Megakaryocytes and erythroid cells are thought to derive from a common progenitor during hematopoietic differentiation. Although a number of transcriptional regulators are important for this process, they do not explain the bipotential result. We now show by gain-and loss-offunction studies that erythroid Krü ppellike factor (EKLF), a transcription factor whose role in erythroid gene regulation is well established, plays an unexpected directive role in the megakaryocyte lineage. EKLF inhibits the formation of megakaryocytes while at the same time stimulating erythroid differentiation. Quantitative examination of expression during hematopoiesis shows that, unlike genes whose presence is required for establishment of both lineages, EKLF is uniquely down-regulated in megakaryocytes after formation of the megakaryocyte-erythroid progenitor. Expression profiling and molecular analyses support these observations and suggest that megakaryocytic inhibition is achieved, at least in part, by EKLF repression of Fli-1 message levels. IntroductionHematopoiesis is the process by which a self-renewing population of stem cells provide a continuous replenishment of differentiated blood cells by generating progeny with sequentially altered gene expression patterns. [1][2][3] Identification of these cells has relied on selective enrichment by cell-surface markers combined with culture and in vivo cellular assays that enable detection of cells at specific stages of differentiation. Although stem cells are multipotent, individual steps of subsequent differentiative decisions are performed by a series of simpler, even bipotential, decisions whereby one cell type gives rise to 2 or 3 descendants of differing character. 4 This has led to a commonly accepted pattern of parent and progeny relations, 2 although variations of it have recently been suggested 5 (but see Forsberg et al 6 ).A large number of genetic, cellular, and gene expression studies point to the critical importance of cytokine pathways 7 and expression patterns of transcription factors 1,[8][9][10] for establishing and maintaining steady state numbers of lymphoid, myeloid, and erythroid cells that, at the same time, can respond quickly to changes in the organismal environment and increase or decrease the cellularity of specific blood cell types. The megakaryocyte and erythrocyte lineages are proposed to derive from a common precursor, the megakaryocyte-erythroid progenitor (MEP) 4,11,12 (reviewed in Pang et al 13 ). Strikingly, these 2 lineages share a number of commonalities with respect to transcription factors that are absolutely required (eg, GATA1,14,15 FOG1, 16 SCL,17, ). At the same time, the protein partners that form with these factors as differentiation proceeds can be significantly different between lineages. 20 However, because these factors are all positively required for both lineages, we are still left with an incomplete picture of how these lineages are differentially established during hematopoiesis. 13 Erythroid Krüppel-like factor (EKLF; KLF1 21 ) is ...
Smad4 functions as a transcription factor in TGF-b signalling. We have investigated the role of Smad4 in the TGF-b 1 cell responses of transformed PDV keratinocytes, which contain a Ras oncogene, and of nontumorigenic MCA3D keratinocytes, by transfecting both cell lines with a dominant-negative Smad4 construct. Smad4 mediates TGF-b 1 -induced up-regulation of p21 Cip1 and growth arrest in MCA3D cells. However, in PDV keratinocytes, Smad4 is only partially involved in TGFb 1 -induced growth inhibition, and does not mediate enhancement of p21 Cip1 levels by the growth factor. TGF-b 1 activates Ras/Erk signalling activity in both cell lines. PD098059, a speci®c inhibitor of MEK, disminishes TGF-b 1 -induced p21 Cip1 levels in PDV but not in MCA3D cells, suggesting an involvement of Erk in upregulation of p21 Cip1 by TGF-b 1 in PDV cells. PDV dominant-negative Smad4 cell transfectants, but not MCA3D transfectants, showed constitutive hyperactivation of the Ras/Erk signalling pathway, increased secretion of urokinase, higher motility properties, and a change to a ®broblastoid cell morphology associated in vivo with the transition from a well di erentiated to a poorly di erentiated tumour phenotype. Infection of MCA3D control and dominant negative Smad4 cell transfectants with retroviruses carrying a Ras oncogene led to enhanced p21 Cip1 and urokinase secreted levels, independently of TGF-b 1 stimulation, that were reduced by PD098059. These results suggest that Smad4 acts inhibiting Ras-dependent Erk signalling activity in Rastransformed keratinocytes. Loss of Smad4 function in these cells results in hyperactivation of Erk signalling and progression to undi erentiated carcinomas. Oncogene (2000) 19, 4134 ± 4145.
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