Stimulation Of Hemoglobin Synthesis Research Articles

Testosterone administration inhibits hepcidin transcription and is associated with increased iron incorporation into red blood cells

Testosterone administration increases hemoglobin levels and has been used to treat anemia of chronic disease. Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially older men. However, the mechanisms by which testosterone increases hemoglobin remain unknown. Testosterone administration in male and female mice was associated with a greater increase in hemoglobin and hematocrit, reticulocyte count, reticulocyte hemoglobin concentration, and serum iron and transferrin saturation than placebo. Testosterone downregulated hepatic hepcidin mRNA expression, upregulated renal erythropoietin mRNA expression, and increased erythropoietin levels. Testosterone-induced suppression of hepcidin expression was independent of its effects on erythropoietin or hypoxia-sensing mechanisms. Transgenic mice with liver-specific constitutive hepcidin over-expression failed to exhibit the expected increase in hemoglobin in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound (58) Fe, the amount of (58) Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to bone morphogenetic protein (BMP)-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. In conclusion, testosterone inhibits hepcidin transcription through its interaction with BMP/Smad signaling. Testosterone administration is associated with increased iron incorporation into red blood cells.

Triticum aestivum (Wheatgrass) formulation: An alternate treatment for the patients with Thalassemia

We have developed the tablet formulation of Triticum aestivum (wheatgrass) and investigated clinically, its effects in patients suffering from b-thalassemia (major) at K. T. Children Hospital, Civil Hospital, Rajkot. The tablets (wheatgrass powder 250 mg.) were given 3 times in a day for 9 months. Blood samples were collected at the start, after 6 months and 9 months and analyzed for various biochemical and hematological parameters. Treatment with wheatgrass formulation for 9 months produced significant decrease in hemoglobin, total RBC, eosinophil and reticulocyte counts. The mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were significantly increased was significantly increased. The serum ferritin was also significantly decreased. There was no influence on serum magnesium, serum iron and Thiobarbituric acid reacting substances (TBARS). Our data indicate that treatment with wheatgrass on patients with <TEX>$\beta$</TEX>-thalassemia (major) may have beneficial effects in the form of a decrease in ineffective erythropoiesis, stimulation of hemoglobin synthesis in RBC, decrease in iron load and decrease in eosinophil count.

Lead-induced upregulation of the heme-regulated eukaryotic initiation factor 2α kinase is compromised by hemin in human K562 cells

Expression and kinase activity of the heme-regulated-eIF-2α kinase or -inhibitor (HRI) are induced during cytoplasmic stresses leading to inhibition of protein synthesis. Using a reporter construct with HRI promoter, we have determined the promoter activity during heat-shock and lead toxicity in human K562 cells. These two conditions induced HRI promoter activity by 2- to 3-fold. Contrary to this, hemin, a suppressor of HRI kinase activity, downregulated HRI promoter activity and stimulated hemoglobin synthesis. Interestingly, when hemin-treated cells were transfected and exposed to lead, hemin compromised lead-effect substantially by downregulating HRI promoter activity, HRI transcription and HRI kinase activity. These results together suggest that heme signaling in relation to translation regulation is not only restricted to the cytoplasm (modulating HRI kinase activity) alone but it also spans to the nucleus modulating HRI expression. Hemin may thus be useful for alleviation of stress-induced inhibition of protein synthesis.

TEL/ETV6 accelerates erythroid differentiation and inhibits megakaryocytic maturation in a human leukemia cell line UT‐7/GM

TEL/ETV6 accelerates erythroid differentiation in the murine erythroleukemia cell line. To clarify the effects of TEL on megakaryocytic maturation as well as erythroid differentiation, we chose the human leukemia cell line UT-7/GM that differentiates into the erythroid and megakaryocytic lineages by treatment with erythropoietin and thrombopoietin, respectively. Upon erythropoietin exposure, overexpressed TEL stimulated hemoglobin synthesis and accumulation of the erythroid differentiation-specific transcripts such as gamma-globin, delta-aminolevulinic acid synthase-erythroid, and erythropoietin receptor. Moreover, the glycophorin A(+)/glycoprotein IIb(-) fraction appeared more rapidly in the TEL-overexpressing cells. Interestingly, overexpression of TEL was associated with lower levels of the megakaryocytic maturation-specific glycoprotein IIb and platelet factor 4 transcripts under the treatment with thrombopoietin. Consistently, the glycophorin A(-)/glycoprotein IIb(+) fraction increased more slowly in the TEL-overexpressing cells. Finally, expression of endogenous TEL proteins in UT-7/GM cells was down-regulated following erythropoietin and thrombopoietin exposure. All these data suggest that TEL may decide the fate of human erythrocyte/megakaryocyte common progenitors to differentiate towards the erythroid lineage and against the megakaryocytic lineage.

PIXE ANALYSIS OF BONE MARROW IN MICE ACQUIRED RADIORESISTANCE AFTER PRE-IRRADIATION WITH SMALL DOSE OF X-RAYS

Pre-irradiation with 0.3-0.5 Gy of X-rays induces radioresistance (decreased bone marrow death) in ICR and C57BL strains of mice 2 weeks afterward. Recovery of blood cell counts of thrombocytes, leukocytes and erythrocytes was enhanced by the pre-irradiation. And the radio-resistance is acquired even in splenectomized C57BL mice. Elements in the bone marrow 11 days after the challenging dose of 5.0 Gy was determined by a PIXE analysis in three groups of mice: intact (with no irradiations), without pre-irradiation (control) and pre-irradiation (experimental) groups. Challenging irradiation decreased Mg , P , S , K , Ca and Zn levels, and the pre-irradiation enhanced the recovery of those levels, i.e. stimulated metabolism in bone marrow of irradiated mice. Increase in Fe in both control and experimental groups would indicate stimulated hemoglobin synthesis after challenging irradiation.

5-Aminolevulinate synthase expression and hemoglobin synthesis in a human myelogenous leukemia cell line.

We examined the effect of hemin, TGF-beta1 and cytosine arabinoside (Ara-C) on the levels of mRNAs for the erythroid-specific 5-aminolevulinate synthase (ALAS-E) and gamma-globin in various human myelogenous leukemia cell lines. Detailed analyses were also made using one of them, YN-1, which was isolated and established in culture from a patient with chronic myelogenous leukemia. Our results demonstrate that gamma-globin protein level and the percentage of benzidine-positive cells in the cell line increased markedly (10- to 30-fold) upon treatment with hemin, TGF-beta1, or Ara-C. In contrast, gamma-globin mRNA was already markedly expressed prior to treatment in 4 out of 9 cell lines examined, including YN-1, and the level increased only marginally after treatment with hemin. ALAS-E mRNA levels were increased in YN-1 cells after treatment with TGF-beta1 and Ara-C, while hemin treatment had little effect. These results indicate that heme supply is insufficient in YN-1 cells and suggest that hemin increases hemoglobin synthesis principally at the post-transcriptional level, whereas TGF-beta1 and Ara-C stimulate hemoglobin synthesis by activating efficient endogenous heme supply in the cells.

Increased Erythropoietin Sensitivity After In Vitro Transformation of Hematopoietic Precursors by RNA Tumor Viruses&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="fn3"&gt;3&lt;/xref&gt;

Two strains of Friend virus differ in their in vivo actions in that one strain induces anemia (FVA), while the other induces polycythemia (FVP). This study characterizes differences in the in vitro effects of these viruses on hematopoietic cells of (BALB/c x DBA/2)F1 mice. Both variants induced erythroid bursts that proliferated and differentiated without added erythropoietin (EPO). However, while the bursts induced by FVP were well "hemoglobinized" (i.e., most cells contained hemoglobin), the cells of FVA-induced bursts contained little or no hemoglobin. The nonhemoglobinized bursts, induced by FVA, were established to be erythroid by cytochemistry, electron microscopy, and hormone sensitivity. FVA-induced cells appeared to be hypersensitive to EPO, since small concentrations of the hormone produced marked increases in hemoglobin production--even when the hormone was added to the culture 3 days post infection. Time-lapse photography documented that EPO stimulated hemoglobin synthesis in virally transformed cells rather than uninfected erythroid precursors. This observation of FVA-induced hypersensitivity prompted the reexamination of the hormone requirements of FVP-induced bursts--previously considered to be EPO-independent. Reduction of the serum in the cultures allowed the demonstration that FVP-induced erythroid cells also were hypersensitive to EPO. Thus FVA and FVP can be readily distinguished in vitro by the relative EPO sensitivity of virus-induced bursts. From these findings, a hypothesis is drawn: i.e., oncogenic transformation may result from increased sensitivity of progenitor cells for natural, physiologic regulators, and transformation is not necessarily accompanied by a block in differentiation. In addition, since hypersensitive virus-induced bursts could be recognized and picked from the cultures, these studies provide a method for obtaining highly purified erythroid precursors for the study of the regulation of terminal differentiation by EPO and other regulatory factors.

Effects of cyclic nucleotides on hemoglobin synthesis in fetal calf liver cells in culture.

Fetal calf liver cells were cultured in a serum-free medium in the presence of dibutyryl cAMP (dbcAMP), dibutyryl cGMP (dbcGMP), or cGMP. After a 20-h incubation with the nucleotides the synthesis of the fetal hemoglobins F0 and F1 was measured by the incorporation of [3H]leucine. The three cyclic nucleotides stimulated hemoglobin synthesis at a concentration of 1 μM. dbcGMP (10 nM) inhibited the synthesis of F0 and F1 but at a concentration of 1 μM significantly stimulated the synthesis of both hemoglobins. dbcAMP (10 μM) preferentially stimulated the synthesis of the transient hemoglobin F1 whereas cCMP (1 μM)stimulated specifically the synthesis of the definitive hemoglobin F0. Prostaglandin E1 (100 nM) stimulated the synthesis of F1 in a manner similar to that observed for dbcAMP. AMP, GMP, CMP, and sodium butyrate did not stimulate hemoglobin synthesis at the concentrations indicated above. The analysis of the α- and γ-globin chains by high pressure liquid chromatography indicated that the synthesis of both chains are stimulated by the three cyclic nucleotides. These changes in hemoglobin synthesis taking place at different concentrations of cyclic nucleotides may be of importance in the maturation of erythroid cells, because in these cells differentiation is normally coupled with cell proliferation. Furthermore, some of these cyclic nucleotides may be second messengers of erythropoietic hormones. We have found that the intracellular levels of cGMP were increased 15 min after addition of step III sheep plasma erythropoietin to the cultures, whereas the amounts of cAMP remained unchanged. It is then possible that erythropoietin itself or a factor present in the erythropoietin preparation may act by a cGMP-dependent mechanism.

The formation of erythrocyte membrane proteins during erythropoietin-induced differentiation.

The effect of erythropoietin on the synthesis of the proteins characteristic of mature erythrocyte membranes was studied in cultures of bone marrow cells from adult, polycythemic rats. Stimulated synthesis of the major glycoprotein, glycophorin, was maximal at 30 h and fell to control level by 66 h. Stimulated synthesis of the major integral membrane protein, band 3, occurred at about 18 h was maximal at 66 h, and fell to control level by 114 h. In contrast, stimulated hemoglobin synthesis did not start until after 24 h, was maximal at 96 h, and was at control level at 114 h. Erythropoietin had, in addition, a transient effect on the synthesis of some membrane proteins found in marrow cells but not in mature red cells.

Stimulation of hemoglobin synthesis in murine erythroleukemia cells by low molecular weight ketones, aldehydes, acids, alcohols and ethers

The effects of short chain (C1-C5) aldehydes, ketones, acids, alcohols and ethers on murine erythroleukemia (MEL) cells were examined to determine which particular chemical moieties and some of their combinations stimulated hemoglobin synthesis in these cells. The C4 series of compounds was active at lower concentrations than homologs of shorter chain lengths. Within an homologous series the potency and efficacy of the alcohol was always less than that of the acid and aldehyde compounds. Though heptanoic acid was found to be an inducer of hemoglobin synthesis in MEL cells, the 4,6-dioxoheptanoic acid analog is a potent inhibitor of hemoglobin synthesis. Analysis of porphyrin content of MEL cells incubated with the inducers 2-butanone, 2-methoxyethanol, acetone and methanol, showed that increased hemoglobin synthesis was always accompanied by the accumulation of porphyrins, most of which was protoporphyrin. These studies suggest that low molecular weight ketones, aldehydes, acids, ethers and alcohols can correct the defect in erythroid differentiation exhibited by MEL cells and they further suggest that the physiological trigger for inducing hemoglobin synthesis in these cells is less discriminating than previously recognized.

Induction of differentiation of murine erythroleukemia cells by aminonucleoside of puromycin and inhibition of this induction by purines and purine derivatives.

The effect of the aminonucleoside of puromycin (AMS) on Friend erythroleukemia cells in culture was investigated, because purines and purine analogues are known to act as inducers of differentiation. After treatment with 20-30 micro M AMS for 4 days, the cultures contained between 80 and 90% benzidine-positive cells. Stimulation of hemoglobin synthesis was dose and time dependent. Inosine had no stimulatory activity; however, when it was added to the medium together with AMS, erythroid differentiation was almost completely inhibited. The inhibitory effect of inosine on this potent inducer was also dose and time dependent. No cytotoxicity was observed with either compound, alone or in combination. Inhibition of AMS stimulation of erythroid differentiation was also observed in the presence of inosine monophosphate and poly(inosinic acid). Hypoxanthine had a dual effect. At high concentrations (500 microgram/ml) it acted as an inducer, but when added at low concentrations (20 microgram/ml) together with AMS it inhibited differentiation. These findings suggest there is a link between purine biosynthesis and the event(s) required to trigger differentiation. Agonist-antagonist activity of closely related biological compounds has thus been revealed in the erythroleukemia cells.

In vitro transformation of mouse bone marrow cells by the polycythemic strain of Friend leukemia virus.

Strains of Friend leukemia virus (FLV) that are associated with polycythemia contain the defective spleen focus-forming virus (SFFV). To determine whether the transforming ability of FLV was affected by the presence of this second agent, DBA/2J mouse bone marrow cells were infected in vitro. Criteria for transformation were the establishment of permanent lines, growth on semisolid agarose, and the production of tumors at the site of inoculation in syngeneic hosts. Two lines of immature hematopoietic cells that grow in suspension originated from the infected cultures. Each has an almost diploid karyotype (38-39 chromosomes) and 3-4 metacentric chromosomes. These transformed cells express gp71 viral envelope glycoprotein and p30 viral core protein antigens. Virus production was measured by reverse transcriptase (RNA-dependent DNA polymerase) activity of the virions released into the medium. The virus, assayed in vivo for infectivity, has SFFV activity but is attenuated for leukemogenicity. The stimulation of hemoglobin synthesis in the cells grown in medium supplemented with dimethyl sulfoxide or hexamethylene bisacetamide indicates that the cells are erythroid in origin. SFFV may have a function analogous to erythropoietin in influencing the process of transformation by FLV.

Induction of erythroid differentiation in friend murine erythroleukemic cells by inorganic selenium compounds

Inorganic selenium compounds are shown to be inducers of hemoglobin synthesis in malignant murine erythroleukemia (MEL) cells. SeO 2 can induce hemoglobin synthesis at 1 20 the concentration of butyric acid and 1 5000 the concentration of dimethylsulfoxide (DMSO), two potent inducers of erythroid differentiation in MEL cells. SeO 2 and H 2SeO 3 showed an equivalent capacity to stimulate hemoglobin synthesis in three different MEL cell lines. The incorporation of 3H-glycine into hemoglobin was demonstrated in lysates of SeO 2-induced MEL cells.

Extraction of erythropoietin from normal kidneys.

Significant amounts of active erythropoietin were extracted from the kidneys of normal rats, cattle, dogs, and rabbits by homogenization of the organs in 0.1 M phosphate buffer. The mean erythropoietin activities of the extracts, as determined by the starved-rat assay, were 0.26 U/g beef kidney, 0.41 U/g dog kidney, and 0.11 U/g rat kidney. The dog kidney extracts had a mean activity of 0.35 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells (in vitro assay) and produced a dose-dependent stimulation of 59Fe incorporation into circulating red cells when assayed in polycythemic mice. Extracts of rabbit kidney cortices had a mean activity of 2.12 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells. When the dog kidney homogenate was fractionated on DEAE-cellulose, all of the erythropoietin activity was adsorbed to the exchanger in the presence of 0.01 M acetate buffer, pH 4.5, and was completely eluted by 0.1 M Na2HPO4-0.5 M NaCl, pH 8. An antibody made against human urinary erythropoietin completely inactivated the erythropoietic factor in the dog kidney extract. Serum from a donor dog had no erythropoietin activity when assayed in the starved rat, suggesting that the factor in the extracts is intracellular erythropoietin rather than that contained in plasma trapped in the renal vasculature. The complete inactivation of the erythropoietic factor in these kidney homogenates by antierythropoietin and its behavior on DEAE-cellulose indicate that this factor is structurally similar to native plasma erythropoietin. The extracts are completely active without being incubated in the presence of serum.

The Effect of Radiation in Vitro on Erythropoietin Action

The effect of ionizing radiation on the response to erythropoietin of cultured rat marrow cells, at constant erythropoietin dose and constant cell density, differed according to the end point analyzed. Erythropoietin-induced RNA synthesis (an early response) and induced glucosamine incorporation into stroma (a late response) were relatively radioresistant. Induced hemoglobin synthesis was characterized by marked radiosensitivity. Inactivation parameters were anti n = 1.04 and D/sub 0/ = 63 R (59 rad) +- 15 R (95 percent confidence interval). The radiosensitivity was not the result of inactivation of secondary enzymic processes. The inactivation curves for stimulated hemoglobin synthesis at different doses of erythropoietin near the physiological range were found to be linear, with anti n values in the range of 0.92 to 1.14. The corresponding log-log erythropoietin dose--response plots at different radiation doses were linear and parallel, a result inconsistent with an all-or-none action of radiation on erythropoietin-sensitive cells. At high cell density, the effect of 60 R on induced hemoglobin disappeared, whereas the effect on induced iron uptake persisted.

Lack of effect of erythropoietin on cyclic adenosine-3',5'-monophosphate levels in rat fetal liver cells.

Abstract The effect of Erythropoietin (Ep) and epinephrine on the cellular concentration of adenosine 3′,5′-cyclic monophosphate (cAMP) was studied using suspensions of fetal liver cells obtained from 14-day pregnant rats. This system was chosen because the cell suspensions were greater than 65 per cent erythroid and very sensitive to Ep. In cells preincubated with theophylline, Ep failed to increase cAMP levels after both 5 minute and 30 minute incubations with the hormone. By contrast, epinephrine stimulated cAMP concentrations 6- to 7-fold indicating that the adenyl cyclase present was capable of responding to an appropriate stimulus. In suspensions first treated with epinephrine to raise cAMP levels, addition of Ep did not lower the concentration of this nucleotide. Heme synthesis experiments employing the same conditions as the cAMP studies show that Ep effectively stimulated 59 Fe incorporation into heme. This indicated that under the conditions of the cAMP studies the hormone was fully capable of interacting with the erythropoietin-responsive cell. The above experiments show that erythropoietin can stimulate hemoglobin synthesis in vitro without producing any detectable increase or decrease in the cellular cAMP concentration. Thus, the data fail to support the hypothesis that the effect of Ep on red-cell production is mediated by a change in cAMP levels.

RNA et biosynthèse de l'hémoglobine.

In 1960, we have observed with G. Schapira and J. C. Dreyfus that when ribosomes from rabbit reticulocytes were incubated with the pH 5 fraction from guinea pig reticulocyte, as well as in the reverse system, both rabbit and guinea pig hemoglobins were synthesized. The discovery of the messenger RNA by Jacob and Monod allowed us to give an explanation of these observations. The reality of these synthesis was ascertained by the method of double labelling and peptide analysis. In order to demonstrate the presence of messenger RNA in reticulocytes we have purified RNA from these cells. Hemoglobin synthesis was stimulated when this RNA was added to a reticulocyte cell free system. This RNA was fractionated on a sucrose gradient. The fraction which sedimented between 18S and 4S, and which was called RNA III, stimulated hemoglobin cell free synthesis. The stimulation is specific, since: u – The other RNA fractions did not stimulate hemoglobin synthesis. – RNA III from reticulocytes from other species were either inactive or inhibitory. These results are in favor of the presence of messenger RNA in this fraction in reticulocytes. RNAs extracted from rabbit, liver, kidney, intestine nuclei, and the fraction III from these RNAs also stimulated hemoglobin synthesis. RNA from cytoplasm, RNA III from liver nuclei from other species did not stimulate hemoglobin synthesis. These results favor the idea that nuclei from differentiated cells contain messenger RNAs which are not translated in these cells. We are now studying mechanims involved in the selection of messenger RNAs in differentiated cells.

On the Mechanism of Erythropoietin-induced Differentiation: IX. INDUCED SYNTHESIS OF 9 S RIBONUCLEIC ACID AND OF HEMOGLOBIN

Erythropoietin induces the synthesis of 9 S RNA and hemoglobin by cultured bone marrow cells derived from polycythemic rats and mice in which hemoglobin synthesis has been suppressed. Synthesis of 9 S RNA starts within 1 hour (rat cells) and 4 hours (mouse cells) of addition of erythropoietin to the medium, while hemoglobin synthesis is not initiated until 8 to 10 hours later. These findings indicate that there is another regulatory step between transcription, initiated by erythropoietin, and translation. Under conditions in which no hemoglobin synthesis occurs, bone marrow cells still synthesize a very small amount of 9 S RNA which may be a messenger for a protein other than globin. Comparison of stimulated hemoglobin synthesis by cells from normal animals with cells from polycythemic animals indicates that erythropoietin has an effect on some differentiated cells in the former population as well as on the undifferentiated cells in the latter population.

The stimulation of hemoglobin synthesis by porphyrins

Several synthetic metalloporphyrins, as well as hemin, stimulate hemoglobin synthesis in isolated nuclei from erythrocytes. The effect of a metalloporphyrin appears to be related principally to the character of the metal component. Hematoporphyrins complexed with transition metals of the fourth period tend to be stimulatory while porphyrins complexed with either smaller or larger metals have no effect or are inhibitory. Evidence is presented that the synthetic metalloporphyrins probably act at the same site as the natural porphyrin, heme.

Erythropoietic activity of steroid metabolites in mice.

The per cent Fe(59) incorporation into circulating erythrocytes as an indicator for new hemoglobin production was used in the polycythemic exhypoxic mouse system to study the effects of certain steroid hormone metabolites on erythropiesis. Enhanced Fe(59) incorporation was observed after the administration of several metabolites with a 5beta-H configuration, while those with a 5alpha-H configuration had no stimulatory effect. The stimulatory effect in avian systems has been shown by others to be due to an increased activity of delta-aminolevulinic acid synthetase, the limiting enzyme in heme biosynthesis. These in vivo studies thus indicate that in this mouse system, as in previously reported studies with avian and human bone marrow cells, some steroid metabolites stimulate hemoglobin synthesis. The observation that the same structure-junction relationship exists in the currently described system as in the avian suggests that these steroids probably induce delta-aminolevulinic acid synthetase in the mouse. The erythropoietic action of these nonandrogenic steroid metabolites may prove to be clinically useful.