Absence Of Rb Research Articles

Rb is dispensable for self-renewal and multilineage differentiation of adult hematopoietic stem cells

Stem cells have been identified as essential for maintaining multiple organ systems, including the hematopoietic system. The distinct cell fates of self-renewal and differentiation of hematopoietic stem cells (HSCs) depend on cell division. Recently, several negative regulators of the cell cycle, such as the cyclin-dependent kinase inhibitors p21(Cip1), p27(Kip1), and p16(INK4a)/p19(ARF), have been demonstrated to have a role in regulating HSC fate decisions, suggesting that regulation of the G(1)-S phase transition can contribute to HSC self-renewal. Because the retinoblastoma protein, Rb, plays a central role in the regulation of the G(1)-S phase cell cycle, we sought to determine whether it has an intrinsic role in the regulation of HSC fate. Surprisingly, we found that HSC function was essentially normal in the absence of Rb. Rb(Delta/Delta) HSCs contributed normally to both myeloid and lymphoid lineages in both primary and secondary recipients, and no evidence of transformation was observed. Additionally, we observed a mild myeloid expansion and decrease in mature B cells within the Rb(Delta/Delta) bone marrow but a similar contribution to phenotypic HSC populations compared with nondeleted bone marrow. The Rb family members p107 and p130 were not deregulated in cells in which Rb had been deleted, as determined by quantitative RT-PCR on the highly enriched stem and primitive progenitor cell lin(-)c-Kit(+)Sca-1(+) population. These studies demonstrate that Rb is not intrinsically required for self-renewal and multilineage differentiation of adult HSCs.

Binding of a Single Rb+ Increases Na+/K+-ATPase, Activating Dephosphorylation without Stoichiometric Occlusion

We used partially purified Na+/K+-ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 microM ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+-ATPase, a reasonable prediction of the experimental results is obtained.

Rb Enhances p160/SRC Coactivator-dependent Activity of Nuclear Receptors and Hormone Responsiveness

The retinoblastoma tumor suppressor protein (Rb) is best known as a repressor of genes involved in cell cycle progression. Rb has also been implicated in activation of transcription, in particular by nuclear receptors (NRs) and by differentiation-related transcription factors, but the relevance of this activity is unclear. We show that Rb and the related proteins p107 and p130 enhance the activity of NRs related to NGFI-B (Nur factors) through direct interactions with NGFI-B and SRC-2. Although recruitment of SRC/p160 coactivators to the NGFI-B AF1 domain is independent of Rb, its presence enhances SRC-dependent transcription. Rb potentiation of SRC coactivators is exerted on a subset (Nur factors, hepatocyte nuclear factor-4 (HNF-4), SF-1, and ER) but not all NRs. The levels of Rb-related proteins modulate hormone responsiveness of the NGFI-B-dependent pituitary proopiomelanocortin gene and HNF-4-dependent transcription during enterocyte differentiation. Increased Rb expression upon cell differentiation may promote differentiated functions, at least in part, by potentiation of NR activity.

Competitive Na(+) and Rb(+) binding in the minor groove of DNA.

Sequence-dependent coordination of alkali ions to the nucleotide bases in the minor groove of AT-tract B-DNA has recently been inferred from X-ray crystallography, solution NMR and computer simulations. Here, we present new (23)Na and (87)Rb magnetic relaxation dispersion (MRD) data that demonstrate competitive and long-lived binding of Na(+) and Rb(+) ions in the minor groove of the B-DNA duplex [d(CGCGAATTCGCG)](2). The Na(+)/Rb(+) selectivity of the minor groove is found to be weak, consistent with local structural flexibility. The ion occupancies derived from the MRD data are substantially higher than previously reported, suggesting that groove-bound ions significantly influence the energetics and structural polymorphism of DNA in vivo. For example, in the presence of 0.20 M Na(+) and 0.56 M Rb(+) at 4 degrees C, the ApT site in the minor groove is occupied by a Rb(+) ion, a Na(+) ion, or a water molecule 40, 10, and 50% of the time, respectively. In the absence of Rb(+), the Na(+) occupancy increases to 50%. At 4 degrees C, the mean residence time of groove-bound ions is 0.2 +/- 0.1 micros for Rb(+) and 10 ns to 100 micros for Na(+). A shorter correlation time of 2 ns is attributed to counterions bridging cross-strand phosphate groups.

Rb regulates proliferation and rod photoreceptor development in the mouse retina.

The retinoblastoma protein (Rb) regulates proliferation, cell fate specification and differentiation in the developing central nervous system (CNS), but the role of Rb in the developing mouse retina has not been studied, because Rb-deficient embryos die before the retinas are fully formed. We combined several genetic approaches to explore the role of Rb in the mouse retina. During postnatal development, Rb is expressed in proliferating retinal progenitor cells and differentiating rod photoreceptors. In the absence of Rb, progenitor cells continue to divide, and rods do not mature. To determine whether Rb functions in these processes in a cell-autonomous manner, we used a replication-incompetent retrovirus encoding Cre recombinase to inactivate the Rb1(lox) allele in individual retinal progenitor cells in vivo. Combined with data from studies of conditional inactivation of Rb1 using a combination of Cre transgenic mouse lines, these results show that Rb is required in a cell-autonomous manner for appropriate exit from the cell cycle of retinal progenitor cells and for rod development.

PPARgamma controls cell proliferation and apoptosis in an RB-dependent manner.

The nuclear receptor PPARgamma is implicated in the control of cell proliferation and apoptosis. However, the molecular mechanisms by which it controls these processes remain largely elusive. We show here that PPARgamma activation in the presence of the retinoblastoma protein (RB) results in the arrest of cells at the G1 phase of the cell cycle, whereas in the absence of RB, cells accumulate in G2/M, endoreduplicate, and undergo apoptosis. Through the use of HDAC inhibitors and coimmunoprecipitations, we furthermore demonstrate that the effects of RB on PPARgamma-mediated control of the cell cycle and apoptosis depend on the recruitment of histone deacetylase 3 (HDAC3) to PPARgamma. In combination, these data hence demonstrate that the effects of PPARgamma on cell proliferation and apoptosis are dependent on the presence of an RB-HDAC3 complex.

The Retinoblastoma-Histone Deacetylase 3 Complex Inhibits PPARγ and Adipocyte Differentiation

The retinoblastoma protein (RB) has previously been shown to facilitate adipocyte differentiation by inducing cell cycle arrest and enhancing the transactivation by the adipogenic CCAAT/enhancer binding proteins (C/EBP). We show here that the peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor pivotal for adipogenesis, promotes adipocyte differentiation more efficiently in the absence of RB. PPARγ and RB were shown to coimmunoprecipitate, and this PPARγ-RB complex also contains the histone deacetylase HDAC3, thereby attenuating PPARγ's capacity to drive gene expression and adipocyte differentiation. Dissociation of the PPARγ-RB-HDAC3 complex by RB phosphorylation or by inhibition of HDAC activity stimulates adipocyte differentiation. These observations underscore an important function of both RB and HDAC3 in fine-tuning PPARγ activity and adipocyte differentiation.

MyoD Activity Upregulates E2F1 and Enhances Transcription from the Cyclin E Promoter in Differentiating Myoblasts Lacking a Functional Retinoblastoma Protein

We investigated the mechanism leading to cyclin E accumulation when cultured mouse myoblasts, lacking functional Rb because of sequestration or deletion, are exposed to differentiating conditions (mitogen subtraction and cell-cell contact), which activate MyoD and normally downregulate factors involved in cell division. After excluding that stabilization might account for the observed cyclin-E mRNA accumulation, we found an induction of the cyclin-E promoter that correlated with E2F activity upregulation and depended on both MyoD activation and Rb inactivation. Analyses of the E2F1-promoter activity, in normal and Rb-deficient fibroblasts converted by MyoD, identified a MyoD function stimulating E2F1 expression. The E2F1 induction was very manifest in the Rb−/− cells, but also detectable, at the early stage of differentiation, in normal cells. Its effects, although not indispensable for myogenesis, presumably contribute to raise the concentration of Rb-E2F1 transcription-repressing complexes, since MyoD strongly induces also Rb in differentiating myocytes. The activity of an E2F1 promoter lacking the E2F sites indicated that E2F1 itself underwent self-repression by such mechanism at late stages of differentiation. In the absence of Rb, however, the induced E2F1 is left with only its activating role, reversing the normal effect of this MyoD function.

Alterations in expression of E2F-1 and E2F-responsive genes by RB, p53 and p21 Sdi1/WAF1/Cip1 expression

RB, p53 and p21 Sdi1/WAF1/Cip1 interact in the induction of G1 arrest. We established osteosarcoma cell lines in which a tetracycline-regulatable promoter controls the induction of RB, p53 and p21. By using these cell lines, we investigated whether RB, p53 or p21 regulates, in the same manner or differently, expression and function of E2F-1 and its responsive genes. E2F-1 gene products and transcripts of the E2F-responsive genes decreased in response to RB. Similar changes occurred to p53 and p21 when RB is present. However, in the absence of RB, some of the E2F-responsive genes decreased in response to p53 but not to p21. Thus, RB is a critical component for regulating the E2F-responsive genes, while p53 alone affects only a subset of these genes.

E2F1 and p53 Are Dispensable, whereas p21Waf1/Cip1 Cooperates with Rb to Restrict Endoreduplication and Apoptosis during Skeletal Myogenesis

We describe temporal and genetic analyses of partially rescued Rb mutant fetuses, mgRb:Rb−/−, that survive to birth and reveal specific defects in skeletal muscle differentiation. We show that in the absence of Rb, these fetuses exhibit increased apoptosis, bona fide endoreduplication, and incomplete differentiation throughout terminal myogenesis. These defects were further augmented in composite mutant fetuses, mgRb:Rb−/−:p21−/−, lacking both Rb and the cyclin-dependent kinase inhibitor p21Waf1/Cip1. Although E2F1 and p53 mediate ectopic DNA synthesis and cell death in several tissues in Rb mutant embryos, both endoreduplication and apoptosis persisted in mgRb:Rb−/−:E2F1−/− and mgRb:Rb−/−:p53−/− compound mutant muscles. Thus, combined inactivation of Rb and p21Waf1/Cip1 augments endoreduplication and apoptosis, whereas E2F1 and p53 are dispensable during aberrant myogenesis in Rb-deficient fetuses.

Bidirectional signaling: Ras and Rb Communicate in Both Directions

The flow of information from the cell surface to the nucleus often involves signaling cascades that include the small GTP-binding protein Ras. The retinoblastoma protein Rb is a downstream nuclear target of Ras activation and both proteins are implicated in regulating cell proliferation and differentiation. Lee et al. show that Rb may also signal back upstream to Ras. Cells lacking Rb had an increase in activated Ras during the G 1 phase of the cell cycle. Ras activation occurred at the level of GTP binding. During the G 1 phase, activation of Ras, in the absence of Rb, also required protein synthesis. Rb deficiency also resulted in a failure of cells to differentiate, but this was not linked to the ability of Rb to regulate transcription. The authors propose that Rb might control the activity of Ras by regulating the expression of a factor that affects GTP loading of Ras. This regulation may be linked to the influence of Rb on differentiation. Lee, K.W., Ladha, M.H., McMahon, C., and Ewen, M.E. (1999) The retinoblastoma protein is linked to the activation of ras. Mol. Cell. Biol. 19 : 7724-7732. [Online Journal]

Optical pumping magnetic resonance in high magnetic fields: Measurement of high field spin exchange cross sections

The polarization of 129Xe gas by spin exchange with optically pumped Rb vapor is examined in high magnetic field. Measurements of the Rb–Xe velocity averaged spin exchange cross section 〈 σ v̄〉 and the 129Xe relaxation time in the absence of Rb, T 1 Xe, are discussed. Comparison of these high field results to low field literature values for 〈 σ v̄〉 show that this parameter is essentially field independent. T 1 Xe is also found to be significantly longer in high field. On the basis of these observations, it is shown that production of large amounts of spin polarized 129Xe gas can be made more efficient by high field operation.

Evidence for the Existence of Two ATP-sensitive Rb+Occlusion Pockets within the Transmembrane Domains of Na+/K+-ATPase

A trypsin-digested Na+/K+-ATPase that has lost ATPase activity and about half of its protein content retains an essentially intact beta-subunit, the 10 transmembrane domains of the alpha-subunit, and the full capacity to occlude Na+ and Rb+ (a congener of K+). When this preparation was incubated at 37 degrees C in the absence of Rb+, it lost half of its Rb+ occluding capacity and two-thirds of its Na+ occluding capacity. Comparison of the Rb+ occlusion-deocclusion kinetics of the digested enzyme before and after partial inactivation indicated that (a) the affinities of the labile and the stable halves of occluded Rb+ were the same; (b) occlusion and deocclusion rates of the stable pool were lower than those of the labile pool; (c) ATP at a low affinity site (K0.5 = 25-300 microM) increased deocclusion rate in the stable pool and occlusion rate in the labile pool; (d) Na+ increased Rb+ deocclusion rate of the sum of the two pools but not that of the stable pool; and (e) occlusion and deocclusion rates of both pools were decreased by ouabain. These findings suggest that (a) the peptide complex of the digested enzyme contains two distinct but interacting cation occlusion pockets, one occluding two Na+ or one Rb+, and the other occluding one Na+ or one Rb+; (b) this peptide complex that is devoid of the catalytic ATP site retains an allosteric ATP site; and (c) the access channels of the two pockets are regulated differently by ATP but similarly by ouabain. Analyses of the gel electrophoretic patterns of the digested enzyme and the N termini of the appropriate bands showed that inactivation of the labile occlusion pocket was accompanied by 60-70% loss of two alpha-fragments containing H3-H4 and H5-H6 transmembrane domains. This and the previously established interactions among the transmembrane helices of alpha- and beta-subunits suggest that one occlusion pocket is associated with H3-H6 domains and that the other is located within a complex of beta-subunit and two alpha-fragments containing H1-H2 and H7-H10 transmembrane domains.

Stimulation of Human Insulin Receptor Gene Expression by Retinoblastoma Gene Product

Multiple cis-acting elements have been defined to be important for the transcriptional regulation of the human insulin receptor (hIR) gene expression. We report here that one of these elements also mediated the stimulation of hIR promoter activity by the retinoblastoma gene product (Rb). The cis-element responsible for Rb stimulation was localized to the GA and GC boxes situated between -643 to -607 of the hIR gene. We have previously demonstrated that these GA and GC boxes bind Sp1 with high affinity and are responsible for E1a activation of hIR promoter activity. Mutation of these sequences completely abolished Rb-dependent enhancement of hIR promoter activity. In addition, we localized three regions in the N-terminal domain of Rb to be involved in stimulation of hIR promoter activity. Our results represent one of the first studies to demonstrate a functional importance assigned to the multiple phosphorylation sites in the N terminus of Rb. Finally, the mechanism by which Rb activates the hIR promoter are presented.

Increased Potassium Absorption Confers Resistance to Group IA Cations in Rubidium-Selected Suspension Cells of Brassica napus

Cell lines of suspension cultures of Brassica napus cv. Jet Neuf were identified for their ability to tolerate 100 millimolar Rb(+), a level which was double the normally lethal concentration. Ten spontaneous isolates were obtained from approximately 5 x 10(7) cells, one of which was reestablished as a cell suspension. This cell line, JL5, was also resistant to the other group IA cations- Li(+), Na(+), K(+), and Cs(+)-and this trait was stable for at least 30 cell generations in the absence of Rb(+) selection pressure. The growth characteristics were similar to those of sensitive cells under nonselective conditions. The selected JL5 cells were shown by analysis to have effected more net accumulation of K(+) and Rb(+) and less of Na(+) than did the unselected cells. JL5 and unselected cells after 14 days of culture in basal medium contained 597.2 and 258.2 micromoles of K per gram dry weight, respectively. Michaelis-Menten kinetic analysis of K(+) influx showed that JL5 possessed an elevated phase 1 V(max), but there was no alteration in its K(m). This is the first time that a plant mutation has been shown to have both increased influx and net absorption of a major essential cation.

Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (Tp R) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (Ap R) on E. coli. The B. subtilis dihydrofolate reductase operon ( dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene ( thyB) and the Tp R-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as dfrA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA ' on pNBLl. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of jS-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RB S, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA ' -lacZ was observed, although the translational efficiency was very low.

Ion Transport and the Effects of Acetic Acid in White Clover

Dunlop, J., Knighton, M. V. and White, D. W. R. 1988. Ion transport and the effects of acetic acid in white clover. II. Potassium absorption.—J. exp. Bot. 39: 89–96. Acetic acid stimulated K+ influx into cells of white clover in suspension culture and net K+ influx by roots of intact white clover plants. At concentrations of acetic acid up to 2·0 mol m−3 the stimulation continued unabated for at least 2 h. However, at higher concentrations the rate of absorption declined to near zero values after 2 h. When acetic acid was removed from the solution there was a net efflux of K+. The stimulation was pH dependent with a maximum at pH 5·0. There was maximum stimulation at an acetic acid concentration of 2·0 mol m−3 Net H+ efflux was reduced by 1·0 mol m−3 acetic acid. When Rb+ uptake and H+ efflux were measured at a range of RbCl concentrations Rb+ uptake increased with concentration whereas H+ efflux was maximum in the absence of Rb+ (and K+) and decreased as RbCl concentration was increased Acetic acid caused a hyperpolarization of the membrane electrical potential difference (ΔE) of about 25 mV. In 1·0 mol m–3 acetic acid the hyperpolarization persisted for at least 2 h whereas at 10 mol m−3 d E subsequently depolarized to values around −80 mV. With a slight lag, the time course of the stimulation of the rate of K+ absorption followed the polarization and depolarization of ΔE. These results imply that the linkage between K+ and H+ movements is probably through ΔE.