Expression of transferrin receptors during differentiation of human placental trophoblast cells in vitro

Placenta(1992), 13,43-53

Expression of Transferrin Receptors Dwing Differentiation of Human Placenta1 Trophoblast Cells in Vitro M. L. KENNEDY”, G. C. DOUGLAS & B. F. KING Department of Cel1 Biology andHuman Anatomy, School ofMedicine, University of Califoornia, Davis, GY 95616-8643 ’ To whom cowespondence should be addressed Paperaccepted 11.6.1991

SUh4MARY In most cel1 types, transfewin receptor expression is cowelated with the proltferation rate, being increased by growth stimulation, or decreased by induction of terminal daflerentiation. In the human placenta the multinucleated syncytiotrophoblast, in direct contact with maternal bloed, is derived by dt~erentiation from mononucleated cytotrophoblast. In this study we examined changes in transfer+ receptor expression during in vitro diferentiation of trophoblast. Cells cultured in Ham ‘s/Waymouth S medium (HWM) remained primarily mononuclear throughout the study, whereas incubation in keratinocyte growth medium (KGM) led to formation of multinucleate masses within 2-3 days of culture. Cel1 surface binding of ‘251-labelled transfer& increasedfivefold between days 1-5 ofculture in both media and surface receptors were saturated at 7-14 pg/ml (90-ZOOm). At saturation, the amount of transfewin bound to syncytiotrophoblast was 37per cent lower than in cytotrophoblast. Scatchard analysis revealed a reduction in the number of surface transferrin receptors in syncytiotrophoblast compared to cytotrophoblast. A cowesponding 29 per cent reduction in the binding of transfwin to intracellular sites was observed in syncytiotrophoblast, Distribution of receptors between surface and intracellular sites was therefore similar in both cytotrophoblast and syncytiotrophoblast. The afinity of transferrin for transfer% receptors was 3.7-fold higher in syncytiotrophoblast when compared to cytotrophoblast. Observed di~erences between the two cel1 types were not due to the presence of growth factors or higher iron levels in KGM. Eqression of a high number of surfae transfèrrin receptors in syncytiotrophoblast (1.5 x 1012/mg protein), along with a high ajìnity of these receptors for iron-saturated transferrin, could help eqlain the eficient transport oflarge amounts of iron from mother tofetus. INTRODUCTION Human placenta1 villi are covered by syncytiotrophoblast in direct contact with maternal blood. Syncytiotrophoblast is a differentiated, multinucleated polarized epithelium that is 0143-4004/92/010043

+ 11 $OS.OO/O

@) 1992 Baillière Tindall Ltd

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Placenta(1992), Vol. 13

derived from undifferentiated, mononucleated cytotrophoblast (Enders, 1965). By virtue of its location, syncytiotrophoblast plays a crucial role in maternal-fetal transport of nutrients, including the transfer of iron to meet fetal demands. Circulating transferrin delivers iron to cells via receptor-medicated endocytosis. Iron-rich transferrin binds to a specific cel1 surface receptor, is internalized and enters into acidic vesicles where iron is then released. Transferrin and transferrin receptors are recycled back to the cel1 surface where transferrin dissociates from the receptor (Huebers and Finch, 1987; Ward, 1987). The presence of high numbers of transferrin receptors on the surface of cells is typically associated with the iron-demanding processes of cel1 proliferation or hemoglobin production. In contrast, differentiated cells usually express low numbers of transferrin receptors (May and Cuatrecasas, 1985; Testa, 1985; Heubers and Finch, 1987, Ward, 1987; Kuhn, 1989). Notable exceptions include mature human macrophages (erythrocyte iron sequestration) and human syncytiotrophoblast (Testa, 1985; Huebers and Finch, 1987); transferrin receptor numbers in syncytiotrophoblast are comparable to transferrin receptor numbers in proliferating cells (Ward, 1987; van Dijk, 1988). Transferrin and transferrin receptors have been shown to be present in syncytiotrophoblast in vivo (reviewed by van Dijk, 1988) and more recently in cytotrophoblast in vitro (Douglas and King, 1990a), but nothing is known about transferrin receptor expression during trophoblast differentiation. The establishment of an in vitro system for inducing differentiation of human cytotrophoblast to syncytiotrophoblast (Douglas and King, 1990b) has provided an opportunity to study transferrin receptor expression during this differentiation process.

MATERIALS

AND

METHODS

Materials Human diferric transferrin was purchased from Boehringer Mannheim, Indianapolis, IN. Carrier-free Na’25I was obtained from the Amersham Corporation, Arlington Heights, IL. Hank’s balanced salt solution was obtained from Gibco, Santa Clara, CA. Waymouth’s MB 752/1 medium, Ham’s F12 medium, bovine serum albumin (essentially globulin free), ferric chloride (dissolved in pH 5-6 distilled water), and an antibiotic/antimycotic mixture were purchased from Sigma Chemical Company, St Louis, MO. Keratinocyte growth medium (KGM; Boyce and Ham, 1983) and keratinocyte basal medium were purchased from Clonetics Corp., San Diego, CA. Fetal bovine serum was purchased from Gemini Bioproducts Inc., Calabasas, CA. Disposable PD-10 columns were obtained from Pharmacia Fine Chemicals, Piscataway, NJ. Preparation of 1251-labelled Transferrin. Human diferric transferrin (0.3 mg) was reacted in a final volume of 1 ml with carrier-free Na12’I (0.3 mCi) in a glass via1 coated with 100 ,ug Iodo-Gen (1,3,4,6-tetrachloro-3,3diphenylglycoluryl, Pierce Chemical Co., Rockford, IL). The reaction was allowed to proceed for 10 min at room temperature after which the mixture was removed from the via1 and passed down a disposable Sephadex G-25 column (PD-10 column) equilibrated with phosphate-buffered saline, pH 7.0. The iodinated product was sterile filtered, bovine serum albumin added to 2 mg/ml, and stored at 4°C no longer than 2 weeks. Specific activity averaged 803 + 93 cpm/ng.

Kennedy et al: Transferrin Receptor Expression Dwing Trophoblast Differentiation

45

Preparation of trophoblast cells Trophoblast cells were isolated from normal, term placentas delivered by caesarian section. The isolation method involved tryptic digestion followed by Percoll gradient separation (see Douglas and King, 1989). A population of mononucleated cells was obtained of which less than 5 per cent were vimentin- and HLA-positive; the remainder were cytokeratin-positive and vimentin- and HLA-negative, typical of villous trophoblast.

Cel1 culture Cells were cultured either in a 1:l mixture of Ham’s F12 and Waymouth’s MB 725-1 medium (HWM) or commercial keratinocyte growth medium (KGM), a differentiationinducing medium consisting of keratinocyte basal medium supplemented with growth factors (epidermal growth factor, insulin, hydrocortisone). Fetal bovine serum was added to a final concentration of 10 per cent. Cells from the Percoll gradient were plated into 96-wel1 plastic multi-dishes (Falcon, Becton Dickinson, Cockeysville, MD) at 2.5 x 10’ cells/well (approximately 4 x 105 cells/cm2) and placed in an air/C02 (95:5) incubator at 37°C. Ifcells were plated longer than 48 h, the medium was removed and fresh medium added. Cel1 viability, as determined by measurements of LDH secretion and trypan blue exclusion, remained >95 per cent throughout 5 days of culture in either HWM or KGM (Douglas and King, 1990a, b). Where indicated, iron concentrations in the media were altered by addition of 17 k\í ferrous sulphate to HWM, or addition of diethylenetriaminepentaacetic acid (Sigma) at a concentration which reduced iron in KGM from 28 to 11 PM.

Binding of radiolabelled transferrin In the following procedures, bovine serum albumin (10 mg/ml) was substituted for fetal bovine serum. Before each experiment, endogenous transferrin was removed by washing cells three times with medium then pre-incubating in the same medium for 30 min at 37X, followed by three further washes in ice-cold medium. For studies requiring cel1 permeabilization, 0.5 per cent saponin was added for 15 min at 4°C prior to experiments (see Douglas and King, 1990a). Binding was measured by first precooling the cells on ice for 5 min, then adding radiolabelled transferrin (amounts are given in the figure legends) and continuing the incubation at 4°C. Non-specific binding was determined by adding a 250-fold excess of unlabelled diferric transferrin to a parallel series of wells (Hollenberg and Cuatreacasas, 1979; Klausner et al, 1983). Non-specific binding increased with increasing transferrin concentration, and accounted for 4-17 per cent and 7-36 per cent of total binding in HWM and KGM, respectively. Incubation volumes were maintained at 0.20 ml/well. The cells were then washed four times with 0.3 ml ice-cold medium, solubilized by the addition of 0.2 ml of 0.5 per cent sodium dodecyl sulphate (SDS) and counted for radioactivity in an LKB Autogamma counter. Addition or reduction/deletion of iron or growth factors did not directly affect the process of ‘251-labelled transferrin binding to the cel1 surface.

Protein determination The protein content of samples was determined using the Lowry method, modified by the inclusion of SDS in the alkaline copper reagent (Bennett, 1982). Bovine serum albumin was used as the reference standard. At al1 days measured, 2.5 x 106 cells were found to contain 1 mg protein in both HWM and KGM.

Placenta (1992), Vol. 13 (bl IO-

0-

7

14

21

28

0

7

14

21

28

Tf (pg/ml)

Tf (pg/rnl)

Figure 1. Binding of ‘251-labelled transferrin

to trophoblast cells. Cells were incubated for 4 days in HWM (a) or KGM (b), then incubated with different amounts (0.5-25 &nl) of 1251-labelled transferrin for 4 h at 4°C. Specific binding was determined as described in the Methods section. Results are mean values from three experiments. -, total; -A-, specific; ---íI---, non-specific. -.

Expression of results Where possible, results are given as means plus or minus the standard deviation for the number of experiments shown. Most experiments represent cells from a different placenta and within an experiment determinations were usually carried out in triplicate.

RESULTS Differentiation in culture The characteristics ofthese cells in culture have been described in detail previously (Douglas and King, 1990b) and are summarized briefly here. At day 1 in culture, cells began to spread and aggregate, forming colonies; there were no differences in morphology between cells grown in HWM or KGM as determined by desmoplakin and nuclear staining. By day 2, however, cel1 diameters were increased and multinucleation was evident in cells grown in KGM; by day 3-4, differentiated syncytiotrophoblast-like cells predominated (Douglas and King, 1990b). The vast major@ of cells grown in HWM remained mononuclear throughout 5 days in culture. Levels of chorionic gonadotropin, a biochemical marker of differentiation, were higher by day 2 of culture in KGM when compared with HWM, and 1 S-20-fold higher by day 4-5 of culture in KGM (see Douglas and King, 1990b). Surface binding Initial studies were designed to determine whether there were any differences in surface transferrin binding between cyto- and syncytiotrophoblast. Cells were cultured for 4 days in each medium before initiation of experiments. Cel1 surface binding, determined by incubating cyto- and syncytiotrophoblast cells with ‘251-labelled transferrin at 4°C reached equilibrium by 1-2 h (data not shown). Binding was concentration dependent, and was saturable at 7-14&ml (Figure 1). Maxima1 specific transferrin binding by syncytiotrophoblast cells was 40 per cent lower than that of cytotrophoblast. Having established a differente in surface binding of transferrin between cyto- and

liennecl~~et al: Tramf&rin Receptor Expression Dwing Trophoblast Differentiation 16

14 -

0

I

2

3 Tlme

4

5

6

(days)

to trophoblast cells during differentiation. Cells were binding of 1251-labelled transferrin incubated for various times (1-5 days) in HWM or KGM, then incubated with ‘251-labelIed transferrin (14 pg/ml) for 4 h at 4°C. Data represent specifïc binding, determined as described in the Methods section. Data are mean ralues from 3-4 experiments. 3, HWM; -. -A- -; KGM.

Figure 2. Surface

syncytiotrophoblast, we sought to determine at what time in culture these changes were occurring. Comparisons of surface transferrin binding between HWM and KGM at a transferrin concentration sufficient for maxima1 specific binding (14 pg/ml), are show-n in Figure 2. Differences in surface binding between cells grown in HWM and KGM were apparent at day 2. By 3-5 days of culture, maxima1 transferrin binding to surface receptor sites was 31 per cent lower in KGM compared to HWM. Further experiments were performed in an attempt to explain these differences. The concentration dependence oftransferrin binding to the cel1 surface was determined in order to provide information about transferrin receptor affinity and numbers of transferrin receptors (Figure 3). Differences in the number of binding sites in HWM versus KGM were apparent by day 2 [Figure 3(a)]. By day 3 and 4 the number of binding sites was 58 per cent lower in KGM compared to HWM. In contrast to changes in numbers of surface binding sites, the binding affinity of the cel1 surface for transferrin increased in cells cultured in KGM, but not until day 3 and 4 ofculture [Figure 3(b)]. In HWM, the Ka (apparent association constant) decreased with time in culture. By day 4, Ka values were 3.7-fold higher in cells grown in KGM when compared with HWM. Intracellular binding Intracellular transferrin binding was measured in saponin-permeabilized cells in order to determine whether there was a change in distribution of transferrin receptors between the cel1 surface and intracellular sites. Maxima1 transferrin binding to intracellular sites decreased with time in culture in KGM compared to HWM (Figure 4). In KGM the number of intracellular binding sites at 3-5 days of culture averaged 29 per cent lower than found for cells grown in HWM. However, the relative distribution of receptors between the surface and

Placenta (1992), Vol. 13

0

I

2

4

3

0

5

l

2

4

3

Time (days)

Tlme (days)

Figwe 3. Changes in cel1 surface binding sites (a) and afhnity (b) for transferrin

during trophoblast differentiation. Cells were incubated for 1 to 4 days in HWM or KGM, then incubated with different amounts (0.5-25 &ml) of ‘2SI-labelled transferrin for 3 h at 4°C. Specific binding data (see Methods section) were analysed by the method of Scatchard, and resulting kinetic values are shown. Data are means for triplicates from two experrments. -8-, HWM; -. -A- -; KGM.

1

z

5

‘)

5

6

Time (doys)

Cells were F&re 4. Intracellular binding of ‘251-labelled transferrin to trophoblast cells during differentiation. incubated for various times (1-5 days) in HWM or KGM, then incubated with 1251-labelled transferrin (14,~g/ml) for 4 h at 4°C. Intracellular binding was measured by subtiaction of surface binding from total binding following treatment with saponin (see Methods section). Data represent specific binding, determined as described in the Methods section. Data are mean values from 3-4 experiments. -8-, HWM; -. -A- -; KGM.

49

líennecl~~et al: Transjèrrin Receptor Eqression Dwing Trophoblast DiffPrentiation

intracellular sites was similar in both HWM and KGM at al1 days measured (Figures 2 and 4); surface binding accounted for 40 per cent of total binding in KGM, and 37 per cent in HWAI. Effect of growth factors and iron KGM contains growth factors (epidermal growth factor, insulin and hydrocortisone) which are not present in HWM, and also has higher iron levels (28 ,LL.V versus 11 PIL%,accounting for 10 per cent fetal bovine serum). Growth factors and iron are potential regulators of transferrin receptor expression (van Dijk, 1988; Huebers and Finch, 1987; Kuhn, 1989). To assess whether these factors accounted for the observed differences in transferrin binding, cells were cultured in modified media in which the concentrations of above agents were adjusted as described under Table 1. NO significant changes in surface or intracellular transferrin binding were observed upon addition or reduction/deletion of either iron or growth factors throughout 4 days of culture (Table 1). Even an iron concentration of 100 ~11 did not affect results (data not shown). Again, surface and intracellular specific binding of transferrin were significantly lower at day 4 in cells cultured in KGM as compared to HWM.

DISCUSSION A reduction in cel1 surface and intracellular transferrin binding was observed when trophoblast cells were cultured in KGM, a medium known to induce differentiation of cytotrophoblast to syncytiotrophoblast (Douglas and King, 1990b). Maxima1 cel1 surface binding of transferrin to transferrin receptors averaged 37 per cent lower in syncytiotrophoblast compared to cytotrophoblast. These results are in agreement with several other reports of reduced cel1 surface and/or total transferrin binding and transferrin receptor numbers during differentiation of a wide variety of cel1 types from several mammalian species (May and Cuatrecasas, 198.5; Testa, 1985; Huebers and Finch, 1987; Ward, 1987; Kuhn, 1989). There are some reports of either an increase or no change in transferrin-transferrin receptor

Table 1. Effect

of growth factors and iron on binding transferrin to trophoblast cells.

HamWWaymouth’s Medium: + growth factors + additional iron Keratinocyte Basal Medium: + growth factors reduced iron

of ‘2’I-labe11ed

Surface (pmoi/mg pro.)

Internal (pmoVmg pro.)

10.1 + 2.6 8.9 rt 2.8 9.9 rf- 1.0

13.6 f 1.4 13.7 + 3.0 12.9 + 1.6

7.0 + 2.2 7.2 k 1.0 6.8 f 1.2

10.4 i 3.2 9.0 * 2.2 10.1 f 1.8

Cells were incubated for 4 days in HWM or KGM. In addition, a parallel series of experiments were performed in which cells were plated and maintained in modified media. Modified HWM was supplemented with iron and growth factors. For modified KGM, the iron concentration was reduced as described in the Methods section, and growth factors were omitted. On day 4, cells were then incubated with 1251-labe11ed transferrin (14&ml) for 4 h at 4°C. Surface binding was distinguished from intracellular binding by comparing cells treated with and without saponin, as described in the Methods section. Results represent mean values for specific binding from 4-5 experiments.

50

Placenta(1992), Vil. 13

binding during cellular differentiation (Hamilton, Weiel and Adams, 1984; Sorokin, Morgan and Yeoh, 1987; Andreesen et al 1988; Leenen et al 1990; van der Ende et al, 1990). Differences observed between cel1 types and during differentiation may be related to differences in cellular iron needs and/or cel1 size. The timing of the changes in surface transferrin binding to trophoblast cultured in differentiation-inducing KGM correlated with biochemical and morphological differentiation (Douglas and King, 1990b). Differences in transferrin binding were first noted at day 2, at which time syncytiotrophoblast-like structures are forming in culture and differences in human chorionic gonadotropin secretion are becoming apparent. Changes in total cel1 transferrin-transferrin receptor binding paralleled those of surface binding. This suggests that the reduced surface binding was not due to an alteration in transferrin receptor distribution between the cel1 surface and the cel1 interior. Some investigations in other cel1 types have also found no change in transferrin receptor distribution during cellular differentiation (Frazier et al, 1982; Hamilton, Weiel and Adams, 1984; Enns et al, 1988). There are some reports of altered transferrin receptor distribution during differentiation of certain tumor-derived cel1 lines, including human choriocarcinoma (Hunt, Ruffin and Yang, 1984; Mulford and Lodish, 1988; van der Ende et al, 1990). The observed differences in transferrin-transferrin receptor binding between cytotrophoblast and syncytiotrophoblast were not due to differences in levels of potential transferrin receptor regulatory factors (iron and growth factors) between the two media. Complete removal or addition of growth factors over 4 days in culture had no effect on transferrin receptor expression; this is in contrast to the stimulatory effect of these same growth factors on chorionic gonadotropin secretion by trophoblast (Douglas and King, 1990b). However, we cannot rule out short-term alterations in trophoblast transferrin receptor expression in the presence of growth factors. In most studies where growth factors (epidermal growth factor or insulin) have been found to affect transferrin receptor expression, the effect occured within 1 h (Wiley and Kaplan, 1984; Davis and Czech, 1986; Ward and Kaplan, 1986). Iron concentrations varying from 11 to 28 +!I had no effect on transferrin binding to transferrin receptor. This may have relevante to the observation that iron transport to the fetus is relatively unaffected by alterations in maternal plasma iron concentrations during pregnancy (van Dijk, 1988). Maternal plasma iron at term is 7-13 ,UL~ versus 18-22 ,UU.~I in non-pregnant controls (van Eijk, 1978; Okuyama et al, 1985). Our laboratory is currently investigating the effects of a wider range of iron and transferrin concentrations on transferrin receptor expression in trophoblast. It is not known why the number of surface and intracellular transferrin receptors in both HWM and KGM increased with time in culture. Several studies have noted that the recovery of transferrin receptors following trypsin exposure was nearly complete within 1 day of culture (Ward, Kushner and Kaplan, 1982). Exposure of trophoblast cells to trypsin during the cel1 isolation procedure is therefore not the sole reason for the increase in transferrin receptor expression over several days of culture. This increase in transferrin receptor numbers was also unlikely to be due to low in vitro extracellular iron levels. Iron at levels higher than those found in plasma did not alter the results. It is possible that transferrin itself directly or indirectly affected transferrin receptor expression (May and Cuatrecasas, 198.5; Kuhn, 1989). The concentration of bovine transferrin in the medium and affinity of bovine transferrin for transferrin receptors were several-fold lower than would be found in human plasma in vivo (Testa, 1985; Huebers and Finch, 1987). This situation could potentially allow higher transferrin receptor synthesis by trophoblast in vitro. An interesting observation was that the affinity of transferrin for transferrin receptors was

h mtze~~,cr al: 7Tun~f>rrin Receptor Expression Dwing Trophoblast Differentiation

.i 1

up to 3.7-fold higher in syncytiotrophoblast when compared with cytotrophoblast. Several studies reported no change in transferrin receptor affinity during cellular differentiation C\.an Bockxmeer and Morgan, 1979; Yeoh and Morgan, 1979; Iacopetta, Morgan and Yeoh, 1982; Chitambar, Massey and Seligman, 1983; Sawyer and Krantz, 1986; Huebers and Finch, 1987; Sorokin, Morgan and Yeoh, 1987); other investigators have reported either an increase (Hunt, Ruffin and Yang, 1984; Leenen et al, 1990) or decrease (Andreesen et al, 1988) in transferrin receptor affinity during differentiation. Leenen et al (1990) proposed the possibility of two structurally different transferrin receptor populations of differing affini-. An increase in affinity of transferrin receptors for transferrin could be advantageous for iron sequestration by syncytiotrophoblast, which is in direct contact with maternal blood and therefore circulating transferrin. The Ka for the binding of human transferrin to syncytiotrophoblast was comparable to the Ka measured for transferrin receptors on brush border membrane vesicles from human placenta (Vanderpuye, Kelley and Smith, 1986; van Dijk, 1988) and in other cel1 types (Thorstensen, 1990), suggesting that the trophoblast receptor is functionally similar. One possible explanation for the high levels of transferrin receptors in trophoblast is for transport of iron unidirectionally from mother to fetus; fetal demands for iron are substantial, particularly in the later stages of human pregnancy where an average of 5 mg of iron per day is transported from mother to fetus (Fletcher and Suter, 1969). It is also possible that the placenta, like the hepatocyte, plays a role in iron storage since both ferritin (human) and transferrin receptor (guinea pig and rabbit) numbers increase during gestation (Okuyama et al, 1985; van Dijk, 1988). Although there are lower numbers of transferrin receptors on the surface of syncytiotrophoblast when compared with cytotrophoblast, it cannot be determined from this studl whether there is an altered distribution of receptors such that, for example, more reside on the apical plasma membrane surface (involved in sequestering transferrin from maternal blood) as opposed to the basal side of syncytiotrophoblast. However, kinetic binding eiiperiments using brush border and basal cel1 membrane vesicles derived from syncytiotrophoblast yielded a fivefold higher number of transferrin receptors on apical cel1 surface when compared to basal surface vanderpuye, Kelley and Smith, 1986). A slightly higher (20 per cent) transferrin receptor affinity was found on the basal membrane vesicles, but this u-as directly attributed to lower levels of isotope dilution when compared to brush border membrane vesicles. Thus, it may be that during syncytiotrophoblast differentiation transterrin receptors of high affinity are concentrated in the apical cel1 membrane where they could function advantageously in the receptor-mediated endocytosis of transferrin from maternal hlootl.

ACKNOWLEDGEMENTS \Ve are grateful to Twanda Thirkill and Hendrik Hakim for expert assistance in cel1 isolation procedures.

Tissue was made available to US through the cooperation of the medical and nursing staff at Sutter Memorial Hospita& Sacramento, C.4. This nork was supported by NIH grand HD 11658 and NIH training grant HD 0713 1.

REFERENCES Andreesen, R., Sephton, R. G., Gadd, S., Atlcins, R. C. & De Abrew, S. (1988) Human macrophage maturation in vitro: Expression of functional transferrin binding sites of high affinity. Blut, 57, 77-83. Bennett, J. P. (1982) Solubilization of membrane-bound enzymes and analysis ofmembrane protein concentration.

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In Techniques in Lipid and Membrane Biochemisty (Eds) Hesketh, T. R., Komberg, H. L., Metcalfe, J. C., Northcote, D. H., Pogson, C. 1. & Tipton, K. F. pp. 1-22. Shannon/New York: Elsevier Biomedical Press. Boyce, S. T. & Ham, R. G. (1983) Calcium-regulated differentiation of normal human enidermal keratinocvtes in Chemically defined clonal culture and serum-free serial culture.JournalofInaestigative~ennatology, 81,33&4Os. Chitambar, C. R., Massey, E. J. & Seligman, P. A. (1983) Regulation of transferrin receptor expression on human leukemie cells during proliferation and induction of differentiation. 3ofozrmalof Clinical Inaestigation, 72, 1314-1325. Davis, R. J. & Czech, M. P. (1986) Regulation of transferrin receptor expression at the cel1 surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor. EMBO-fournal, 5,653-658. Douglas, G. C. & King, B. F. 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(1988) Endocytosis of the transferrin receptor is altered during differentiation of murine erythroleukemic cells.3oumal ofBiological Chemistry, 263, 5455-5461. Okuyama, T., Tawada, T., Furuya, H. & Villee, C. (1985) The role of transferrin and ferritin in the fetalmaternal-placenta1 unit. AmericanJoumal cf Obstetricsand Gynecology,152, 344-350. Sawyer, S. T. & Krantz, S. B. (1986) Transferrin receptor number, synthesis, and endocytosis during erythropoietin-induced maturation of Friend virus-infected erythroid cells. Jmrmal of BiologicalChemistry, 261, 9187-9195. Sorokin, L. M., Morgan, E. H. & Yeoh, G. C. T. (1987) Transferrin receptor numbers and transferrin and iron uptake in cultured chick muscle cells at different stages of development.Jouma/ of Cellular Physiologv,13 1,342353. Testa, U. (1985) Transferrin receptors: Structure and function. Current Topicsin Hematology,5, 127-16 1. Thorstensen. K. & Romslo. 1. (1990) The role oftransferrin in the mechanism of cellular iron uutake. 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