The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor ␤ (TGF-␤)͞SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4؊͞؊ breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMADmediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-␤ response element in the region ؊496͞؊444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-␤-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA͞protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

We have previously shown that transforming growth factor-␤ (TGF-␤) increases type VII collagen gene (COL7A1) expression in human dermal fibroblasts in culture (Mauviel, A., Lapiè re, J.-C., Halcin, C., Evans, C. H., and Uitto, J. (1994) J. Biol. Chem. 269, 25-28). To gain insight into the molecular mechanisms underlying the up-regulation of COL7A1 by this growth factor, we performed transient cell transfections with a series of 5-deletion promoter/chloramphenicol acetyltransferase reporter gene constructs. We identified a 68-base pair region between nucleotides ؊524 and ؊456, relative to the transcription start site, as critical for TGF-␤ response. Using electrophoresis mobility shift assays (EMSAs) with an oligonucleotide spanning the region from ؊524 to ؊444, we discovered that a TGF-␤-specific protein-DNA complex was formed as early as 11 min after TGF-␤ stimulation and persisted for 1 h after addition of the growth factor. Deletion analysis of the TGF-␤-responsive region of the COL7A1 promoter by EMSA identified segment ؊496/؊444 as the minimal fragment capable of binding the TGF-␤-induced complex. Furthermore, two distinct segments, ؊496/؊490 and ؊453/؊444, appeared to be necessary for TGF-␤-induced DNA binding activity, suggesting a bipartite element. Supershift experiments with a pan-Smad antibody unambiguously identified the TGF-␤-induced complex as containing a Smad member. This is the first direct identification of binding of endogenous Smad proteins to regulatory sequences of a human gene.

4-Methylumbelliferone (MU) inhibits the cell surface hyaluronan (HA) formation, and that such inhibition results in suppression of adhesion and locomotion of cultured melanoma cells. Here, we examine the effect of MU on melanoma cell metastasis in vivo. MU-treated melanoma cells showed both decreased cell surface HA formation and suppression of liver metastasis after injection into the mice. Oral administration of MU to mice decreased tissue HA content. These HA knockdown mice displayed suppressed liver metastasis. Thus, both cell surface HA of melanoma cells and recipient liver HA can promote liver metastasis, indicating that MU has potential as an anti-metastatic agent.

Hyaluronan (HA) is a ubiquitous, major component of the pericellular matrix and is necessary for various physiological processes. It plays a very important role in biological barriers. We previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and pericellular HA matrix formation in cultured human skin fibroblasts, Streptococcus equi FM100, and B16F10 melanoma cells. We hypothesized that MU-mediated inhibition of HA synthesis and pericellular HA matrix formation would increase the efficacy of anticancer drugs. We have already demonstrated in vitro, using a sandwich binding protein assay and a particle exclusion assay, that MU inhibits HA synthesis and formation of the pericellular HA matrix, respectively, in human KP1-NL pancreatic cancer cells. AlamarBlue assay revealed that the anticancer effect of gemcitabine in KP1-NL cells was increased by pretreatment with MU. In vivo simultaneous administration of MU and gemcitabine to tumor-bearing mice with severe combined immunodeficiency disease (SCID) decreased the size of the primary and metastatic tumors more than did gemcitabine alone. These data strongly suggest that a combination of MU and gemcitabine is effective against human pancreatic cancer cells. MU may have potential as a chemosensitizer and may provide us with a new anticancer strategy.

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