CgCREM regulates haemocyte proliferation and inflammatory factor expression in the Pacific oyster Crassostrea gigas

The cAMP response element modulator (CREM) is a regulatory transcription factor downstream of cAMP signaling, functioning either as a transcriptional activator or repressor in regulating the proliferation and differentiation of immune cells. In the present study, CgCREM with a conserved pKID domain and a BRLZ domain was identified from Pacific oyster Crassostrea gigas. The mRNA transcripts of CgCREM were found to be highly expressed in embryonic stages, especially in the blastula and trochophore. In adult oyster, CgCREM exhibited the highest expression level in haemolymph, which was 157.3-fold (p < 0.05) of that in hepatopancreas. By immunofluorescence analysis, CgCREM was found to mainly localize in the nucleus of oyster haemocytes, especially in semi-granulocytes. After Vibrio splendidus stimulation, the mRNA expression of CgCREM in haemocytes was significantly up-regulated at 3, 6, and 12 h. The binding of CgCREM to the promotes of downstream CgRunx1 and CgCDC-45 was significantly enhanced after V. splendidus stimulation, which was 10.43-fold (p < 0.001) and 3.16-fold (p < 0.01) of that in control group, respectively. After the expression of CgCREM was knocked-down by RNAi, the expression levels of cell proliferation-related factors (CgRunx1, CgBMP7 and CgGATA-3), and cell cycle regulatory factors (CgCDC-45, CgCDK-2 and CgCDC-6) in haemocytes were significantly reduced after V. splendidus stimulation. Simultaneously, the ratio of EdU⁺ positive haemocytes, granulocytes and semi-granulocytes were significantly reduced in CgCREM knocked-down oysters, concomitent with the down-regulated expressions of inflammatory factors (CgIL17-1, CgIL17-2 and CgIL17-3). All these results collectively suggested that CgCREM, as a transcription factor, was involved in regulating expressions of haemocyte proliferation related genes and inflammatory factors in oyster C. gigas, highlighting the pivotal role of CREM in regulating immune response in mollusks.