The role and regulation of p21(WAF1/CIP1) in myelopoiesis
Elevated levels of the molecular adaptor protein p21 waf1/cip1 (p21) and of the IL-3 receptor α chain are correlated with chemoresistance and poor prognosis in acute myeloid leukemia (AML). p21 is a core regulator of many biological functions including cell cycle control, apoptosis and differentiation. Our laboratory has demonstrated a decrease in p21 expression levels during cytokine-induced granulocytic differentiation, leading us to hypothesize that p21 antagonizes granulopoiesis. The proliferative cytokine IL-3 has been shown to prevent granulocytic differentiation of murine and human myeloid progenitor cells. We also hypothesized that IL-3 inhibition of differentiation is mediated in part by p21, and tested this in murine 32Dcl3 myeloblasts that are used to model granulopoiesis. Our findings demonstrated that p21 antagonized differentiation by promoting apoptosis of cells exposed to the differentiation inducer G-CSF. We also showed that p21 prevented premature expression of primary granule proteins and contributed to maintenance of the myeloblast phenotype. Furthermore, p21 knockdown accelerated morphologic differentiation of 32Dcl3 cells stimulated to differentiate with G-CSF. We then determined how IL-3 maintains p21 expression in myeloblast cells. We showed that IL-3 stabilized p21 mRNA in myeloblasts leading to high levels of p21 protein. This effect mapped to the 3' untranslated region (UTR) of the p21 transcript. p21 transcript stabilization by IL-3 was independent of PI3-kinase and ERK pathway signaling. In vitro binding assays provided evidence that distinct sets of RNA:protein interactions occur within the proximal 303 nucleotides of the p21 3' UTR and are regulated by IL-3 and G-CSF signaling. Association of a 60-65 kDa protein with p21 riboprobes correlated with IL-3 mediated p21 mRNA stabilization, whereas binding by a 40-42 kDa protein was associated with destabilization of p21 transcripts in 32Dcl3 cells undergoing G-CSF-induced differentiation. These findings provide the first evidence for IL-3-mediated stabilization of mRNA transcripts in myeloid progenitor cells. The finding that p21 antagonized granulopoiesis is also novel. Because high levels of the IL-3 receptor and high p21 expression have separately been linked to poor outcomes in AML, IL-3 mediated p21 mRNA stabilization may contribute to differentiation blockade during AML pathogenesis.