Brief Critical Reviews
November 1992: 329-337
New Insights into Placental Iron Transport The transport of iron from mother to fetus presents a number of challenges, none more perplexing than determining how the flow can be unidirectional, yet avoid mixing the mother's serum proteins with the fetal system. Studies with cultured cell lines suggest a possible mechanism by which this may be achieved.
Fetal Iron Availability
All fetuses, regardless of species, require vast quantities of iron for normal development. In species such as the badger, cat, sheep, and seal (see ref. 1 and references therein), the placenta is involved in active phagocytosis of maternal erythrocytes. However, in most mammalian species, fetal iron is derived from maternal transferrin.* To be absorbed by the fetus, iron must penetrate the placental trophoblasts (the syncytiotrophoblast), a polarized layer of fused cells that segregates the maternal and fetal circulations, and move to the fetal side of the memb ~ - a n e Placental .~ trophoblasts contain large numbers of transferrin receptors on both the maternalfacing apical surface and the fetal-facing basal plasma membrane3 as well as an internal transferrin receptor po0L4 Iron delivery is a two-step mechanism in which holo transferrin is first endocytosed and then returned as apotransferrin to the cell surface. A transcytotic movement of protein could endanger the fetus if the mother's transferrin were to emerge with membrane proteins on the fetal side. Experimental data, however, suggest that only the iron, and not the maternal iron-transporting protein, passes from mother to fetus.' How, then, is maternal transferrin kept from commingling with fetal transferrin, and what prevents maternal transferrin from exiting on the fetal side? Why are fetal transferrin receptors so much more numerous than maternal receptors? These are mysteries of placental iron transport that have yet to be resolved. Placental epithelial cells fit the phenotype of most epithelial cells, having both apical and basolateral surfaces. This design allows unidirectional transport of iron (and other nutrients). The amount This review was prepared by Edward D. Harris, Ph.D., at the Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843. Nutrition Reviews, Vol. 50, No. 11
of iron that crosses the placenta depends on two factors: the number of transferrin receptors on the apical side6 and the level of ferritin within the placental cells.' High cellular iron results in downregulation of transferrin membrane receptors, whereas low cellular iron increases expression of receptors. Thus, placental iron availability is regulated by the iron needs of the fetus.6 Down-regulation of receptor also protects a fetus against iron overload from the mother. In a similar way, placental ferritin in cytotrophoblasts and fetal endothelial cells carefully regulates the inward flux of iron into the fetus's circulation. Both mechanisms would seem to be designed to keep maternal and fetal iron circulations in tune with one another, maintaining a continual supply of iron in proper amounts for fetal development.
'
Mechanisms of Placental Iron Transport
Investigators looking at the mechanism of iron transport have turned to cell cultures for answers. BeWo cells, a human chorion carcinoma cell line first described by Pattilo and Gey ,' have been used extensively. These cells display the phenotypic properties of placental trophoblasts, including receptor-mediated uptake and release of iron.' BeWo cells achieve a unique polarity when grown on polycarbonate membrane filters.' Their morphology is typical of placental trophoblasts in that they have microvilli on the apical membrane surface, with large nuclei in the center and Golgi stacks distributed in the perinuclear cytoplasm. Results of studies using '251-transferrin' have shown that BeWo cells display receptors for transferrin on both the apical and basolateral surfaces; the ratio was 1:2, with the basolateral surface (fetus) having more. Both receptors showed identical binding constants for transferrin. Maintaining a separation between fetal and maternal proteins is a function of the placental barrier. Segregation, however, is rendered more difficult by having both fetal and maternal transferrins deliver iron to the same cell, but from opposite sides. The situation is further complicated by having both transferrins penetrate the membrane. It would seem inevitable that fetal and maternal transferrins must meet somewhere in the cell, but this does not happen. Cerneus and van der Ende' recently showed 329
that BeWo cells cycle transferrin through two noninteracting pools in the placenta. They found no evidence for mixing despite accessing of transferrins from both sides. Thus, a solution to the dilemma seems to be for cells to have two separate transferrin cycles. A hypothetical model for such a dual system is suggested in Figure 1. In the scheme, transferrin (abbreviated as Tf) with iron enters the apical side of the placental trophoblast. After the iron is released from the transferrin, the Tf (as apotransferrin) is recycled to the cell surface and released. Iron released within the cell is transferred to ferritin, from which it can readily become assimilated into fetal apotransferrin. This fetal transferrin then exits via the basolateral surface. The proposed scheme suggests that one ferritin pool may be accessible to both maternal and fetal transferrins. It should be noted that Vanderpuye et al.3 found the affinity of placental receptors at neutral pH to be much higher for diferric transferrin than for apotransferrin, making entry of apotransferrin from either side of the cell an unlikely occurrence. Nonetheless, an important key to the mechanism that needs investigation is whether maternal transferrin represents an entering pool of iron and fetal transferrin represents an exiting pool. A possible role for the monoferric
transferrin species in this event is also in need of clarification. If there are two transferrin cycles operating, what keeps the two cycles separate? A growing body of evidence supports the concept that secretory and polarized cells use specific routing and sorting signals to effect vectorial movement of molecules to different locations within the cell. For example, whether a newly made protein is retained in the endoplasmic reticulum" or is excreted from the cell" depends on specific sorting signals attached to the protein or its membrane-bound receptor. One of the more familiar signals is the mannose 6-phosphate signal that targets delivery of an unsuspecting protein passenger to lysosomes.12 A second discriminating factor is in the structure that anchors the protein to the membrane. Proteins bound via glycosyl-phosphatidylinositol (GPI) tend to be directed to the apical surfaces and are forcibly prevented from appearing at basolateral surfaces. l3 If one fuses a GPI anchor to a basolateral surface protein, that protein is automatically redirected to the apical Thus, routing is determined in part by the structure of the receptor that binds the protein. Future research should help to determine whether membrane-directing sorting signals exist for the transferrin receptors on the basolateral and apical surfaces. If such signals do exist, how do they differ, and how do they affect movement of transferrin and iron? Placental Transport of Other Minerals
Figure 1. Hypothetical model for unidirectional iron transport. Maternal iron is delivered to the apical surface as transferrin. The transferrin molecule is moved inside and the dissociated iron binds to fenitin. The iron-free (apo) transfenin departs the cell, completing the cycle on the maternal side. On the basolateral surface, apotransferrin enters and engages ferritin iron before departing as diferric transferrin. Femtin iron represents the point of communication between the two cycles. Unidirectionality is assured by having the fetal cycle remove the iron deposited by the maternal cycle. 330
The successes gained by studying placental iron transport have encouraged nutritionists to investigate placental transport of other essential metals. A basic question is whether all essential metals fit the same scenario as iron. For example, are there specific proteins that convey zinc, copper, and selenium through the placental barrier separating mother from fetus? Does a transferrin-like cycle exist for these metals? The data are far from complete, but there are suggestions that copper may have a maternal-fetal interacting transport system. Moreover, placental copper transport may mimic copper transport in other cells. This conclusion is based on observing that female mice with the mutant brindled allele (Mobr), which affects copper transport in intestine and kidney, have impaired placental transport of aCu.15 It is still not known why the gene for ceruloplasmin, a copper transport protein, is expressed in choroid plexus, yolk sac, and placenta of rats.I6 Human ceruloplasmin mRNA has been isolated from placentas, " and ceruloplasmin protein has been found in placental extracts.I8 Does this mean that placental copper transport relies on a plasma copper-binding protein synthesized in plaNutrition Reviews, Vol. 50, No. 11
centa? If so, how does placental ceruloplasmin integrate with plasma ceruloplasmin? Ceruloplasmin mRNA has also been found in the decidual layer, a layer of cells from the uterine endometrium that surrounds the implanted fetus. l9 Placental transport of iron has given us insights into the workings of placental cells, but much remains to be learned regarding what some may consider the premiere event in nutrition-nourishment of the developing fetus. Although iron, copper, zinc, and selenium are essential elements for fetal development, their presence can also be detrimental to fetal health if they are supplied in excessive amounts or in the wrong chemical form. All of these complexities must be considered in the development of a comprehensive model of placental transport of minerals. 1. Dumartin B, Canivenc R. Placental iron transfer regulation in the haemophagous region of the badger placenta: ultrastructural localization of ferritin in trophoblast and endothelial cells. Anat Embryo1 19923 85:17!5-9 2. van Dijk JP. Regulatory aspects of placental iron transfer: a comparative study. Placenta 1988;9: 215-26 3. Vanderpuye OA, Kelley LK, Smith CH. Transferrin receptors in the basal plasma membrane of the human placental syncytiotrophoblast. Placenta 1986;7:391-403 4. Douglas GC, King BF. Uptake and processing of '251-labelledtransferrin and 59Fe-labelledtransferrin by isolated human trophoblast cells. Placenta 1990;11:41-57 5. Contractor SF, Eaton BM. Role of transferrin in iron transport between maternal and fetal circulations of perfused lobule of human placenta. Cell Biochem Funct 1986;4:69-74 6. Bierings MB, Baert MRM, van Eijk HG, van Dijk JP. Transferrin receptor expression and the regulation of placental iron uptake. Mol Cell Biochem 1991;100:31-8 7. Pattilo RO, Gey GO. The establishment of a cell line of human synthesizing cells in vitro. Cancer Res 1968;28:1231-6
8. van der Ende A, du Maine A, Simmons CF, Schwartz AL, Strous GJ. Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron. J Biol Chem 1987;262: 8910-6 9. Cerneus DP, van der Ende A. Apical and basolatera1 transferrin receptors in polarized BeWo cells recycle through separate endosomes. J Cell Biol 1991 ;114: 1149-58 10. Jackson MR, Nilsson T, Peterson PA. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. Embo J 1990;9:3153-62 11. Munro S, Pelham HRB. A C-terminal signal prevents secretion of luminal ER proteins. Cell 1987; 48:899-907 12. Kornfeld S. Trafficking of lysosomal enzymes. FASEB J 1987;1:462-8 13. Lisanti MP, Caras IW, Davitz MA, RodriguezBoulan E.A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J Cell Biol 1989;109:2145-56 14. Brown DA, Crise B, Rose JK. Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science 1989;245: 1499-1 501 15. Mann JR, Camakaris J, Danks DM. Copper metabolism in mottled mouse mutants. Defective placental transfer of 64Cu to foetal brindled (Mobr) mice. Biochem J 1980;186:629-31 16. Aldred AR, Grimes A, Shreiber G, Mercer JFB. Rat ceruloplasmin. Molecular cloning and gene expression in liver, choroid plexus, yolk sac, placenta, and testis. J Biol Chem 1987;262:287!j-8 17. Yang F, Friedrichs WE, Cupples RL, et al. Human ceruloplasmin. Tissue-specific expression of transcripts produced by alternative splicing. J Biol Chem 1990;265:10780-5 18. Mas A, Sarkar B. The metabolism of metals in rat placenta. Biol Trace Ele Res 1988;18:191-9 19. Thomas T, Schreiber G. The expression of genes coding for positive acute-phase proteins in the reproductive tract of the female rat: high levels of ceruloplasmin mRNA in the uterus. FEBS Lett 1989;243:381-4
Glutamine: A Conditionally Essential Nutrient or Another Nutritional Puzzle Malnutrition and infection are common among patients receiving bone marrow transplant. A recent randomized, double-blind study was conducted to determine whether the addition of glu-
This review was prepared by Sohrab Mobrahan, M.D., at Loyola Medical Center, Maywood, IL 60153. Nutrition Reviews, Vol. 50, No. 11
tamine to standard total parenteral nutrition solutions would improve nitrogen retention and reduce hospital morbidity in a group of 45 bone marrow transplant patients. The results showed improved nitrogen balance and reduced incidence of clinical infection in the glutaminesupplemented patients.
331
November 1992: 329-337
New Insights into Placental Iron Transport The transport of iron from mother to fetus presents a number of challenges, none more perplexing than determining how the flow can be unidirectional, yet avoid mixing the mother's serum proteins with the fetal system. Studies with cultured cell lines suggest a possible mechanism by which this may be achieved.
Fetal Iron Availability
All fetuses, regardless of species, require vast quantities of iron for normal development. In species such as the badger, cat, sheep, and seal (see ref. 1 and references therein), the placenta is involved in active phagocytosis of maternal erythrocytes. However, in most mammalian species, fetal iron is derived from maternal transferrin.* To be absorbed by the fetus, iron must penetrate the placental trophoblasts (the syncytiotrophoblast), a polarized layer of fused cells that segregates the maternal and fetal circulations, and move to the fetal side of the memb ~ - a n e Placental .~ trophoblasts contain large numbers of transferrin receptors on both the maternalfacing apical surface and the fetal-facing basal plasma membrane3 as well as an internal transferrin receptor po0L4 Iron delivery is a two-step mechanism in which holo transferrin is first endocytosed and then returned as apotransferrin to the cell surface. A transcytotic movement of protein could endanger the fetus if the mother's transferrin were to emerge with membrane proteins on the fetal side. Experimental data, however, suggest that only the iron, and not the maternal iron-transporting protein, passes from mother to fetus.' How, then, is maternal transferrin kept from commingling with fetal transferrin, and what prevents maternal transferrin from exiting on the fetal side? Why are fetal transferrin receptors so much more numerous than maternal receptors? These are mysteries of placental iron transport that have yet to be resolved. Placental epithelial cells fit the phenotype of most epithelial cells, having both apical and basolateral surfaces. This design allows unidirectional transport of iron (and other nutrients). The amount This review was prepared by Edward D. Harris, Ph.D., at the Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843. Nutrition Reviews, Vol. 50, No. 11
of iron that crosses the placenta depends on two factors: the number of transferrin receptors on the apical side6 and the level of ferritin within the placental cells.' High cellular iron results in downregulation of transferrin membrane receptors, whereas low cellular iron increases expression of receptors. Thus, placental iron availability is regulated by the iron needs of the fetus.6 Down-regulation of receptor also protects a fetus against iron overload from the mother. In a similar way, placental ferritin in cytotrophoblasts and fetal endothelial cells carefully regulates the inward flux of iron into the fetus's circulation. Both mechanisms would seem to be designed to keep maternal and fetal iron circulations in tune with one another, maintaining a continual supply of iron in proper amounts for fetal development.
'
Mechanisms of Placental Iron Transport
Investigators looking at the mechanism of iron transport have turned to cell cultures for answers. BeWo cells, a human chorion carcinoma cell line first described by Pattilo and Gey ,' have been used extensively. These cells display the phenotypic properties of placental trophoblasts, including receptor-mediated uptake and release of iron.' BeWo cells achieve a unique polarity when grown on polycarbonate membrane filters.' Their morphology is typical of placental trophoblasts in that they have microvilli on the apical membrane surface, with large nuclei in the center and Golgi stacks distributed in the perinuclear cytoplasm. Results of studies using '251-transferrin' have shown that BeWo cells display receptors for transferrin on both the apical and basolateral surfaces; the ratio was 1:2, with the basolateral surface (fetus) having more. Both receptors showed identical binding constants for transferrin. Maintaining a separation between fetal and maternal proteins is a function of the placental barrier. Segregation, however, is rendered more difficult by having both fetal and maternal transferrins deliver iron to the same cell, but from opposite sides. The situation is further complicated by having both transferrins penetrate the membrane. It would seem inevitable that fetal and maternal transferrins must meet somewhere in the cell, but this does not happen. Cerneus and van der Ende' recently showed 329
that BeWo cells cycle transferrin through two noninteracting pools in the placenta. They found no evidence for mixing despite accessing of transferrins from both sides. Thus, a solution to the dilemma seems to be for cells to have two separate transferrin cycles. A hypothetical model for such a dual system is suggested in Figure 1. In the scheme, transferrin (abbreviated as Tf) with iron enters the apical side of the placental trophoblast. After the iron is released from the transferrin, the Tf (as apotransferrin) is recycled to the cell surface and released. Iron released within the cell is transferred to ferritin, from which it can readily become assimilated into fetal apotransferrin. This fetal transferrin then exits via the basolateral surface. The proposed scheme suggests that one ferritin pool may be accessible to both maternal and fetal transferrins. It should be noted that Vanderpuye et al.3 found the affinity of placental receptors at neutral pH to be much higher for diferric transferrin than for apotransferrin, making entry of apotransferrin from either side of the cell an unlikely occurrence. Nonetheless, an important key to the mechanism that needs investigation is whether maternal transferrin represents an entering pool of iron and fetal transferrin represents an exiting pool. A possible role for the monoferric
transferrin species in this event is also in need of clarification. If there are two transferrin cycles operating, what keeps the two cycles separate? A growing body of evidence supports the concept that secretory and polarized cells use specific routing and sorting signals to effect vectorial movement of molecules to different locations within the cell. For example, whether a newly made protein is retained in the endoplasmic reticulum" or is excreted from the cell" depends on specific sorting signals attached to the protein or its membrane-bound receptor. One of the more familiar signals is the mannose 6-phosphate signal that targets delivery of an unsuspecting protein passenger to lysosomes.12 A second discriminating factor is in the structure that anchors the protein to the membrane. Proteins bound via glycosyl-phosphatidylinositol (GPI) tend to be directed to the apical surfaces and are forcibly prevented from appearing at basolateral surfaces. l3 If one fuses a GPI anchor to a basolateral surface protein, that protein is automatically redirected to the apical Thus, routing is determined in part by the structure of the receptor that binds the protein. Future research should help to determine whether membrane-directing sorting signals exist for the transferrin receptors on the basolateral and apical surfaces. If such signals do exist, how do they differ, and how do they affect movement of transferrin and iron? Placental Transport of Other Minerals
Figure 1. Hypothetical model for unidirectional iron transport. Maternal iron is delivered to the apical surface as transferrin. The transferrin molecule is moved inside and the dissociated iron binds to fenitin. The iron-free (apo) transfenin departs the cell, completing the cycle on the maternal side. On the basolateral surface, apotransferrin enters and engages ferritin iron before departing as diferric transferrin. Femtin iron represents the point of communication between the two cycles. Unidirectionality is assured by having the fetal cycle remove the iron deposited by the maternal cycle. 330
The successes gained by studying placental iron transport have encouraged nutritionists to investigate placental transport of other essential metals. A basic question is whether all essential metals fit the same scenario as iron. For example, are there specific proteins that convey zinc, copper, and selenium through the placental barrier separating mother from fetus? Does a transferrin-like cycle exist for these metals? The data are far from complete, but there are suggestions that copper may have a maternal-fetal interacting transport system. Moreover, placental copper transport may mimic copper transport in other cells. This conclusion is based on observing that female mice with the mutant brindled allele (Mobr), which affects copper transport in intestine and kidney, have impaired placental transport of aCu.15 It is still not known why the gene for ceruloplasmin, a copper transport protein, is expressed in choroid plexus, yolk sac, and placenta of rats.I6 Human ceruloplasmin mRNA has been isolated from placentas, " and ceruloplasmin protein has been found in placental extracts.I8 Does this mean that placental copper transport relies on a plasma copper-binding protein synthesized in plaNutrition Reviews, Vol. 50, No. 11
centa? If so, how does placental ceruloplasmin integrate with plasma ceruloplasmin? Ceruloplasmin mRNA has also been found in the decidual layer, a layer of cells from the uterine endometrium that surrounds the implanted fetus. l9 Placental transport of iron has given us insights into the workings of placental cells, but much remains to be learned regarding what some may consider the premiere event in nutrition-nourishment of the developing fetus. Although iron, copper, zinc, and selenium are essential elements for fetal development, their presence can also be detrimental to fetal health if they are supplied in excessive amounts or in the wrong chemical form. All of these complexities must be considered in the development of a comprehensive model of placental transport of minerals. 1. Dumartin B, Canivenc R. Placental iron transfer regulation in the haemophagous region of the badger placenta: ultrastructural localization of ferritin in trophoblast and endothelial cells. Anat Embryo1 19923 85:17!5-9 2. van Dijk JP. Regulatory aspects of placental iron transfer: a comparative study. Placenta 1988;9: 215-26 3. Vanderpuye OA, Kelley LK, Smith CH. Transferrin receptors in the basal plasma membrane of the human placental syncytiotrophoblast. Placenta 1986;7:391-403 4. Douglas GC, King BF. Uptake and processing of '251-labelledtransferrin and 59Fe-labelledtransferrin by isolated human trophoblast cells. Placenta 1990;11:41-57 5. Contractor SF, Eaton BM. Role of transferrin in iron transport between maternal and fetal circulations of perfused lobule of human placenta. Cell Biochem Funct 1986;4:69-74 6. Bierings MB, Baert MRM, van Eijk HG, van Dijk JP. Transferrin receptor expression and the regulation of placental iron uptake. Mol Cell Biochem 1991;100:31-8 7. Pattilo RO, Gey GO. The establishment of a cell line of human synthesizing cells in vitro. Cancer Res 1968;28:1231-6
8. van der Ende A, du Maine A, Simmons CF, Schwartz AL, Strous GJ. Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron. J Biol Chem 1987;262: 8910-6 9. Cerneus DP, van der Ende A. Apical and basolatera1 transferrin receptors in polarized BeWo cells recycle through separate endosomes. J Cell Biol 1991 ;114: 1149-58 10. Jackson MR, Nilsson T, Peterson PA. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. Embo J 1990;9:3153-62 11. Munro S, Pelham HRB. A C-terminal signal prevents secretion of luminal ER proteins. Cell 1987; 48:899-907 12. Kornfeld S. Trafficking of lysosomal enzymes. FASEB J 1987;1:462-8 13. Lisanti MP, Caras IW, Davitz MA, RodriguezBoulan E.A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J Cell Biol 1989;109:2145-56 14. Brown DA, Crise B, Rose JK. Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science 1989;245: 1499-1 501 15. Mann JR, Camakaris J, Danks DM. Copper metabolism in mottled mouse mutants. Defective placental transfer of 64Cu to foetal brindled (Mobr) mice. Biochem J 1980;186:629-31 16. Aldred AR, Grimes A, Shreiber G, Mercer JFB. Rat ceruloplasmin. Molecular cloning and gene expression in liver, choroid plexus, yolk sac, placenta, and testis. J Biol Chem 1987;262:287!j-8 17. Yang F, Friedrichs WE, Cupples RL, et al. Human ceruloplasmin. Tissue-specific expression of transcripts produced by alternative splicing. J Biol Chem 1990;265:10780-5 18. Mas A, Sarkar B. The metabolism of metals in rat placenta. Biol Trace Ele Res 1988;18:191-9 19. Thomas T, Schreiber G. The expression of genes coding for positive acute-phase proteins in the reproductive tract of the female rat: high levels of ceruloplasmin mRNA in the uterus. FEBS Lett 1989;243:381-4
Glutamine: A Conditionally Essential Nutrient or Another Nutritional Puzzle Malnutrition and infection are common among patients receiving bone marrow transplant. A recent randomized, double-blind study was conducted to determine whether the addition of glu-
This review was prepared by Sohrab Mobrahan, M.D., at Loyola Medical Center, Maywood, IL 60153. Nutrition Reviews, Vol. 50, No. 11
tamine to standard total parenteral nutrition solutions would improve nitrogen retention and reduce hospital morbidity in a group of 45 bone marrow transplant patients. The results showed improved nitrogen balance and reduced incidence of clinical infection in the glutaminesupplemented patients.
331
