Stk40 links the pluripotency factor Oct4 to the Erk/MAPK pathway and controls extraembryonic endoderm differentiation

Abstract

Self-renewal and differentiation of embryonic stem cells (ESCs) are controlled by intracellular transcriptional factors and extracellular factor-activated signaling pathways. Transcription factor Oct4 is a key player maintaining ESCs in an undifferentiated state, whereas the Erk/MAPK pathway is known to be important for ESC differentiation. However, the manner in which intracellular pluripotency factors modulate extracellular factor-activated signaling pathways in ESCs is not well understood. Here, we report identification of a target gene of Oct4, serine/threonine kinase 40 (Stk40), which is able to activate the Erk/MAPK pathway and induce extraembryonic-endoderm (ExEn) differentiation in mouse ESCs. Interestingly, cells overexpressing Stk40 exclusively contribute to the ExEn layer of chimeric embryos when injected into host blastocysts. In contrast, deletion of Stk40 in ESCs markedly reduces ExEn differentiation in vitro. Mechanistically, Stk40 interacts with Rcn2, which also activates Erk1/2 to induce ExEn specification in mouse ESCs. Moreover, Rcn2 proteins are specifically located in the cytoplasm of the ExEn layer of early mouse embryos. Importantly, knockdown of Rcn2 blocks Stk40-activated Erk1/2 and ESC differentiation. Therefore, our study establishes a link between the pluripotency factor Oct4 and the Erk/MAPK signaling pathway, and it uncovers cooperating signals in the Erk/MAPK activation that control ExEn differentiation.

Fig. 1.

Stk40 is a direct target of Oct4 and is negatively regulated by Oct4. (A) The schematic diagram of the Stk40 protein showing a serine/threonine protein kinase domain (S_TKc) in the middle (aa35–332). (B) Analysis of Oct4 and Stk40 dynamic expression patterns in ZHBTc4 mouse ESCs treated with Tc by qPCR. All values are shown as means ± SD of results from three independent experiments. *P < 0.05; **P < 0.01. (C) Luciferase assays of the 2-kb upstream fragment of Stk40 reporter in ZHBTc4 ESCs treated with or without Tc for 72 h. ZHBTc4 cells were pretreated with Tc for 24 h before transfection. p, 0.5 kb of putative promoter; p+2k, 2kb upstream regulatory sequence. *P < 0.05. (D) Luciferase assays of the 2-kb upstream fragment of Stk40 reporter with or without mutations introduced in the octamer motif in CGR8 ESCs. p, promoter; wt, wild type; mut, mutant. Luciferase activities are shown as in C. *P < 0.05. (E) ChIP assays in CGR8 ESCs using rabbit IgG or the specific antibody against Oct4. The representative result of PCR amplification reactions of Stk40 (Upper) or Rex1 (Lower) is shown. Mock, no template; input, 10% of total genomic DNA from the nuclear extract.

Fig. 2.

Stk40 induces ESCs to differentiate into ExEn lineages. (A) Morphological changes in ESCs induced by Stk40 overexpression. E14T ESCs were transfected with either pPyCAGIP (vector as a control) or pPyCAGIP-Stk40 (Stk40) and cultured for 10 days in the presence of puromycin. (Scale bar: 100 μm.) (B) Effect of Stk40 on expression of differentiation-related markers using qPCR. Data are shown as the ratio of values for Stk40 overexpressing cells over control cells. All values are shown as means ± SD of results from three independent experiments. (C) Representative images showing expansion of ExEn-like cells in EBs aggregated from Stk40 overexpressing ESCs. (a and b) Light microscopic images. (c and d) Hematoxylin/eosin-stained sections of EBs. (e and f) Costained with Dab2 antibody by the DAB (3,3′-Diaminobenzidine) method (brown signal). (Scale bar: 100 μm.) (D) Surface positioning of Stk40-induced differentiated ESCs in the chimeric EB formation assay. (Scale bar: 50 μm.) (E) Immunofluorescence staining of Dab2 and Gata4 in EBs from Stk40^+/+ or Stk40^−/− ESCs. (Scale bar: 100 μm.) (F) Quantification of ExEn–marker expression by qPCR in EBs aggregated from Stk40^+/+ and Stk40^−/− ESCs at the indicated time points. All values are shown as means ± SD of results from four independent experiments. (G) Contribution of injected Stk40/E14Tg cells to the ExEn lineage in E6.5 (Left) and E7.5 (Right) chimeric embryos. (Scale bar: 250 μm.)

Fig. 3.

Forced expression of Stk40 induces ESC differentiation through activation of the Erk/MAPK pathway. (A) Phase–contrast images of vector- or Stk40 overexpressed E14T cells treated with DMSO or the Mek1/2 inhibitor, U0126 (Magnification: ×20). (B) Phase–contrast images of E14T cells stably expressing vector, Stk40, or Ras constitutively active form (RasT35S) or coexpressing Stk40 and Ras dominant negative form (RasS17N). (Scale bar: 100 μm.) (C) Western blot analysis of GST-RBD–bound activated Ras in E14T ESCs stably expressing vector, Stk40, or RasT35S. Asterisk, nonspecific bands recognized in GST-RBD–bound groups. (D) Western blot analysis of pErk1/2 in E14T cells stably expressing vector, Stk40, or RasT35S with or without U0126 treatment as indicated. WB, Western blot; WCL, whole cell lysate.

Fig. 4.

Rcn2 is a Stk40-interacting protein implicated in Stk40-induced Erk1/2 activation and ExEn differentiation. (A) Rcn2 associates with Stk40 directly. (Top) Reaction products from GST pull-down assays were analyzed by Western blotting with anti-His antibody. (Bottom) Coomassie blue staining of SDS/PAGE gel containing proteins included in the reaction. The experiment was repeated three times. (B) Flag-tagged Stk40 forms protein complexes with endogenous Rcn2 in E14T ESCs. The representative result of three coimmunoprecipitation experiments is shown. (C) Overexpression of Rcn2 induces ESC differentiation. Phase–contrast images of vector- or Rcn2-expressed E14T ESCs are shown. (Scale bar: 100 μm.) (D) Knockdown of Rcn2 blocks Stk40-induced ESC differentiation. Phase–contrast images of vector- or Stk40 over-expression in the stable EGFP or Rcn2 RNAi E14T ESCs are shown. (Scale bar: 100 μm.) (E) Gene expression levels in the cells described in D were analyzed by RT-PCR. (F) Rcn2 can activate Erk1/2 but not Ras. The representative result of three independent Western blot analyses of GST-RBD–bound activated Ras and pErk1/2 in EGFP-, Stk40-EGFP–, or Rcn2-EGFP–expressed E14T cells is shown. (G) Western blot analysis reveals that knockdown of Rcn2 abrogates Stk40-activated Erk1/2. The representative result of three independent experiments is shown.

Fig. 5.

Rcn2 is a developmentally regulated protein required for ExEn differentiation. (A) Quantification of dynamic Rcn2 mRNA levels in EBs aggregated from E14T cells for indicated days. All values are shown as means ± SD of results from four independent experiments. (B) Western blot analysis of Rcn2 protein levels in the cells described in (A). A representative result of three independent experiments is shown. (C) Rcn2 proteins are detected in frozen sections of mouse embryos at E6.5. Immunofluorescent images of Rcn2, Gata4, and Oct4 proteins are shown. (Scale bar: 75 μm.) (Inset) Enlarged view at the distal part of ExEn layers covering the epiblast. (Scale bar: 25 μm.) (D) Knockdown of Rcn2 by RNAi in E14T cells reduces induction of ExEn–marker expression during EB formation. Values of qPCR results from three independent experiments are shown as means ± SD. (E) A proposed model for the function of Stk40 in ExEn differentiation.