Melatonin modulates the hypothalamic-pituitary neuroendocrine axis to regulate physiological color change in teleost fish.

Melatonin (MT) is a crucial neuroendocrine regulator of various physiological activities in vertebrates, especially in circadian or seasonal rhythm control. In the present study, the large yellow croaker (Larimichthys crocea), a marine bony fish with circadian body color change behavior, is chosen for functional investigation on teleost MT signaling systems that remain uncharacterized. All five melatonin receptors (LcMtnr1a1, LcMtnr1a2, LcMtnr1b1, LcMtnr1b2, and LcMtnr1c) were significantly activated by MT, triggering extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation through different G protein coupling signaling pathways, with exclusive Gαi-dependency for LcMtnr1a2 and LcMtnr1c, and Gαq-dependency for two LcMtnr1b paralogs, whereas LcMtnr1a1 activated Gαi and Gαs dual-dependent signaling pathways. A comprehensive model of the MT signaling system in the hypothalamic-pituitary neuroendocrine axis was further constructed based on ligand-receptor interaction analysis using single-cell RNA-seq data, as well as spatial expression patterns of Mtnrs and related neuropeptides in central neuroendocrine tissues. A novel regulatory pathway of MT/melanin-concentrating hormone (MCH) and MT/(tachykinin precursor 1 (TAC1)+corticotropin-releasing hormone (CRH))/melanocyte-stimulating hormone (MSH) was discovered that functions in chromatophore mobilization and physiological color change and was further validated by pharmacological experiments. Together, our findings define multiple intracellular signaling pathways mediated by L. crocea melatonin receptors and provide the first in-depth evidence that uncover the upstream modulating roles of the MT signaling system in the hypothalamic-pituitary neuroendocrine axis of a marine teleost species, particularly in chromatophore mobilization and physiological color change.

Branched-chain amino acid catabolism breaks glutamine addiction to sustain hepatocellular carcinoma progression.

Branched-chain amino acid (BCAA) catabolism is related to tumorigenesis. However, the underlying mechanism and specific contexts in which BCAAs affect tumor progression remain unclear. Here, we demonstrate that BCAA catabolism is activated in liver cancer cells without glutamine. Enhanced BCAA catabolism leads to BCAA-derived carbon and nitrogen flow toward nucleotide synthesis, stimulating cell-cycle progression and promoting cell survival. Mechanistically, O-GlcNAcylation increases under glutamine-deprivation conditions and stabilizes the PPM1K protein, leading to dephosphorylation of BCKDHA and enhanced decomposition of BCAAs. Dephosphorylation of BCKDHA and high expression of PPM1K promote tumorigenesis in vitro and in vivo and are closely related to the poor prognosis of clinical patients with hepatocellular carcinoma (HCC). Inhibition of BCAA and glutamine metabolism can further retard HCC growth in vivo. These results not only elucidate a mechanism by which BCAA catabolism affects tumorigenesis but also identify pBCKDHA and PPM1K as potential therapeutic targets and predictive biomarkers.

Non-canonical phosphoglycerate dehydrogenase activity promotes liver cancer growth via mitochondrial translation and respiratory metabolism.

Phosphoglycerate dehydrogenase (PHGDH) is a key serine biosynthesis enzyme whose aberrant expression promotes various types of tumors. Recently, PHGDH has been found to have some non-canonical functions beyond serine biosynthesis, but its specific mechanisms in tumorigenesis remain unclear. Here, we show that PHGDH localizes to the inner mitochondrial membrane and promotes the translation of mitochondrial DNA (mtDNA)-encoded proteins in liver cancer cells. Mechanistically, we demonstrate that mitochondrial PHGDH directly interacts with adenine nucleotide translocase 2 (ANT2) and then recruits mitochondrial elongation factor G2 (mtEFG2) to promote mitochondrial ribosome recycling efficiency, thereby promoting mtDNA-encoded protein expression and subsequent mitochondrial respiration. Moreover, we show that treatment with a mitochondrial translation inhibitor or depletion of mtEFG2 diminishes PHGDH-mediated tumor growth. Collectively, our findings uncover a previously unappreciated function of PHGDH in tumorigenesis acting via promotion of mitochondrial translation and bioenergetics.

Genome-wide CRISPR screen identifies synthetic lethality between DOCK1 inhibition and metformin in liver cancer.

Metformin is currently a strong candidate anti-tumor agent in multiple cancers. However, its anti-tumor effectiveness varies among different cancers or subpopulations, potentially due to tumor heterogeneity. It thus remains unclear which hepatocellular carcinoma (HCC) patient subpopulation(s) can benefit from metformin treatment. Here, through a genome-wide CRISPR-Cas9-based knockout screen, we find that DOCK1 levels determine the anti-tumor effects of metformin and that DOCK1 is a synthetic lethal target of metformin in HCC. Mechanistically, metformin promotes DOCK1 phosphorylation, which activates RAC1 to facilitate cell survival, leading to metformin resistance. The DOCK1-selective inhibitor, TBOPP, potentiates anti-tumor activity by metformin in vitro in liver cancer cell lines and patient-derived HCC organoids, and in vivo in xenografted liver cancer cells and immunocompetent mouse liver cancer models. Notably, metformin improves overall survival of HCC patients with low DOCK1 levels but not among patients with high DOCK1 expression. This study shows that metformin effectiveness depends on DOCK1 levels and that combining metformin with DOCK1 inhibition may provide a promising personalized therapeutic strategy for metformin-resistant HCC patients.

Molecular characterization and biological function of CXCR1 in Nocardia seriolae-infected largemouth bass (Micropterus salmoides).

Interleukin-8 (IL-8, CXCL8), a pro-inflammatory chemokine secreted by a variety of cell types, plays a critical role in the development of various immune diseases. Interactions between IL-8 and its receptor CXC receptor 1/2 (CXCR1/2) are known to promote chemotaxis and phagocytosis in many immune responses. In this study, we report the molecular characteristics and pharmacological activity of CXCR1 (MsCXCR1) in largemouth bass (Micropterus salmoides) and evaluated the functional involvement of MsCXCR1 in individuals infected with the pathogen Nocardia seriolae. MsCXCR1 was cloned into the pEGFP-N1 plasmid and the subcellular localization of MsCXCR1 on the cell membrane was verified in MsCXCR1-EGFP-expressing HEK293 cells. Following observation of receptor internalization and intracellular signaling detection, we further determined the functional interaction of secreted interleukin-8 (LcIL-8, the ligand for CXCR1 in large yellow croaker) and MsCXCR1 was further determined, and the ERK phosphorylation signal activation mediated by MsCXCR1 was demonstrated. Quantitative real-time PCR assays were conducted to analyze the transcriptional distribution of MsCXCR1 in various tissues of healthy and diseased largemouth bass. These results illustrate the significant elevation of MsCXCR1 expression in the head kidney, spleen and liver of M. salmoides, suggesting that MsCXCR1 was involved in the immune response in N. seriolae-infected largemouth bass and potentially affects the digestive function of this species.

Identification and characterization of a novel 5-hydroxytryptamine receptor in the sea cucumber Apostichopus japonicus (Selenka).

Serotonin (5-hydroxytryptamine [5-HT]) receptors (5-HTRs) mediate neuroendocrine signaling via interactions with the ligand serotonin (5-HT). The 5-HT signaling system has been well studied in vertebrates, but rarely known in invertebrate animals, especially in the marine invertebrates. In this study, we identified and characterized a novel 5-HTR from the sea cucumber Apostichopus japonicus (Aj5-HT4/6 ). The cloned Aj5-HT4/6 open reading frame comprised 1290 bp and encoded 429 amino acids. Bioinformatic analysis of the receptor indicated that it was a member of the class A of the G protein-coupled receptor family. Further experiments using Aj5-HT4/6 -transfected HEK293 cells demonstrated that treatment with 5-HT could induce rapid internalization of Aj5-HT4/6 fused with enhanced green fluorescent protein from the cell surface into the cytoplasm and triggered a significant increase in levels of the second messenger cAMP as well as mitogen-activated protein kinase phosphorylation in a 5-HT dose-dependent manner. Quantitative real time-polymerase chain reaction demonstrated that Aj5-HT4/6 was predominantly expressed in the muscle and respiratory tree, and its expression was significantly decreased during estivation. Taken together, these results imply that Aj5-HT4/6 is potentially involved in the movement and metabolism of the sea cucumber.

Genome-wide prediction and comparative transcriptomic analysis reveals the G protein-coupled receptors involved in gonadal development of Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is dioecious, with seasonal reproduction. G protein-coupled receptor (GPCR)-mediated signaling systems might play critical roles in the reproductive control of A. japonicus. Here, we classified GPCR from the genome in silico and used transcriptomic analyses to further mine those that function in gonadal-development control. Totally, 487 GPCRs were predicted from A. japonicus, and 183 of these were further annotated to molecular pathways. Transcriptome analysis revealed 327 GPCRs expressed in gonads, and these were classified into four families and 19 subfamilies. Three pathways were apparently associated with reproduction, including neuroactive ligand-receptor interaction, the mTOR and Wnt signaling pathways. Seven and eight ovary- and testis-specific GPCRs were filtered, and the gene expression profiles were determined in multiple tissues and gonads at different developmental stages by qPCR. These results provide new insights into the discovery of GPCR-mediated signaling control in sea cucumber reproduction, especially in gonadal development control.