Erythroid progenitor renewal versus differentiation: genetic evidence for cell autonomous, essential functions of EpoR, Stat5 and the GR.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis.

Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes v-ErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal differentiation under appropriate conditions, were established from fetal livers of p53-/- mice. Expression and activation of the EGF-receptor (HER-1/c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal differentiation. Upon inhibition of ErbB function, differentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5- and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms.

Reliability of mRNA profiling: verification for samples with different complexities.

Normalization of mRNA profiling data remains an open issue, which turns critical when comparing divergent samples or mRNA populations with different complexities. To address this question, we generated samples with different RNA amounts and complexities by subcellular fractionation of cytoplasmic RNA into the mutually exclusive ribosome-free and polysome-bound RNA pools. For each of the 563 mRNAs analyzed, the hybridization signal corresponding to the cytoplasmic sample equals the sum of signals from the ribosome-free plus the polysome-bound targets (cytoplasmic mRNA = ribosome-free mRNA + polysome-bound mRNA). This intuitive equation was fulfilled only after data normalization following "spiking" of the samples with an exogenous RNA. This is the first demonstration that spiking allows one to correct not only for differences in reaction efficiencies but also to reflect the variations in amount and complexity between the initial mRNA populations.

Translation control: bridging the gap between genomics and proteomics?

mRNA profiling enables the expression levels of thousands of transcripts in a cell to be monitored simultaneously. Nevertheless, analyses in yeast and mammalian cells have demonstrated that mRNA levels alone are unreliable indicators of the corresponding protein abundances. This discrepancy between mRNA and protein levels argues for the relevance of additional control mechanisms besides transcription. As translational control is a major mechanism regulating gene expression, the use of translated mRNA in profiling experiments might depict the proteome more closely than does the use of total mRNA. This would combine the technical potential of genomics with the physiological relevance of proteomics.

Regulation of ferritin mRNA translation in primary erythroblasts: exogenous c-Kit plus EpoR signaling mimics v-ErbA oncoprotein activity.

In general, translation efficiency of ferritin mRNAs is modulated by variations in iron supply. In primary avian erythroblasts undergoing short-term proliferation, however, ferritin heavy chain (ferH) mRNA is repressed at all iron levels. Yet, expression of v-ErbA oncoprotein is sufficient to reinduce ferH mRNA utilization at physiological iron concentrations. Since overexpression of the receptor tyrosine kinase c-Kit and erythropoietin receptor (EpoR) stimulates long-term proliferation of primary erythroblasts like v-ErbA, we analyzed the impact of cooperation between c-Kit and EpoR on the regulation of iron storage. Whereas endogenous c-Kit in combination with exogenous EpoR had no significant effect, ectopic overexpression of both receptors abolished translational repression of ferH mRNA upon iron administration. Thus, high-intensity signaling through c-Kit plus EpoR pathways mimics the v-ErbA-mediated regulatory phenotype.

Isolation of translationally controlled mRNAs by differential screening.

Translationalregulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation-regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome-free and polysome-bound mRNAs were prepared from quiescent and activated T cells and used as templates to synthesize four cDNA pools. These in turn were used as probes to hybridize four identical replicas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals from the ribosome-free fraction in resting T cells to the polysome-associated fraction in activated T cells. Translational repression corresponded to the opposite hybridization pattern. Fifty-two cDNAs were identified as translationally controlled by screening 472 genes in a cDNA array; 12 additional ones were obtained by screening a cDNA library. Several of the transcripts corresponded to mRNAs previously reported to be translationally controlled, thus validating the method. For the majority, however, such regulation had not yet been described. Translational control was verified for representative examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation. Our strategy therefore provides an efficient tool to directly isolate or identify translationally controlled mRNAs in a variety of physiological situations. Moreover, differential screening using arrays enables simultaneous analysis of both transcriptional and translational regulation, further enhancing the power of gene expression analysis.

Erythroid cell development and leukemic transformation: interplay between signal transduction, cell cycle control and oncogenes.

Studies using genetically modified mice and ex vivo tissue culture of erythroid progenitors converge to show that generation of mature erythroid cells depends on the interplay between specific transcriptional regulators and intracellular signals controlled by cytokines and growth factors. These studies also show that terminal differentiation in the erythroid lineage is unusual since the acquisition of the phenotypic traits of mature cells occurs while the cells are still actively dividing. Furthermore, under specific stress conditions, a massive and sustained self-renewal of committed erythroid progenitors can take place to replenish the pool of terminally differentiated cells. We review here how the erythroid genetic program and its interplay with specific cytokines, growth factors and hormones controls survival, proliferation and differentiation of erythroid progenitors both in normal and stress conditions. Special emphasis is laid on our present understanding of the differences in cell cycle control, which result either in self-renewal of erythroid progenitors or in the particular cell divisions which accompany terminal differentiation. Finally, we discuss how deregulation of the various aspects of the physiological control of erythroid progenitor survival, proliferation and differentiation can lead to erythroblast transformation and erythroleukemia.

Impaired ferritin mRNA translation in primary erythroid progenitors: shift to iron-dependent regulation by the v-ErbA oncoprotein.

In immortalized cells of the erythroid lineage, the iron-regulatory protein (IRP) has been suggested to coregulate biosynthesis of the iron storage protein ferritin and the erythroid delta-aminolevulinate synthase (eALAS), a key enzyme in heme production. Under iron scarcity, IRP binds to an iron-responsive element (IRE) located in ferritin and eALAS mRNA leaders, causing a block of translation. In contrast, IRP-IRE interaction is reduced under high iron conditions, allowing efficient translation. We show here that primary chicken erythroblasts (ebls) proliferating or differentiating in culture use a drastically different regulation of iron metabolism. Independently of iron administration, ferritin H (ferH) chain mRNA translation was massively decreased, whereas eALAS transcripts remained constitutively associated with polyribosomes, indicating efficient translation. Variations in iron supply had minor but significant effects on eALAS mRNA polysome recruitment but failed to modulate IRP-affinity to the ferH-IRE in vitro. However, leukemic ebls transformed by the v-ErbA/v-ErbB-expressing avian erythroblastosis virus showed an iron-dependent reduction of IRP mRNA-binding activity, resulting in mobilization of ferH mRNA into polysomes. Hence, we analyzed a panel of ebls overexpressing v-ErbA and/or v-ErbB oncoproteins as well as the respective normal cellular homologues (c-ErbA/TRalpha, c-ErbB/EGFR). It turned out that v-ErbA, a mutated class II nuclear hormone receptor that arrests erythroid differentiation, caused the change in ferH mRNA translation. Accordingly, inhibition of v-ErbA function in these leukemic ebls led to a switch from iron-responsive to iron-independent ferH expression.

Post-transcriptional control via iron-responsive elements: the impact of aberrations in hereditary disease.

Tight regulation of iron metabolism is crucial to avoid formation of deleterious radicals and is mainly executed at the post-transcriptional level. The regulatory loops are exerted by trans-acting iron regulatory proteins (IRPs) and cis-acting stem-loop motifs, termed iron-responsive elements (IREs), located in the untranslated regions (UTRs) of target mRNAs. Iron scarcity induces binding of IRPs to a single IRE in the 5'-UTR of ferritin, eALAS, aconitase and SDHb mRNAs, which specifically suppresses translation initiation. Simultaneous interaction of IRPs with multiple IREs in the 3'-UTR of transferrin receptor (TfR) mRNA selectively causes its stabilization. The pattern is reverted under iron overload: IRP-mRNA binding affinity is reduced, which results in efficient protein synthesis of target transcripts harboring IREs in the 5'-UTR and rapid degradation of TfR mRNA. Although multiple evidences support this model, several studies reported massive alterations in the regulation of iron homeostasis under specific physiological conditions, raising the possibility for additional regulatory events. Intensive analysis of the palindromic IRE consensus sequence revealed the critical elements for the formation of a functional structure and demonstrated the consequences of IRE mutations in IRP binding. Recent investigations indicated the involvement of naturally occurring IRE mutations of the ferritin L subunit in the hyperferritinemia-cataract syndrome, a hereditary disorder. This review summarizes the apparent links between iron-dependent post-transcriptional control and its abnormalities, governed by the properties of a single mRNA stem-loop structure.

Translational control: a general mechanism for gene regulation during T cell activation.

Distributional changes of individual mRNAs between free ribonucleoprotein particles (mRNP) and ribosome-bound transcripts are used to assess translational control. Simultaneous analysis of many mRNA species is required to estimate the overall contribution of translation to the regulation of gene expression. To this purpose, total cytoplasmic RNA was fractionated in sucrose step gradients and poly(A)+ RNA was prepared from mRNP and ribosome-bound fractions. Since direct, simultaneous analysis of a profusion of mRNAs is not feasible, distribution of their in vitro translation products was examined after separation in 2-dimensional gels, followed by computer-based analysis of autoradiographs. When this analysis was applied to antigenically stimulated T cells, 36% of in vitro translation products showed a greater than 10-fold increase in intensity, suggesting transcriptional activation of the corresponding mRNAs. In comparison, 7.9% of individual mRNAs (54 of 685 species) were translationally activated. They were redistributed from free mRNP to ribosome-associated fractions; 4.7% (32 species) were translationally repressed, as indicated by the opposite pattern. The differential recruitment of 12.6% of mRNA species demonstrates specificity and the general significance of translational control during T cell activation, which implies that translation may play a similar role in regulating gene expression in a variety of physiological processes.

Dynamics of cell cycle regulators: artifact-free analysis by recultivation of cells synchronized by centrifugal elutriation.

Studies on the molecular properties of cell cycle regulators in animal cells require cell preparations highly enriched in particular cell cycle phases. Centrifugal elutriation is frequently used to synchronize cells because this technique was thought to cause only minimal distortions in protein expression or metabolic functions. However, in primary chicken erythroblasts, we consistently observed artefacts in mitotic cyclin mRNA expression and p70 S6 kinase activity, which were clearly caused by the elutriation procedure. Therefore, we modified the standard protocol by reseeding various elutriated fractions into preconditioned medium, a process termed recultivation, and harvesting after an appropriate amount of time. This avoided the pleiotropic effects caused by stress and lack of growth factor supply during elutriation. Using this recultivation procedure, highly synchronous progression starting from any given cell cycle phase could be achieved for a variety of cell types, including primary, factor-dependent cells of hematopoietic origin. Mitotic cyclin expression and S6 kinase activity was found to be normal again in recultivated cultures, as opposed to elutriated ones. Finally, monitoring of mitosis-specific cyclin A degradation in recultivated G2 phase cells showed that recultivation provided an excellent tool to follow cells through M phase into G1 without the requirement for a chemical cell cycle block.

Structure and function of the iron-responsive element from human ferritin L chain mRNA.

We report the cloning and functional characterization of the iron responsive element (IRE) of human ferritin light (L) chain mRNA from a cDNA library of primary human T lymphocytes. Comparison of this palindromic cDNA element to the IRE predicted from the reported genomic sequence revealed significant differences, resulting in a stem-loop structure with lower stability than the IRE of the heavy (H) chain mRNA. Nevertheless, the L subunit IRE mediated efficient binding of the iron regulatory protein (IRP) in a manner comparable to that of human ferritin H chain mRNA in vitro. In accordance with previous observations on H form transcripts, the cis-acting regulatory IRE motif of human ferritin L chain mRNA was capable of repressing translation under iron deprivation but permitted mobilization of the transcripts into polysomes following iron repletion in vivo.

Mouse thymidine kinase stability in vivo and after in vitro translation.

Using a combination of centrifugal elutriation and recultivation of synchronised cell populations we could show that murine thymidine kinase (TK) is rapidly degraded during mitosis in polyoma virus-transformed mouse fibroblasts, in parallel to the time-course for loss of cyclin A. Transformation is no prerequisite for the instability phenotype since artificial overexpression of TK under the control of a constitutive promoter in normal mouse fibroblasts also resulted in rapid turnover of TK during mitosis. The decay of TK protein could be partially mimicked in vitro with enzymatically active protein translated in a rabbit reticulocyte lysate: full length polypeptide was lost slightly more rapidly in the presence of G2/M cytosolic extracts than with G1/S preparations. In addition, an enzymatically active C-terminal truncation of 37 amino acids at Gln-196 was completely stable under the conditions tested, confining the instability domain between residues 196 to 233. These experiments also indicated the border for intact TK since translation products up to Tyr-189 or less were completely inactive. This was also confirmed by a mutant TK protein from mouse F9tk- teratocarcinoma cells which harboured a similar deletion.

Overexpression of thymidine kinase mRNA eliminates cell cycle regulation of thymidine kinase enzyme activity.

Expression of thymidine kinase (TK) enzyme activity and mRNA is strictly S phase-specific in primary cells. In contrast, DNA tumor virus-transformed cells have enhanced and constitutive levels of TK mRNA during the whole cell cycle. Their TK protein abundance, however, still increases at the G1-S transition and stays high throughout G2 until mitosis. Therefore, post-transcriptional control must account for the decoupling of TK mRNA from protein synthesis in G1. To characterize the underlying mechanism, we studied the consequences of TK mRNA abundance on the cell cycle-dependent regulation of TK activity in nontransformed cells. Constitutive as well as conditional human and mouse TK cDNA vectors were stably transfected into mouse fibroblasts, which were subsequently synchronized by centrifugal elutriation. Low constitutive TK mRNA expression still resulted in a fluctuation of TK activity with a pronounced maximum in S phase. This pattern of cell cycle-dependent TK activity variation reflected the one in primary cell but is caused by post-transcriptional control. Increasing overexpression of TK transcripts after hormonal induction compromised this regulation. At the highest constant mRNA levels, regulation of enzyme activity was totally abolished in each phase of the cell cycle. These data indicate that post-transcriptional regulation of TK is tightly coupled to the amount of mRNA; high concentrations apparently titrate a factor(s) required for repressing TK production during G1 and presumably also G2.

Terminal differentiation of normal chicken erythroid progenitors: shortening of G1 correlates with loss of D-cyclin/cdk4 expression and altered cell size control.

Detailed knowledge is available about the molecular makeup of the cell cycle clock in dividing cells. However, comparatively little is known about cell cycle regulation during terminal differentiation. Here we describe a primary cell system in which this question can be addressed. Normal avian erythroid progenitors undergo continuous self-renewal in suspension culture in the presence of growth factors and hormones, allowing us to obtain large cell numbers (10(10)-10(11)). By replacing these "self-renewal factors" with erythropoietin and insulin, the cells can be induced to synchronous, terminal differentiation. During the first 72 h, the cells undergo five cell divisions. Thereafter, they arrest in G1 and complete their maturation into RBC without further divisions. Sixteen to 24 h after induction of differentiation, the cell cycle length decreased from about 20 to 12 h. This shortened doubling time was due to a drastic reduction of G1 (from 12 to 5 h), while S- and G2-phase lengths were not affected. At the same time, the differentiating cells underwent an extensive and concerted switch in their gene expression pattern. During the subsequent four cell divisions, the cell volume decreased from about 300 to less than 70 femtoliters, but the rate of protein synthesis normalized to cell volume remained constant. Interestingly, the shortening of G1 was accompanied by a rapid down-regulation of D-type cyclins and their partner, cyclin-dependent kinase type 4 (cdk4), while expression of S- and G2-M-associated cell cycle regulators (cyclin A and cdk1/cdc2) remained high until the cells arrested in G1 72-96 h after differentiation induction. We conclude that concerted reprogramming of progenitor gene expression during erythroid differentiation is accompanied by profoundly altered cell cycle progression involving the loss or alteration of cell size control at the restriction point.