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Abstract: The number of epidermal growth factor receptors (EGFRs) and their ligands are highly expressed in malignant tumor cells. The EGF signaling pathway is also activated in up to one-third of patients with breast cancer. In this study, we investigated the novel function of the JAK3 inhibitor, WHI-P131, on EGF-induced MMP-9 expression and the regulatory mechanism of EGF-induced MMP-9 expression in SKBR3 cells. We observed that EGF increased MMP-9 mRNA and protein expression in a dose-dependent manner. EGF also induced the phosphorylation of EGFR, ERK, and STAT-3, and these effects were inhibited by the EGFR inhibitor, AG1478. To investigate the involvement of the STAT-3 pathway on EGF-induced MMP-9 expression, we pretreated SKBR3 cells with JAK1, JAK2, and JAK3 inhibitors prior to EGF treatment. The results showed that the JAK3 inhibitor, WHI-P131, as well as JAK3 siRNA transfection, but not the JAK1 and JAK2 inhibitors, significantly decreased EGF-induced MMP-9 expression. In addition, EGF-induced STAT-3 phosphorylation was only inhibited by WHI-P131. We then transfected cells with adenoviral STAT-3 (Ad-STAT-3), followed by treatment with EGF. Interestingly, EGF-induced MMP-9 expression was decreased by Ad-STAT-3 overexpression in a dose-dependent manner, while it was significantly increased by STAT-3 siRNA transfection. Our results also showed that basal levels of MMP-9 expression were significantly increased by constitutive active-MEK (CA-MEK) overexpression. EGF-induced ERK phosphorylation was prevented by WHI-P131, but not by JAK1 and JAK2 inhibitors. On the other hand, EGF-induced MMP-9 expression was decreased by the MEK1/2 inhibitor, UO126. Therefore, for the first time, we suggest that the JAK3 inhibitor, WHI-P131, inhibits EGF-induced STAT-3 phosphorylation as well as ERK phosphorylation. The JAK3/ERK pathway may play an important role in EGF-induced MMP-9 expression in SKBR3 cells. [Copyright &y& Elsevier]

Abstract: Caveolin-1 (Cav-1) is a major structural protein of caveolae and plays an important role as a negative regulator of various signaling pathways such as the transforming growth factor-β (TGF-β)/smad pathway. In this study, we investigated the role of cav-1 on basal and TGF-β1-induced expression of type I procollagen in human dermal fibroblasts. Our results demonstrated that basal and TGF-β1-induced expression of type I procollagen were significantly increased by adenoviral cav-1 (Ad-cav-1) overexpression, while the basal level of type I procollagen was decreased by cav-1 siRNA. Overexpression of cav-1 inhibited TGF-β1-induced phosphorylation of smad3 and transcription of 3TP-Lux and SBE luciferase reporters, suggesting that cav-1 may inhibit the TGF-β1/smad signaling pathway. We observed that TGF-β1-induced type I procollagen expression was decreased by smad3 siRNA transfection. However, the reduction of TGF-β1-induced type I procollagen expression by smad3 siRNA was reversed by cav-1 overexpression. In addition, our results also showed that TGF-β1 treatment increased the phosphorylation of Akt, and Ad-cav-1 infection augmented this TGF-β1-induced phosphorylation of Akt. Ad-myr-Akt infection significantly increased the basal expression of type I procollagen. In contrast, TGF-β1-induced type I procollagen expression was decreased by Akt siRNA transfection and the PI3-kinase inhibitor, LY294002, inhibited the TGF-β1-induced type I procollagen expression and also inhibited the cav-1-induced expression of type I procollagen. In conclusion, our results suggest that cav-1 increases the basal and TGF-β1-induced expression of type I procollagen by regulating two opposite signaling pathways: inhibiting TGF-β1/smad signaling and activating a PI-3 kinase/Akt/mTOR-dependent pathway in human dermal fibroblasts, ultimately resulting in increased type I procollagen expression. [Copyright &y& Elsevier]