Phenotype anchoring in zebrafish reveals a potential role for matrix metalloproteinases (MMPs) in tamoxifen's effects on skin epithelium

Abstract

The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen - a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥10 µM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen’s bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustly misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3–7 fold), consistent with AP-1 activity - a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen’s effect is the result of perturbation of the MMP system in skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen’s non-classical mode of action and implicates a possible role for MMPs in tissues such as skin.

Focal adhesion kinase regulates tractional collagen remodeling, matrix metalloproteinase expression, and collagen structure, which in turn affects matrix‐induced signaling

“…Upon cell adherence to the ECM, FAK is one of the first molecules to be recruited to adhesions, where it undergoes autophosphorylation (Rajshankar, Wang, & McCulloch, 2017) and provides critical control of adhesion-dependent signaling processes (Ilic et al, 1995). FAK regulates cell migration, proliferation, and survival (Klingberg et al, 2014) and is involved in sensing the mechanical properties of the ECM, a process that contributes to the regulation of cell migration (Bugel, Wehmas, La Du, & Tanguay, 2016;Jiang et al, 2016). While FAK is abundant in cell adhesions, it may also function in the nucleus (Jiang et al, 2016) to regulate gene transcription (Lim, 2013).…”