Tenascin can inhibit apoptosis induced by copper in silver pomfret (Pampus argenteus)

Silver pomfret, an economically important fish species, encounters significant challenges posed by copper pollution in wild and culturing environment. High level of copper has the potential to accumulate in aquatic organisms over prolonged exposure periods. The extracellular matrix, which is highly susceptible to copper, encompasses various proteins, among which tenascin (TN) might play a crucial role in potentially regulating copper-induced apoptosis. To investigate this hypothesis in fish, we initially identified six TN family members in silver pomfret, namely TNCa, TNCb, TNR, TNXB, TNXBa and TNN. Phylogenetic analysis showed four gene subfamilies, namely TNC, TNR, TNN, and TNX, through comparative analysis with TNs from seven other teleost fish species. Subsequently, we analyzed transcriptome of silver pomfret under copper stress and found differential expression of TN family genes under varying levels of copper stress, particularly TNCa. KEGG enrichment analysis demonstrated a significant enrichment of TN downstream pathway, specifically the Pi3K-AKT signaling pathway. RT-qPCR results indicated up-regulation of TNs and apoptosis inhibition-related genes and down-regulation of apoptosis promotion-related genes within this pathway. Hence, we speculated that TN inhibits copper-induced apoptosis by modulating this particular pathway. To further validate this hypothesis, we established a copper stress cell model using a silver pomfret kidney cell line (PaK) and utilized siRNA to knock down TNCa expression within this cellular model. Comparing RNAi-Cu group to NC-control group, we observed significantly reduced cell viability and intensified apoptosis signals in the former. Additionally, RNAi-Cu group displayed down-regulation of apoptosis inhibition-related genes and up-regulation of apoptosis-related genes in Pi3K-AKT signaling pathway compared to NC-Cu group. These findings indicated that TN might be able to inhibit copper-induced apoptosis through Pi3K-AKT signaling pathway. Ultimately, this study offers foundational knowledge for exploring heavy metal toxicity effect on fishes and some references for utilization of copper in parasite control.