Role of CDK1 in human stem cell biology

Abstract

Self-renewal and pluripotency of stem cells (SC) is maintained by well-characterized transcriptional factors that assemble a core regulatory network, although less defined auto- and cross-regulatory signaling pathways are also critical. Accumulating evidence indicates that cell cycle regulators play an important role in control of pluripotency and differentiation that goes beyond their roles in cell cycle regulation. Cyclin-dependent kinase 1 (CDK1) is essential for cell cycle progression and embryonic development, and has recently been implicated in a signaling network with pluripotency proteins. In this study, we investigated the potential role of CDK1 in regulation of pluripotency and differentiation. CDK1 expression is enriched in the pluripotent state of human embryonic stem cells (hESCs) and down regulated during differentiation. shRNA knockdown of CDK1 or a selective CDK1 inhibitor (RO-3306) promoted hESC differentiation, shown by the reduced expression of the pluripotency markers Oct4, Nanog, and Sox2, and the up-regulation of the mesendoderm transcripts Brachyury, Eomes, Goosecoid, and MixL1. Interestingly, down-regulation of CDK1 by both shRNA and RO-3306 could not block ES cells at G2/M or other phases of the cell cycle, suggesting that relatively milder knockdown of CDK1 did not induce hESC cell cycle arrest or impair overall proliferation but was enough to promote differentiation. Knockdown of CDK1 also decreased phosphorylation of Akt at Thr308 and Ser473, one of the key regulators of pluripotency signaling. Further, JNJ-770621, a dual inhibitor of CDK1/CDK2 and Aurora kinases but not Akt or Erk, induced human embryonal carcinoma cell (NCCIT) differentiation accompanied by reduced phosphorylation of Akt. Addition of UO126 and SB431542, which inhibit MEK/ERK and TGFβ/Smad2/ respectively (downstream of PI3K/Akt signaling), reversed the differentiation induced by shCDK1 or RO-3306 treatment. Moreover, phosphorylation of PDK1 (upstream of Akt) was notably reduced in CDK1 knockdown cells but not in serum-induced differentiation, indicating that the inhibition of activation of PDK1 or Akt could be the primary effect of CDK1 down-regulation. The results suggest that threshold levels of CDK1 might direct- or cross-regulate key signaling pathways, such as that operating through PI3K-PDK1-Akt. In summary, CDK1 is required for maintenance of stem cell pluripotency, in a manner that is distinct from its role in regulation of cell cycle progression, potentially through direct or indirect cross-regulation of fundamental pluripotency signaling of PI3K/PDK1/Akt-ERK-GSK3β and TGFβ/Smad.