The cyclin-dependent kinase 2 (CDK2) mediates hematopoiesis through G1-to–S transition in Chinese mitten crabEriocheir sinensis

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Abstract

Cyclin-dependent kinases (CDKs), a family of cell cycle-related serine/threonine kinases, participate in various biological processes, and play crucial roles in the innate immunity. In the present study, a CDK2 (designed as EsCDK2) with a serine/threonine protein kinase catalytic domain was identified from Chinese mitten crab (Eriocheir sinensis). The full-length cDNA sequence of EsCDK2 was of 2405 bp with an open reading frame (ORF) of 909 bp. EsCDK2 shared 66%–81% sequence similarities with previously identified CDK2s. It was clustered with the CDK2 from Penaeus monodon in the invertebrate branch of the phylogenetic tree. The mRNA transcripts of EsCDK2 were highly expressed in hematopoietic tissue (HPT) and gonad, while lower in hemocytes, heart, gills, and muscle. EsCDK2 protein distributed in both cytoplasm and nucleus of HPT cells. The expression of EsCDK2 mRNA in HPT was significantly up-regulated and peaked at 3 h post stimulations with Aeromonas hydrophila (2.31-fold, p < 0.05) and Lipopolysaccharide (LPS) (2.02-fold, p < 0.05). After exsanguination, the total hemocyte counts (THC) decreased significantly to 0.42 × 107/ml (0.39-fold, p < 0.05) at 0.5 h, then returned to a normal level at 6 h, while the mRNA expression of EsCDK2 in HPT cells was up-regulated at the early phase from 0.5 h to 6 h. After injection of EsCDK2-dsRNA, the mRNA expression level of EsCDK2 in HPT and THC both decreased to 0.53-fold (p < 0.01) and 0.78-fold (p < 0.05) at 24 h, respectively, and the percentage of new-born hemocytes in HPT also decreased significantly from 37.7% to 16.3% (0.43-fold, p < 0.01). After knocking down of EsCDK2, THC decreased dramatically at 6 h (0.65-fold, p < 0.01) post exsanguination, while returned normal at 6 h in PBS group. After interference of EsCDK2 mRNA expression, the percentage of G0-G1 phase cells significantly increased to 85.01% (1.26-fold, p < 0.01), while S phase and G2-M phase cells significantly decreased to 7.92% (0.46-fold, p < 0.01) and 7.07% (0.43-fold, p < 0.01) respectively, indicating that the cell cycle of HPT cells arrested at G1 phase. These results collectively demonstrated that EsCDK2 participated in the regeneration of hemocytes or hematopoiesis by regulating the transition from G1 to S phase in the cell cycle, and involves in the innate immune responses of E. sinensis.

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