Stk40 deletion elevates c-JUN protein level and impairs mesoderm differentiation

Mesoderm development is a finely tuned process initiated by the differentiation of pluripotent epiblast cells. Serine/threonine kinase 40 (STK40) controls the development of several mesoderm-derived cell types, its overexpression induces differentiation of mouse embryonic stem cells (mESCs) towards the extraembryonic endoderm, and Stk40 knockout (KO) results in multiple organ failure and is lethal at the perinatal stage in mice. However, molecular mechanisms underlying the physiological functions of STK40 in the mesoderm differentiation remain elusive. Here, we report that Stk40 ablation impairs mesoderm differentiation both in vitro and in vivo. Mechanistically, STK40 interacts with both the E3 ubiquitin ligase mammalian constitutive photomorphogenesis protein 1 (COP1) and the transcriptional regulator proto-oncogene c-Jun (c-JUN), promoting c-JUN protein degradation. Consequently, Stk40 knockout leads to c-JUN protein accumulation, which, in turn, apparently suppresses the WNT signaling activity and impairs the mesoderm differentiation process. Overall, this study reveals that STK40, together with COP1, represents a previously unknown regulatory axis that modulates the c-JUN protein level within an appropriate range during mesoderm differentiation from mESCs. Our findings provide critical insights into the molecular mechanisms regulating the c-JUN protein level and may have potential implications for managing cellular disorders arising from c-JUN dysfunction. The ESCs were maintained on inactivated MEF feeders in the N2/B27 medium with 2 mM Glutamax (Gibco), 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol (Sigma), 1000 U/ml LIF (Millipore), 3 uM CHIR99021, and 1 uM PD0325901 (STEMCELL Technologies) . Cell colonies were digested into single cells for regular passaging using Accutase (STEMCELL Technologies). For induced mesoderm differentiation from mESCs, cells were passaged onto gelatin-coated cell culture plates to remove feeder cells prior to the initiation of differentiation. After one passage, ESCs were dissociated with Accutase, and 150000 cells were seeded in one well of 6-well plates pre-coated with gelatin, and maintained in the N2B27 medium with addition of 1% Knock-out Serum Replacement (KSR, Gibco), 0.1% bovine serum albumin (Gibco) and BMP4 (R&D) at 10 ng/ml for 2 days. Then, the medium was replaced by a DMEM-based medium with 15% KSR, 1 uM CHIR99021 (STEMCELL Technologies), and 0.5% DMSO (Sigma) for one additional day (Chal, Jerome, et al. 2015).