Inhibition of erythropoiesis by Smad6 in human cord blood hematopoietic stem cells

► Smad6 is expressed in human CD34+ cord blood hematopoietic stem cells (HSCs). ► HSC-specific expression of Smad6 does not regulate stemness. ► Smad6 prevents erythropoiesis in HSCs. ► Smad6 represses the genes essential for erythropoiesis, such as KLF1 and GATA-2. Bone morphogenetic proteins (BMPs) that belong to the transforming growth factor-β (TGF-β) superfamily cytokines, play crucial roles in hematopoiesis. However, roles of Smad6 in hematopoiesis remained unknown in contrast to the other inhibitory Smad (I-Smad), Smad7. Here we show that Smad6 inhibits erythropoiesis in human CD34+ cord blood hematopoietic stem cells (HSCs). Smad6 was specifically expressed in CD34+ cord blood HSCs, which was correlated with the expression of BMP2/4/6/7 and BMP type I receptor (BMPRI). BMP-specific receptor-regulated Smads (R-Smads), Smad1 and Smad5 in cooperation with Smad4 induced transcription of the Smad6 gene. Instead of affecting cell cycle, apoptosis, self-renewal, and stemness of CD34+ cells, Smad6 knockdown enhanced, whereas Smad6 overexpression suppressed erythropoiesis in stem cell culture and colony formation assay. Consistently, Smad6 suppressed the expression of the genes essential for erythropoiesis, such as Kruppel-like factor 1 (erythroid) (KLF1/EKLF) and GATA binding protein 2 (GATA-2). Promoter analyses showed that Smad6 repressed Smad5/4-induced transcription of the Klf1 gene. Thus, our data suggest that Smad6 indirectly maintains stemness by preventing spontaneous erythropoiesis in HSCs.