Activation of Stat5 is frequently found in leukemias. To study the mechanism and role of Stat5 activation, we introduced a constitutively activated Stat5a mutant, cS5F, into murine bone marrow (BM) cells. BM transplantation with cS5F-transfected cells caused development of multilineage leukemias in lethally irradiated wild-type or nonirradiated Rag2(-/-) mice. The leukemic cells showed strongly enhanced levels of cS5F tetramers but unchanged cS5F dimer levels in a DNA binding assay. Moreover, Stat5a mutants engineered to form only dimers, but not tetramers, failed to induce leukemias. In addition, Stat5 tetramers were found to accumulate in excess compared to dimers in various human leukemias. These data suggest that Stat5 tetramers are associated with leukemogenesis.

Expression profiling with cDNA arrays is an excellent tool for molecular analysis of complex processes such as terminal erythroid differentiation. The shortcomings of the currently available erythroid in vitro differentiation models, however, severely impaired the usefulness of this approach to study erythropoiesis. Here, we describe a novel, murine erythroid cell system closely corresponding to in vivo erythroid progenitors. Mortal, long‐term proliferating erythroid progenitors of fetal liver or immortal strains of p53‐deficient erythroblasts were established in culture. Both cell types proliferated in serum‐free medium and were strictly dependent on physiologically relevant cytokines and hormones, stably retaining a diploid set of chromosomes. If exposed to physiological differentiation factors (erythropoietin plus insulin), cells synchronously recapitulated the normal in vivo differentiation program to mature terminally into enucleated erythrocytes and expressed stage‐specific erythroid transcription factors in the expected temporal order. Using cDNA arrays, we found a large number of genes differentially expressed at time points during differentiation. Already 6 h after differentiation induction, 17% of the expressed genes showed significant alterations in mRNA abundance, increasing to 53% (12% up‐regulated, 41% down‐regulated genes) by 48 h. Cluster analysis of mRNA expression kinetics during differentiation identified six distinct expression patterns. All genes on the array with a known function in erythropoiesis showed the expected variations in expression. The genes identified also allowed first insights into the sequence of events within the regulatory network responsible for erythroid maturation. In mortal wild‐type as well as immortal p53‐/‐ erythroblasts, changes in mRNA abundance of several well‐regulated gene products was verified at the protein level. Taken together, this novel hematopoietic cell system faithfully executes essential steps of normal erythropoiesis and allows us to dissect and characterize molecular mechanisms involved in erythropoiesis.

During erythroid maturation, Epo regulates red cell numbers via apoptosis inhibition, caused by Epo-dependent upregulation of the antiapoptotic protein Bcl-X(L). This allows "default" terminal differentiation of apoptosis-protected, committed erythroblasts, independent of any exogenous signals.

Mice carrying a hepatocyte-specific inactivation of the glucorticoid receptor (GR) gene show a dramatic reduction in body size. Growth hormone signaling mediated by the Stat5 transcription factors is impaired. We show that Stat5 proteins physically interact with GR and GR is present in vivo on Stat5-dependent IGF-I and ALS regulatory regions. Interestingly, mice with a DNA-binding-deficient GR but an unaltered ability to interact with STAT5 (GR dim/dim ) have a normal body size and normal levels of Stat5-dependent mRNAs. These findings strongly support the model in which GR acts as a coactivator for Stat5-dependent transcription upon GH stimulation and reveal an essential role of hepatic GR in the control of body growth. Received September 8, 2003; revised version accepted January 27, 2004. Glucocorticoids (GCs), which are synthesized in the adrenal cortex, act in many if not all cells of the body and play important roles in development and homeostasis. The effects of the hormone are mediated by the glucocorticoid receptor (GR) that is ubiquitously expressed and by the mineralocorticoid receptor that displays a more restricted expression profile. GR activity depends on the binding of its ligand, which is released on physiological, circadian, and stress stimuli, and thus GR participates in coordinating the organism's responses to the environment. GR can both activate and repress transcription of target genes via DNA binding to glucocorticoid responsive elements (GREs) or cross-talk with other transcription factors (Beato et al. 1995;Tronche et al. 1998). Because the liver is a major target organ for GCs in control of glycogen metabolism and gluconeogenesis, we wished to address the function of hepatic GR by genetic means. As shown previously, loss of the receptor leads to lethality (Cole et al. 1995;Tronche et al. 1998). We therefore have generated cell/tissue-specific and functionselective mutations using the Cre/loxP system Tronche et al. 1999). To define the role of GR in hepatocytes, we expressed the recombinase in hepatocytes under the control of the albumin promoter and the albumin and ␣-fetoprotein enhancers to generate mutant mice with selective inactivation of GR in these cells only (GR AlfpCre mice; Kellendonk et al. 2000). This approach allowed us to partially circumvent the perinatal lethality of GR null mutants. Surprisingly, adult mice with the hepatocyte-specific loss of GR have a severe reduction in body weight. Analysis of these mice revealed not only a novel function of GR in growth control, but also an unprecedented mode of activity. Results and DiscussionApproximately 50% of the homozygous mutants died within 48 h after birth, likely due to the metabolic consequences of the hepatocyte-specific GR knock-out (data not shown). No increase in mortality was observed at later stages. Mice lacking GR in hepatocytes were indistinguishable from their littermates until 3-4 wk of age, but later displayed a severe growth deficit that was more pronounced in adult males than in females (reduction by 32%...