TC-1 and NIH3T3 cell lines. We also assessed galectin-3 and MMP-1 expression in the B16F1 and B16F10 melanoma cell lines and found that the non-metastatic cell line (B16F1) did not express galectin-3 while the metastatic cell line (B16F10) did. A previous report indicated that galectin-3 regulates the metastasis of various types of cancer via different mechanisms (Liu and Rabinovich, 2005). However, the mechanisms underlying the involvement of galectin-3 in mouse cancer cell motility remain largely unknown. Our findings seem to indicate that one possible mechanism of melanoma cell motility in mice involves a putative relationship between galectin-3 and MMP-1 expression. We observed that galectin-3 knockdown drama- tically suppressed the motility, migration, and invasion of melanoma cells. In parallel, MMP-1 knockdown also significantly reduced the cell motility. Knockdown of galectin-3 reduced the expression of MMP-1 but not vice versa, and it is suggested that galectin-3 is an upstream regulator of MMP-1. This finding sheds new light on the mysterious relationships between galectin-3 and MMP-1. To understand how galectin-3 regulates MMP-1 expression, we determined the interaction between galectin-3 and the AP-1 complex (Song et al., 2005). The AP-1 family of transcription factors is composed of the homodimers or heterodimers of the Jun (JunB, JunD, c-Jun) and Fos (c-Fos, Fos B, fra-1, fra-2) onco-proteins (Lee et al., 1987). Among them, c-Jun plays important roles in regulating cancer invasion and metastasis. For instance, the c-Jun dominant-negative mutant, TAM67, inhibits the effects of AP-1 on the growth of breast cancer (Moore-Carrasco et al., 2006), as well as on the invasion and metastasis of murine osteosarcomas (Leaner et al., 2009). Fra-1, another component of the AP-1 family, plays a key role in MMP-1 transcription in A2058 melanoma cells (Tower et al., 2003). Here, we provide evidence for the association of galectin-3 with c-Jun and fra-1 and regulation of AP-1 transcription activity. Interestingly, c-Jun and fra-1 bound to the promoter regions of MMP-1 in the presence of galectin-3, but not in its absence. This is the first case reported in which galectin-3 contributes to MMP-1 promoter activity by forming a complex with c-Jun and fra-1 in melanoma cells. The role of galectin-3 in melanoma metastasis was also supported by the in vivo experimental results. In a mouse pulmonary metastasis model, animals treated with galectin-3 shRNA-transfected B16F10 melanoma cells showed considerable inhibition of lung metastasis compared with the untreated control group. In conclusion, the promotion of galectin-3 expression resulted in melanoma invasion/metastasis due at Galectin-3 increases MMP-1 expression 391 least in part to increased MMP-1 expression via the transcriptional activation of AP-1. These findings combined with the recent report by Kim et al, for the involvement of galectin-3 in gastric cancer cell motility (Kim et al., 2011), suggest that galectin-3 plays a role in various cancers’ metastasis through a common molecular mechanism. Methods Cell culture and siRNAs transfection B16F1, B16F10, 4T-1, TC-1 and NIH3T3 cell lines ob- tained from the Korea Cell Line Bank were cultured in DMEM or RPMI 1640 medium with 5% fetal bovine serum (FBS) and 1% Antibiotics (Invitrogen). Both galectin-3 and MMP-1 siRNAs were purchased from Invitrogen and trans- fection was performed with Lipofectamine RNAiMAX re- agent (Invitrogen) following the manufacturer’s instructions. The sequences of mouse galectin-3 and MMP-1 siRNAs were 5'-AUGAUUGUGAUCAGCAUGCTT-3' and 5'-GCCA GAACTTCCCAACCAT-3', respectively. RNA isolation and RT-PCR Total RNA was extracted from mouse melanoma cells with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. RT was carried out using a Reverse Transcription system (Promega), and PCR was performed with Ex-taq DNA polymerase (TaKaRa). The sequences of primers were as follows: mMMP-1; 5'-CCTGGAATTGGCAACAAAGT-3' (sense) and 5'-TAGCACGCAAGAATCAGGTG-3' (anti-sense); mGalectin-3: 5'-CAGTGCTCCTGGAGG CTATC-3' (sense) and 5'-AAGGGGAAGGCTGACTGTCT-3' (anti-sense); mβ- actin: 5'-AGCCTCGCCTTTGCCGA-3' (sense) and 5'-CTG GTGCCTGGGGCG-3' (anti-sense). Western blot analysis Western blot analysis was carried out as described pre- viously (Kim et al., 2010). Briefly, cells were lysed in RIPA buffer (Biosesang) containing a protease inhibitor cocktail (Sigma), followed by sonication on ice. The cell lysate was centrifuged and the supernatant was collected. Twenty μg of proteins were subjected to SDS-PAGE and transferred to PVDF transfer membranes (Amersham). After being blocked with 5% skim milk for 1 h, the membrane was in- cubated with primary antibody dissolved in 5% BSA over- night at 4oC. The membrane was then incubated with sec- ondary antibody for 1 h, followed by detection with an ECL kit (Amersham). The following antibodies were used: an- ti-β-actin (Santa Cruz); anti-galectin-3 (Santa Cruz); an- ti-MMP-1 (Calbiochem, Santa Cruz). Immunoprecipitation assays Cell lysate containing 750 μg of protein was pre-cleared by incubation with 40 μl protein-A/G linked agarose beads (Santa Cruz) for 1 h at 4oC. After the beads were spun