Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Abstract

Members of the transforming growth factor-␤ superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-␤ pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling. Mouse embryonic stem (ES)2 cells are derived from the inner cell mass of the early blastocyst. It is from the inner cell mass that the germ layers arise to produce all cell types of the adult. The capacity of ES cells to either self-renew or differentiate into cells of the three germ layers provides an excellent tool for studying early embryonic development, including self-renewal and pluripotency (1). Some of the essential factors controlling ES cell pluripotency have been identified; however, additional work is necessary to identify other functional signaling pathways that impinge upon the ES cell phenotype, to determine genes that are regulated by these pathways, and to discover how the various pathways interact. Applications of this work will yield insights into the promising use of stem cells for regenerative therapies.ES cells can be maintained as pluripotent cells when grown on mitotically inactivated embryonic fibroblasts or when grown in media supplemented with leukemia inhibitory factor (LIF (2, 3)). Additionally, under serum-free culture conditions, signaling through the bone morphogenetic protein (BMP) pathway is essential for maintaining mouse ES cell pluripotency (4, 5). The combination of LIF and BMP4 inhibits multiple differentiation-inductive signals, a capability that...

Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells

“…Our Western blot data (Fig. 6F) confirmed that SB431542 specifically inhibits SMAD2 phosphorylation and causes a downregulation of LEFTY1 and LEFTY2 expression in iPSCs (ϳ50-fold) and in ESCs (ϳ20-fold), similar to that previously observed by Galvin et al [43]. After 10 days of SB431542 exposure, iPSCs/ESCs presented a flattened monolayer morphology (Fig.…”

Smad2 mediates Activin/Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells

“…This may be attributed to incomplete deprivation of Smad2 expression by RNA interference, or Smad3 but not Smad2 plays a dominant role in this process. A recent study showed that inhibition of Activin/Nodal signaling by SB431542 can increase BMP signaling and thus reinforce mouse ES cell pluripotency [24], consistent with our notion that endogenous Activin/Nodal signaling is not essential for self-renewal maintenance.…”

“…To determine whether Smad2-DNA association in undifferentiated ES cells accounts for the transcriptional outputs of Activin/Nodal signaling, we compared our Smad2-DNA binding data with the Activin-or SB431542-treated mouse ES cell expression array data obtained by Galvin et al [24] by Parametric Analysis of Gene Set Enrichment (PAGE) analysis (Supplementary information, Table S2). PAGE is a statistical method to detect whether a prior defined gene set shows statistically significant differences between two samples or states [26].…”

“…Activin/Nodal signaling has been reported to promote mouse ES cell proliferation [22]. A recent report also suggested that endogenous Nodal signaling indirectly modulates pluripotency via negative regulation of BMP signaling [24]. When ES cells are induced to differentiate, both branches of the TGF-β family signals are implicated in various lineage commitment, serving as either inducers or repressors in a contextdependent manner [5][6][7].…”

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

“…These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad7 [14][15][16][17] 6. Count cells with a hemocytometer or cell counter and plate 6,000-10,000 cells/ml in a 10 cm Petri plate (non-tissue culture treated).…”