Transcriptional Regulation of Seprase in Melanoma by the Canonical Transforming Growth Factor- (TGF- ) Signaling Pathway
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The tumor invasive phenotype driven by seprase expression/activity has been widely examined in an array of malignant tumor cell types, however very little is known about the regulatory mechanisms governing this critical protease. Human seprase (also named fibroblast activation protein-alpha (FAPΑ); antiplasmin-cleaving enzyme (APCE); dipeptidyl prolyl peptidase 5 (DPP5) is a 170 kDa homodimeric glycoprotein consisting of two 97 kDa subunits. Seprase is expressed at high levels by tumor cells and stromal fibroblasts in a variety of invasive carcinomas, but is essentially absent or undetectable in normal tissues except during active tissue remodeling in embryogenesis, and in the early stages of wound healing. Despite such confined expression to only highly invasive cell types, essentially nothing is known about the transcriptional regulation of the seprase gene. The specific aims of this dissertation were firstly to determine if seprase was transcriptionally regulated in metastatic melanoma cell lines, and secondly to decipher which signaling pathway(s) and/or transcription factor(s) were involved in said regulation should it be occurring. In my dissertation research, a significant portion of the human seprase promoter has been cloned. The transcriptional regulation of the gene has also been demonstrated in a pair of invasive melanoma cell lines with differential expression of the gene. In addition, a crucial TGF-Β-responsive cis-regulatory element has been identified in the proximal seprase promoter region, which enabled robust transcriptional activation of the promoter as determined in promoter mutagenesis/reporter studies. Furthermore, treatment of melanoma cell lines with TGF-Β1 caused a rapid and profound up-regulation of endogenous seprase mRNA, which coincided with an abolishment of the negative regulator c-Ski, and an increase in binding of Smad3/4 to the seprase promoter in vivo. Genetic blockade of TGF-Β signaling by either stable overexpression of the transcriptional repressor c-Ski, or transient expression of a dominant negative form of the TGF-Β Type II receptor in melanoma cells significantly reduced seprase mRNA levels. Consistent with genetic blockade of TGF-Β/Smad signaling, treatment of cultured cell lines with the TGF-Β RI chemical inhibitor SB-431542, or with a neutralizing antibody against the TGF-Β ligand also severely impaired TGF-Β-dependent seprase transcription. Together, these data suggest that seprase is transcriptionally regulated in human melanoma cells via the canonical TGF-Β signaling pathway. Our findings support the roles of both TGF-Β and seprase in tumor invasion and metastasis.