1a induced IkB kinase (IKKa/b) phosphorylation, IkB phosphorylation, p65 Ser 536 phosphorylation, and kB-luciferase activity. Furthermore, the SDF-1a-mediated increasing kB-luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKa and IKKb mutants. Taken together, these results suggest that the SDF-1a acts through CXCR4 to activate MEK and ERK, which in turn activates IKKa/b and NF-kB, resulting in the activations of avb3 integrins and contributing the migration of human osteosarcoma cells. J. Cell. Physiol. 221: 204–212, 2009. ß 2009 Wiley-Liss, Inc. Osteosarcoma is a high-grade malignant bone neoplasm that occurs primarily in children and adolescents. The prognosis of these patients has improved substantially recently due to the development of various adjuvant and chemotherapies. However, a significant number still relapse. Recurrence usually occurs as pulmonary metastases or, less frequently, metastases to distant bones or as a local recurrence (Arndt and Crist, 1999; Bacci et al., 2001; Kempf-Bielack et al., 2005). Thus, a novel strategy that would efficiently inhibit metastasis, especially to the lung, from the primary osteosarcoma site is highly desirable. Decades of scrutiny into the molecular bases of cancer have largely focused on what causes oncogenic transformation and the incipient emergence of tumors (Gupta and Massague, 2006). The invasion of tumor cells is a complex, multistage process. To facilitate cell motility, invading cells need to change cell-cell adhesion properties, rearrange the extracellular matrix (ECM) environment, suppress anoikis and reorganize their cytoskeletons (Woodhouse et al., 1997). Integrins are a family of transmembrane adhesion receptors comprising of 19a and 8b subunits that interact noncovalently to form up to 24 different heterodimeric receptors. The combination of Abbreviations: SDF-1, stromal-derived factor-1; MEK, MAPK kinase; ERK, extracellular signal regulated kinase; IKK a/b,IkB kinase a/b; ECM, extracellular matrix; siRNA, small interference RNA; qPCR, quantitative real-time PCR. Contract grant sponsor: National Science Council of Taiwan; Contract grant number: NSC97-2320-B-039-031-MY3. Contract grant sponsor: China Medical University; Contract grant number: CMU97108. Contract grant sponsor: China Medical University Beigang Hospital; Contract grant number: CMUBH R970004. *Correspondence to: Yi-Chin Fong, School of Chinese Medicine, China Medical University, Taichung, Taiwan. E-mail: yichin.fong@mail.cmu.edu.tw **Correspondence to: Chih-Hsin Tang, Department of Pharmacology, College of Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung, Taiwan. E-mail: chtang@mail.cmu.edu.tw Received 9 February 2009; Accepted 29 April 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 3 June 2009. DOI: 10.1002/jcp.21846 ORIGINAL ARTICLE 204 Journal of Journal of Cellular Physiology Cellular Physiology ß 2009 WILEY-LISS, INC.