Regulation of transferrin, transferrin receptor, and ferritin genes in human duodenum

GASTROENTEROLOGY

1992;102:802-809

Regulation of Transferrin, Transferrin Receptor, and Ferritin Genes in Human Duodenum ANTONELLO GIOVANNA MARIO

PIETRANGELO, CASALGRANDI,

PERINI,

EZIO

EMIL10 ROCCHI, GIAMPIERO RIGO,

VENTURA,

and GAETANO

ALBERT0

FERRARI,

CAIRO

Clinica Medica III e Terapia Medica, Cattedra di Gastroenterologia, University of Modena, Modena, and Centro di Studio della Patologia Cellulare de1 Consiglio Nazionale delle Ricerche, Milan, Italy

To gain insights at the molecular level into the expression of iron-regulated genes [transferrin (Tf), transferrin receptor (TfR), and ferritin H and L subunits] in human intestinal areas relevant to iron absorption, the steady-state levels of specific messenger RNAs (mRNAs) were analyzed in gastric and duodenal samples obtained from 6 normal subjects, or 10 patients with anemia, 14 patients with untreated iron overload, and 6 patients with various gastrointestinal disorders. No Tf mRNA was detected in human gastroduodenal tissue, confirming earlier findings in the rat. In normal subjects, although higher levels of ferritin H- and L-subunit mRNAs were consistently found in duodenal than in gastric samples, no differences in the content of TfR transcripts were detected. However, a dramatic increase in TfR mRNA levels was specifically found in duodenal samples from subjects with mild iron deficiency but severe anemia. This response of the TfR gene is presumably secondary to decreased cellular iron content due to its accelerated transfer into the bloodstream, as also indicated by the low levels of ferritin subunit mRNAs found in the same tissue samples, and is not linked to faster growth rate of mucosal cells because no changes in duodenal expression of histone, a growth-related gene, were detected. In patients with secondary iron overload, a down-regulation of duodenal TfR gene expression and a concomitant increase in ferritin mRNA content were documented. On the contrary, a lack of TfR gene down-regulation and an abnormally low accumulation of ferritin H- and L-subunit mRNAs were detected in the duodenums of subjects with idiopathic hemochromatosis. Whether these molecular abnormalities in idiopathic hemochromatosis are relevant to the metabolic defect(s) of the disease is presently unknown.

ransferrin (Tf), transferrin receptor (TW), and ferritins are key proteins in iron metabolism involved in serum transport, cellular uptake, and tissue storage of the metal, respectively.’ Iron uptake involves Tf-iron binding to its specific cellular receptor (TW), Tf-TfR complex entering the cell by endocytosis, and apotransferrin-TfR recycling after iron release.24 Inside the cell, iron is stored as soluble ferritin, a multimeric protein composed of heavy (H) and light (L) chains5 A great deal of data are available on the expression of Tf, TW, and ferritin genes in different cell types under normal or abnormal iron-balance conditions.5-8 However, little is known about the regulation of their expression in the intestinal (e.g., duodenal) epithelial cell, which, as the main physiological site of iron absorption, has a central role in governing body iron homeostasis.g*‘O The Tf/TfR cycle, although not involved in the uptake of iron at the luminal membrane,“~‘2 is likely to be actively functioning at the basolateral membrane of the enterocyte, where the TfR has been specifically localyzed.13 A possible role for mucosal ferritin in the control of iron absorption has been also envisaged. In fact, as originally proposed by Hahn et a1.14 and Granick,15 duodenal ferritin might function as an iron acceptor and block absorption of unwanted iron. Although this model remains unproved,16*17 the hypothesis that the amount of mucosal ferritin dictates the extent of iron absorbed by the enterocyte is still intriguing.‘* Recent immunological and immunohistochemical studies examined separately TfR1g*20and ferritin21*22 expression in intestinal tissue obtained from sub-

T

0 1992 by the American Gastroenterological Association 0018-5085/92/$3.00

March 1992

jects with normal or altered iron status. However, no molecular biological studies are available regarding the expression of all iron-regulated genes in the small intestines of individual subjects. The molecular hybridization approach offers some advantages such as the use of highly sensitive techniques and the possibility of directly measuring early gene products [i.e., messenger RNAs (mRNAs)]. This may help us understand the molecular basis for metabolic processes as well as mechanisms responsible for changes in the amount of final gene products (i.e., protein), which is particularly important in studying the expression of genes, like ferritin, undergoing a multistep pretranslational, translational, and posttranslational regulation.5-8*23 The availability of cloned molecular probes allowed us to analyze the steady-state levels of Tf, TfR, and ferritin-subunit mRNAs in the same duodenal samples obtained from normal subjects and patients with various disorders of iron metabolism (anemia or primary or secondary iron overload). Materials and Methods Subjects Thirty-eight subjects undergoing upper gastrointestinal endoscopy were selected for the study. All subjects gave informed consent. The study was in accordance with ethical committee guidelines of the University of Modena and was approved by the committee. The subjects studied were in four groups. Experimental groups. GROUP A (N = 10)(ANEMIA). Group A included patients with iron-deficiency anemia (IDA) (5 male and 5 female) suffering from menorrhagia (n = 4), peptic ulceration (n = l), duodenal polyp (n = l),or bleeding from esophageal varices (n = z), duodenal ulcer (n = l),or duodenal leiomyoma (n = 1). GROUPB(N = 14)(IRONOVERLOAD). SubjectsingroupB had idiopathic hemochromatosis (IH) (8 male) or secondary hemochromatosis (SH) (4 male and 2 female; porphyria cutanea tarda, n = 3, or alcoholic liver disease, n = 3). In these patients, liver iron content was also determined either morphologically by staining liver biopsy specimens for iron (Perls’ Prussian blue) or by atomic absorption spectrophotometry, as previously described.24 The diagnosis of IH was based on absence of known causes of secondary iron overload and on iron status evaluation in serum and liver samples (Table 1).25 Hepatic iron content was 267 + 61 kmol/g dry weight in subjects with IH and 57 + 18 pmol/g dry weight in patients with SH. The reference range for liver iron content was O-24 pmol/g dry weight, as evaluated in four subjects undergoing minor abdominal surgery without abnormalities of iron metabolism. Porphyria cutanea tarda was confirmed in subjects with typical photocutaneous lesions by uroporphyrin analysis using a high-pressure liquid chromatography method.z6 No patient studied was receiving phlebotomy therapy at the time of molecular analyses. Control groups. GROUPc (N = 8) (GASTROINTESTINAL

DUODENAL EXPRESSION OF IRON GENES

DISORDERS). Because

803

some patients in groups A and B suf-

fered from different gastrointestinal disorders, biopsy specimens were also taken from patients with various gastrointestinal diseases (6 male and 2 female) but normal iron balance (chronic liver disease, n = 4; gastric ulcer, n = 2; duodenal ulcer, n = 2). GROUP D (N = 6)[CONTROL).This group included normal volunteers (4 male and 2 female) with negative endoscopic examination results and with no abnormalities of iron metabolism.

Methods Processing of biopsy specimens. Multiple biopsy specimens were taken from normal-appearing mucosa of the stomach and distal duodenum of each patient and either dropped in liquid nitrogen and stored at -80°C before RNA extraction (see below) or fixed in a 4% solutton of paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) and embedded in paraffin. Five-micrometer sections were subsequently cut and stained with H&E for histopathological examination. RNA extraction and Northern blot analysis. Total RNA was obtained using the acid guanidium thiocyanatephenol-chloroform extraction described by Chomczynski and Sacchi.‘? Ten micrograms of total RNA was heat denatured for 5 minutes at 65°C in buffer containing 50% formamide, 2.4 mol/L formaldehyde, and 1X MOPS and applied to a 1.2% agarose/formaldehyde gel made up in 1X MOPS [10X MOPS is 0.2 mol/L 3-N-(morpholino)propanesulfonic acid, 50 mmol/L sodium acetate, and 10 mmol/L Na,-ethylenediaminetetraacetic acid), pH 7.4. The gel was run in 1X MOPS, pH 7.4, at 80 V for 4 hours. The gel was subsequently transferred to Hybond C extra filters (Amersham, Little Chalford, Bucks, England) in 10X SSC (20X SSC is 3 mol/L NaCl and 0.3 mol/L sodium citrate) by capillary action and baked at 80°C for 4 hours. The blots were prehybridized for 6 hours at 42°C in 50% formamide, 5X SSC, 50 mmol/L sodium phosphate, pH 6.5, 1X Denhardt’s solution (100X Denhardt’s solution is 2% bovine serum albumin; 2% polyvinylpirolidone, mol wt 360,000; and 2% ficoll, mol wt 400,000) and 100 pg/mL denatured salmon-sperm DNA. Hybridizations were performed in the same solutions with 2-3 X lo6 cpm/mL of probe for 20 hours at 42°C. The DNA probes were labeled by nick translation (Amersham), according to the manufacturer’s directions, using [32P]dCTP (specific activity, 3000 Ci/ mmol) to obtain a specific activity of 4-8 X 10’ cpm/pg DNA. After hybridization, filters were washed three times at room temperature in 2X SSC, 0.1% sodium dodecyl sulfate (SDS), for 5 minutes, then once at 42°C in 0.2X SSC, 0.1% SDS, for 30 minutes, and finally twice at 42°C in 0.1X SSC, 0.1% SDS, for 30 minutes. After washing, filters were exposed to autoradiography at -80°C on Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY), using Du Pont Lightening Plus intensifier screens (Du Pont Company, Wilmington, DE). For quantitative determinations, autoradiograms were scanned using a laser densitometer (LKB, Ultroscan; LKB Diagnostics, Piscataway, NJ), making sure that the exposure of the films was in the linear range. The values were corrected by the amount of ribosomal RNA

804 PIETRANGELO ET AL.

Table

1. Duodenal

Subjects Group A IDA Hb > loo g/L Hb < 70 g/L Group B IH SH Group C Gastrointestinal diseases Group D Normal

Expression

GASTROENTEROLOGY Vol. 102, No. 3

of TfR and Ferritin

L- and H-Subunit

Hb

Serum iron

n

Age (yr)

(g/L)

bg/dL)

10 6 4

42 f 11 30 f12 53 +13

90 * 2ob 109-t8' 61 Z!I 6b

8 6

46 f12 61+19

8 6

Genes in Relation

to Iron Status

Tf saturation (%I

Serum ferritin Gug/Ll

28.9f lib 26.6f Sb 32.2f 15b

14 f4a 1224" 17 + 2

16.6f lob 9.8f 6b 23.7f 7b

2.82zk1.2' 0.50+ 0.3" 0.42-t0.3' 1.87+ 0.1' 0.28+ O.lb 0.21+ O.lb 4.25f 0.6b 0.73zk0.2O 0.71f 0.3"

135 L 21 123 +lO

190f43b 149 f14b

89+10b 61f12b

2800 f 1460b 497 +194b

1.91* 0.3" 0.40+ 0.3b 0.41+ 0.3b 0.53f o.3a 2.08-+0.3b 1.89+ 0.3b

47 *15

126+15

70.2f 18

21 + 5

91.8+ 21

1.10* 0.3

1.13_+0.4

1.20+ 0.6

49 +19

134 +11

80.6+ 5

26f8

126k 38

1.17+ 0.1

1.10+ 0.2

1.19f 0.1

NOTE. Values are mean f SD. Significantly

H

(arbitrary densitometric

different from control: “P < 0.05; bP < 0.001(Kruskal-Wallis

(rRNA) in each lane as determined by rehybridization of the same blot to the pXCR7 probe for rRNA. Probes. The pFr and Fr 3 clones for the L and H subunits, respectively, of human ferritin are almost-fulllength complementary DNAs (cDNAs) that have been shown to recognize specifically ferritin mRNAs.28*2gThe Tf 23 cDNA for human transferrin encodes the protein from aa 98 through the C terminus reaching the poly A tai1.30,31 The pTRl0 clone is a 3.7-Kb cDNA in the pEMBL 8 vector, reconstructed from overlapping TfR cDNAs, which encompasses the complete TfR-coding region.32 The pXCR7 probe contains a Xenopous rDNA unit.33 The histone H3 cDNA recognizes specifically histone H3 mRNA.34 The ferritin cDNAs, the Tf and TfR clones, the probe for rRNA, and the histone H3 probe were generous gifts of R. Cortese (Heidelberg, Germany), S. Sidoli (Milan, Italy), C. Schneider (Trieste, Italy), I. Bozzoni (Rome, Italy), and S. Ferrari (Modena, Italy), respectively. Assessment ofiron status. In all subjects, hemoglobin, serum iron, total iron-binding capacity, and transferrin saturation were assessed using standard automated laboratory methods. Ferritin was measured by a commercial radioimmunoassay kit using 1311-ferritin as a tracer (Becton-Dickinson & Co., New York, NY).

L

TfR

units)

test).

of RNA samples from subjects representative of each group are shown in Figures 2 and 3. Quantitative analyses of all densitometric data are summarized in Table 1. No appreciable differences in the expression of Tf, TfR, and ferritin genes were detected between subjects in groups C and D. Tf Gene Expression No signal for Tf mRNA was detected after hybridization of total RNA extracted from gastric and duodenal tissue specimens of subjects with normal or abnormal iron balance conditions. Figure 1A shows that no signal for Tf mRNA is found in gastrointestinal samples, whereas a strong signal is detectable in human liver RNA. Hybridization of the same filter with a probe for rRNA showed that equal

A

6

a!?

Statistical Analysis Biochemical ble 1 are expressed

and densitometric

data shown

in Ta-

as means f SD. Significant differences in any mean values between control and patient populations were evaluated using the Kruskal-Wallis test with multiple comparisons.35

Results Histological examination of the biopsy specimens did not show appreciable abnormalities in the subjects selected for the study except for mild chronic superficial gastritis or duodenitis in a few patients in groups A, B, and C. The biochemical parameters of subjects included in the study are listed in Table 1.The Northern blots

L

S

D

L

S

D

Figure 1. Tf mRNA steady-state gastric tissue obtained

level in liver, duodenal, and from normal subjects. Total cellular RNA

(10 pg) isolated from liver, duodenal, and gastric tissue samples was electrophoresed for 4 hours at 80 V in a 1.2% agarose gel under denaturing conditions and blotted to nitrocellulose filters. (A)Autoradiograms of the blot hybridized with the Tf23 cDNA probe for human Tf. (B)Autoradiograms of the same blot used in

A rehybridized

with the pXCR7 cDNA probe for the 18 S rRNA. L, liver; S, stomach; D, duodenum.

DUODENAL

March 1992

amounts of undegraded lane (Figure 1B).

EXPRESSION

OF IRON GENES

in the control population. Among iron-overloaded patients, an increase in ferritin L mRNA was detected in subjects with SH but an abnormally low level of ferritin L transcripts was found in patients with IH (Figure 3). Variations of ferritin H gene expression followed a trend similar to that of ferritin L gene expression in all groups. Changes of ferritin subunit mRNA content in gastric samples appeared to be similar to those found in duodenal specimens.

RNA were loaded in each

TfR Gene Expression Table 1 and Figure 2 show an increase in the steady-state level of duodenal TfR mRNA in patients with IDA compared with the control population. If iron-deficient subjects are grouped according to hemoglobin level, the highest TW gene activation is appreciable in those patients with hemoglobin levels of ~70 g/L (Table 1).Interestingly, the activation of TfR gene expression in these patients is evident only in duodenal samples (Figure 2). In fact, no significant differences in gastric TfR mRNA were found among the various groups except for a lower level in patients with iron overload (Figure 2). In the latter group, patients with IH had more duodenal TfR mRNA than controls and, particularly the SH group (P < O.O05), who showed a down-regulation of TfR gene expression (Figure 2).

Histone Gene Expression Because of the strong correlation between TfR gene expression and cell proliferation,36 we analyzed the expression of a cell-cycle-related gene in the same tissue samples to address whether the difference in TfR mRNA levels found in anemic patients could arise from changes in the growth rate of the intestinal cells. Histone H3 mRNA levels were unchanged in all samples except for a strong increase in the gastric sample of a patient with gastric ulcer (Figure 2).

Ferritin H and L Gene Expression In the present study, normal subjects showed a typical pattern of ferritin L gene expression in the upper gastrointestinal tract and a consistently higher mRNA level in the duodenum (Figure 3). The same was true for ferritin H-subunit mRNA. Ferritin L mRNA accumulation was significantly lower in duodenal samples of patients with IDA than

Discussion In this study we found similar expression of TfR gene in gastric and duodenal samples obtained from normal subjects. On the contrary, higher levels of ferritin subunit mRNAs were consistently de-

TfR Figure 2. TfR and histone H3 mRNA steady-state levels in gastric and duodenal tissue obtained from normal, anemic, and siderotic subjects. Total cellular RNA (10 pg) isolated from gastric (l-6) and duodenal (7-12) biopsy specimens was electrophoresed for 4 hours at 80 V in a 1.2% agarose gel under denaturing conditions and blotted to nitrocellulose filters. Autoradiograms of the blots hybridized with specific probes, as noted on the left, are shown. 1 and 7, control: 2 and 8, gastric ulcer; 3 and 9, IDA (Hb > 100 g/L); 4 and 10, IDA (Hb < 70 g/L); 5 and 11, IH; 6 and 12, SH.

1

2

3' 4

5

6

7

8

7

8

9

9

ICI II 12

at I

H3 12

3

4

805

5

6

loll

12

28s

806

PIETRANGELO ET AL.

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2

3

GASTROENTEROLOGY Vol. 102, No. 3

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91011

12

-18s

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tected in tissue samples obtained from distal duodenum. This may indicate that under basal conditions, only ferritin gene activity is sensitive to the increased duodenal iron traffic due to the absorptive process. In this context, mucosal ferritin may represent a “passive” compartment complexing iron not rapidly transported to the bloodstream. In a recent immunohistochemical study examining gastrointestinal TfR expression,l’ a higher intensity of duodenal villous staining for TfR in patients with IDA was detected. However, hemoglobin levels of anemic patients ranged from 55 to 183 g/L in that study, and individual TfR patterns of iron-deficient subjects with normal or abnormal hemoglobinemia were not shown. In the present study, most of the activation of TfR gene expression found in iron-deficient patients is accounted for by subjects with severe anemia. In this context, it is known that conditions associated with anoxic hypoxia or anemia induced by blood loss are typically associated with a dramatic increase in iron absorption.3741 Moreover, experimental evidence suggests that enterocytes can respond directly to the hypoxic stimulus independently of changes in plasma iron turnover.” This

910

II12

Figure 3. Ferritin L- and Hsubunit mRNA steady-state levels in gastric and duodenal tissue obtained from normal, anemic, and siderotic subjects. legend to Figure 2 for details.

See

would indicate that TfR activity is preferentially enhanced in conditions with increased iron absorption due to severe anemia. However, it appears unlikely that TfR has a direct role in the uptake of iron at the luminal membrane as proposed,42 because TfR is absent in the brush border of human duodenal epithelial cells’3,43 and conflicting data have been presented on the ability of 5QFe-transferrin to donate its iron to the mucosal cells.44*45It appears more likely that TW, preminently localized in the basal-lateral membrane region of crypt and villous cells,‘3~1Q~20~43 might facilitate entry of iron into the cell from plasma by internalizing plasma-derived diferric Tf. Although Tf has been identified in human duodenal mucosal cells,22*46our molecular analyses were unable to detect by “Northern blot” any mRNA population specific for Tf, thus excluding an appreciable local production of the protein. This finding confirms earlier evidence in the rat.47 Thus, Tf is probably derived from the plasma and has a role in transferring iron into the mucosal cell from the bloodstream. What is the factor(s) responsible for TfR activation in anemia? Because TfR induction is associated with cellular proliferation,3” higher expression of TfR in

DUODENAL EXPRESSION

March 1992

anemia might serve to supply iron for the growth and turnover of proliferating epithelial cells in the crypt, thus representing an “index” of increased proliferating activity of mucosal cells.1g*20However, the lack of difference in the level of histone H3 mRNA, whose expression parallels the percentage of actively proliferating cells, between anemic and control population suggests that no major differences exist in the growth rates of intestinal cells among the subjects studied. On the other hand, it is likely that during anemia, the expression of TfR still depends on the cellular iron status: while iron absorbed in greater amounts by the enterocyte is rapidly diverted to the bloodstream, the reduction of the intracellular iron pool might activate TfR gene expression in an attempt to restore cellular iron balance. In our patients with severe anemia due to bleeding from the gastrointestinal tract (hemoglobin < 70 g/L), in concomitance with a dramatic activation of TfR gene expression, we found a decrease in ferritin mRNAs less severe than that found in chronically iron-deficient patients (Hb > 100 g/L). A possible explanation for this finding is that the hemorragic state induces very rapid changes in the intracellular “labile” iron pool, which rapidly affects the steady-state level of the short-lived TfR mRNA48 but not that of the relatively stable ferritin mRNA population.4g’50 Results obtained in iron-overload states support this interpretation of the mechanisms responsible for TfR activation in anemia. A down-regulation of duodenal TfR expression was observed in subjects with SH. In the same intestinal samples, significantly higher ferritin H- and L-subunit gene expression was appreciable. This represents the usual response activated by epithelial cells when a cellular iron load must be faced: the self-control of cellular iron metabolism is accomplished by a concerted translational regulation of mRNAs which encode two metabolically related proteins (i.e., TfR and ferritin) to further decrease uptake of iron and to complex the metal in a nontoxic form.51 No decrease in TfR mRNA was seen in IH, although it was expected on the basis of the increased body iron store. This finding is in agreement with the results of previous biochemical studies.‘g~20 Under these conditions, we also detected a low accumulation of ferritin transcripts, whereas immuno logical analyses found that ferritin content is unchanged’l or even increased” in duodenal homogenates It is possible that the higher sensitivity of the molecular hybridization technique, which, for instance, makes it possible to detect ferritin gene products in normal gastric mucosa whereas other biochemical techniques do not,** and the use of an “internal” control for hybridization (e.g., rRNA signal) also enhanced the accuracy of quantitative eval-

OF IRON GENES 807

uations. Furthermore, our results better agree with the reported absence of ferritin staining in duodenal absorptive cells in IH as evaluated by immunohistochemistry.” In conclusion, our molecular data support the implication that in subjects with IH, duodenal ferritin and TfR mRNAs are still modulated in concert, which may indicate that in IH these genes maintain their iron-dependent regulation mainly conferred by a common structural motif in the untranslated region of mRNAs [iron-regulatory elements (IREs)] and exerted by IRE-binding proteins51 In fact, the lack of TfR down-regulation as well as the reduced level of ferritin mRNAs may be secondary to the lower mucosal iron content caused by accelerated transfer of the metal into the bloodstream, as occurs in anemic patients. In this case, other biochemical defects (membrane iron carrier?) would primarily mediate such an accelerated flux of iron through the duodenal cell in IH, thus causing a “paradoxical” mucosal iron deficiency. On the other hand, it is possible that a tissue-specific abnormality might prevent iron from being “sensed” properly by the metabolically responsive IRE-binding proteins, thus “presenting” the cellular iron status to this regulatory system as iron deficient. This same abnormality could also be responsible, directly or indirectly, for an increased duodenal iron uptake or accelerated transfer of iron. Further studies on the role of membrane iron carriers or on the interplay of the cytosolic regulatory factors on a tissue-specific basis in IH may lead us to a greater understanding of the metabolic abnormalities of the disease. References 1.

2.

3.

4.

5. 6.

7.

8.

Aisen P, Listowsky I. Iron transport and storage proteins. Annu Rev Biochem 1980;49:357-393. Bleil JD, Bretscher MS. Transferrin receptor and its recycling in HeLa cells. EMBO J 1982;1:351-355. Klausner RD, Ashwell G, van Renswoude J, Harford JB, Bridges KR. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Nat1 Acad Sci USA 1983;83:2263-2366. Dautry-Varsat A, Ciechanover A, Lodish HF. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Nat1 Acad Sci USA 1983;80:2258-2262. Drysdale JW. Human ferritin gene expression. Progr Nucl Acids Res Mol Biol 1988:35:127-155. Arosio P, Cairo G, Levi S. The molecular biology of iron-binding proteins. In: de Sousa M, Brock JH, eds. Iron in immunity, cancer and inflammation. New York: Wiley, 1989:55-79. Cairo G, Tacchini L, Schiaffonati L, Rappocciolo E, Ventura E, Pietrangelo A. Translational regulation of ferritin synthesis in rat liver. Effects of chronic iron overload. Biochem J 1989;264:925-928. Pietrangelo A, Rocchi E, Schiaffonati L, Ventura E, Cairo G.

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Received January 28,199l. Accepted August 5,1991. Address requests for reprints to: Antonello Pietrangelo, M.D., Ph.D., Clinica Medica III e Terapia Medica, Policlinico Universitario, Via de1 Pozzo 71, 41100 Modena, Italy. This work was made possible by a generous gift from Professor Mario Coppo, Emeritus of Medicine, University of Modena. It was supported by grants from the Progetto Nazionale “Cirrosi epatica ed epatiti virali” (Unit of Modena) of the Minister0 dell’Universita’ e della Ricerca Scientifica e Tecnologica and from Consiglio Nazionale delle Ricerche. Presented in part at the Annual Meeting of the American Gastroenterological Association, New Orleans, Louisiana, May 1991, and published in abstract form (Gastroenterology 1991;100:700A). The authors thank Dr. P. Aisen for reading the manuscript and for helpful discussion.