Cellular Responses to Cold Stress in Pampus Argenteus: Involvement of Dusp5 in MAPK Signaling, Apoptosis Suppression, and Organelle Protection.

The silver pomfret (Pampus argenteus) represents a commercially vital marine fish species. However, its aquaculture potential, especially in temperate northern regions, is critically constrained by pronounced cold sensitivity. Building upon preliminary investigations which demonstrated a significant inverse correlation between dusp5 expression levels and ambient water temperature, this study aimed to elucidate the functional mechanism of dusp5-a cold-inducible dual-specificity phosphatase affecting the MAPK pathway-in mediating cold stress adaptation. Utilizing cellular models subjected to dusp5 knockdown (KO) and overexpression (OE), we systematically evaluated its impact on cold stress responses. Functional analysis revealed that dusp5 OE conferred significant cytoprotection: it effectively suppressed cold-induced DNA fragmentation indicative of apoptosis (quantified by TUNEL assay), ameliorated mitochondrial dysfunction (assessed via JC-1 staining and TEM ultrastructural examination), and diminished oxidative stress (indicated by reduced malondialdehyde/MDA levels). Subsequent qPCR profiling demonstrated that dusp5 OE modulated the expression of selected genes associated with MAPK signaling cascades, apoptotic pathways, endoplasmic reticulum (ER) stress response, and mitochondrial functional integrity under cold duress. Furthermore, employing dual-luciferase reporter assays, we identified atf2, atf4, jund, and stat1 as putative regulators of dusp5 promoter activity. Complementary histidine affinity chromatography facilitated the identification of candidate dusp5-associated proteins predominantly localized to the ER and mitochondrial compartments, with functional annotations implicating roles in ER homeostasis, autophagic processes, and peroxisome metabolism. Collectively, this work provides the preliminary mechanistic insights of dusp5 in promoting cold tolerance within P. argenteus, yielding novel and fundamental insights into the molecular regulators underpinning low-temperature adaptation mechanisms in marine teleosts.