- Email: [email protected]
Effect of altered thyroid hormone status on rat brain ferritin H and ferritin L mRNA during postnatal development
Developmental Brain Research 119 Ž2000. 105–109 www.elsevier.comrlocaterbres
Research report
Effect of altered thyroid hormone status on rat brain ferritin H and ferritin L mRNA during postnatal development Cathy W. Levenson ) , Cheryl A. Fitch Program in Neuroscience and Department of Nutrition, Food and Exercise Sciences, Florida State UniÕersity, Tallahassee, FL 32306-4340, USA Accepted 12 October 1999
Abstract The iron binding protein ferritin is a heterogeneous mix of 24 heavy ŽH. and light ŽL. subunits. The H subunit is associated with iron utilization, while the L subunit is responsible for iron storage. Examination of the developmental pattern of mRNA abundance in rat brain revealed that ferritin L mRNA is highest at birth and declines during the first postnatal week. A similar decline was seen in ferritin H mRNA, but was followed by an increase in ferritin H mRNA in the second postnatal week which continued through postnatal day 21. The pattern of H mRNA regulation is similar to that in previous reports of total ferritin protein in the developing rat brain and is consistent with the fact that brain ferritin is predominately ferritin H. The effect of thyroid hormone on the developmental regulation of ferritin mRNAs was examined by the subcutaneous injection of a single dose of exogenous thyroxine ŽT4 ; 2 mgrg. on postnatal day 1. Hypothyroidism was induced in pregnant dams with propylthiouracil ŽPTU; 0.05% in drinking water. from gestational day 7. Northern analysis from postnatal days 2–21 showed that T4 increased ferritin H mRNA throughout development, while ferritin L mRNA was decreased compared to age-matched controls. PTU treatment decreased ferritin H and increased L mRNA in the later stages Ždays 14–21. of development. Given the distinct functions of ferritin H and L this suggests a role for thyroid hormone in the ability of the brain to regulate stored vs. utilizable iron during critical periods of development. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Thyroxine; Hyperthyroidism; Hypothyroidism; Iron
1. Introduction The iron-binding protein ferritin is a heterogenous assembly of 24 heavy ŽH. and light ŽL. subunits w1x that are transcribed from distinct genes on separate chromosomes w9x. The heavier of the two subunits ŽH. has an approximate molecular weight of 21 kDa, and the lighter subunit ŽL. is around 19 kDa w1,11x. These two subunits appear to have different functions. The L subunit is closely associated with iron storage w8x, while the H subunit is associated with cellular iron utilization w2x. In fact, the functions of the two subunits are so distinct that the abundance of one subunit in a particular cell type or organ is a good indicator of the iron utilization and storage pattern w7x. For example, the L subunit is predominant in liver and spleen which are organs known to participate significantly in body iron storage w10x. In ) Corresponding author. Florida State University, 237 Biomedical Research Facility, Tallahassee, FL 32306-4340, USA. Fax: q1-850-6440989; e-mail: [email protected]
contrast, organs with high iron utilization requirements, such as brain, synthesize ferritin that is largely composed of H subunits w10,17x. Brain iron utilization is important for normal brain development, myelination, electron transport, and the activity of a number of enzymes responsible for neurotransmitter synthesis, such as tyrosine hydroxylase and tryptophan hydroxylase w5x. Brain development is accompanied by changes in ferritin protein abundance in both humans w30x and rodentsw15,19x. Brain ferritin is high at birth w16,31x and declines over the first two postnatal weeks w31x. After postnatal week 3 there is an increase in ferritin that continues throughout adulthood w16,31x. The distribution of ferritin in the brain also appears to be altered during development. In the early postnatal period brain ferritin is predominately localized to the microglia. By postnatal day 30 ferritin is found most abundantly in oligodendrocytes w11x. Thyroid hormone has been shown to be a significant regulator of brain development w23x. For example, hyperthyroidism accelerates myelin deposition in the central
0165-3806r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 1 6 3 - 7
106
C.W. LeÕenson, C.A. Fitch r DeÕelopmental Brain Research 119 (2000) 105–109
nervous system ŽCNS. w29x, while hypothyroidism delays the developmental pattern of myelination w29,34x and the expression of a number of myelin-associated proteins such as myelin basic protein and 2X ,3X-cyclic nucleotide phosphodiesterase w34x. Given the unique developmental pattern of brain ferritin protein that is characterized by an initial decrease followed by an increase to adult levels w11,16,31x, the current study was designed to explore the developmental regulation of the mRNA for the individual ferritin subunits, ferritin H and ferritin L. Furthermore, given the role of thyroid hormone in brain development, we hypothesized that thyroid hormone may play a role in the developmental regulation of the ferritin subunits, particularly with respect to mRNA abundance. Thus, we tested whether alterations in thyroid hormone Žhyperthyroidism and hypothyroidism. during the early neonatal period would alter the developmental pattern of H andror L gene expression in the rat.
2. Materials and methods 2.1. Animals Sprague–Dawley rats ŽCharles River Labs, Raleigh, NC. were used for all experiments. Pregnant dams were housed individually in standard cages with bedding and maintained on a 12 hr12 h lightrdark cycle and fed a standard rodent diet Ž5001; Ralston-Purina, St. Louis, MO. and distilled water ad libitum. The Institutional Animal Care and Use Committee ŽIACUC. approved all experiments. Treatment groups were similar to those described previously w24x, with each group consisting of at least three animals. Neonatal hyperthyroidism was induced by administration of thyroxine ŽT4 ; Sigma, St. Louis, MO. to pups on postnatal day 1 Žthe day of birth was considered day 0.. The T4 was dissolved in normal saline with 0.05 mM NaOH and administered subcutaneously Ž2 mgrg body weight.. Vehicle-treated pups served as controls and were injected with the same volume of salinerNaOH as T4treated pups. Hypothyroidism was induced in developing rats using propylthiouracil ŽPTU; Sigma. administered in the drinking water Ž0.05% wrv. to pregnant dams from day 7 of gestation. After parturition, dams remained on PTU water to insure that pups would develop hypothyroidism. Pups from dams given distilled water served as controls. Whole brain and trunk blood were collected for isolation of total cellular RNA and serum, respectively. 2.2. Serum T4 measurement Serum T4 was measured by radioimmunoassay ŽRIA. using a commercially prepared kit ŽDiagnostic Systems Labs, Webster, TX.. Briefly, 10 ml serum, 25 ml standards, and 200 ml I 125-labeled T4 were added to antibody-
coated tubes and incubated at room temperature for 1 h. After decanting, tubes were rinsed and left to dry for several minutes. Tubes were then counted on a gamma counter ŽICN Micromedic Systems, Costa Mesa, CA.. The sensitivity of the assay was 10 ngrml, with an intra-assay coefficient of variation of - 5%. 2.3. Northern analysis of ferritin mRNA Total cellular RNA was isolated from whole brain using the guanidinium – isothiocyanate – phenol– chloroform method w12x. RNA Ž20 mg. from each pup was subjected to agarose gel Ž1% agarose with 0.66 M formaldehyde. electrophoresis and transferred to a nylon membrane ŽGeneScreen, NENrDupont, Boston, MA.. Northern blots were hybridized to 32 P-labeled cDNA probes specific for rat ferritin H, ferritin L w28x and 28S rRNA ŽRandom Primer-Labeling System, Gibco BRL, Gaithersburg, MD. overnight at 658C. After exposure of hybridized blots to X-ray film at y808C, densitometry was used to measure relative amounts of bound cDNA probes. Blots were stripped by boiling Ž1% SDS, 15 mM NaCl, 1 mM NaH 2 PO4 , 0.1 mM EDTA. between hybridizations. Complete removal of the ferritin H probe was confirmed before hybridization with the ferritin L probe. Relative ferritin mRNA abundances Žboth H and L. were determined by computer evaluated densitometry ŽQuantity One Quantification. created by Protein and DNA Imaging ŽPDI, Boston, MA. and expressed as a function of 28S rRNA Žto control for loading.. Results are reported as mean " S.D. Ž n s 3.. Time points with fewer than three animals were eliminated. ANOVA and a post hoc t-test were used to determine significance between treatment groups. Differences were considered statistically significant at p F 0.05.
3. Results Northern analysis of normal ferritin H and L mRNA during postnatal development revealed that ferritin H mRNA abundance decreased between day 1 and day 7, after which time there was a gradual increase through the end of the developmental study, day 21 ŽFig. 1.. Ferritin L mRNA abundance declined from peak levels at day 1 during the first postnatal week. Low levels of ferritin L mRNA were maintained in the brain after the end of the first week. ŽFig. 1.. Early neonatal administration of T4 resulted in a transient, but highly significant increase in serum T4 levels. Serum T4 levels rose after injection and remained elevated Ž p - 0.001. through postnatal day 5 ŽFig. 2.. Administration of water containing PTU to pregnant dams resulted in significantly reduced Ž p - 0.05. serum T4 levels compared to age-matched control pups. Pups remained hypothyroid throughout the developmental period ŽFig. 2..
C.W. LeÕenson, C.A. Fitch r DeÕelopmental Brain Research 119 (2000) 105–109
Fig. 1. Developmental regulation of rat brain ferritin H and ferritin L mRNA. Total brain RNA Ž15 mg. was collected from brains of normal rat pups Ž ns 3. on postnatal days 2–21 and separated by agarose gel electrophoresis, transferred to a nylon membrane and hybridized to 32 P-labeled cDNA probes for ferritin H, ferritin L and 28S rRNA Žloading control.. Data are expressed as a function of 28S rRNA abundance. Inset shows representative autoradiograph of ferritin H and L mRNA as well as 28S rRNA control from postnatal days 2–21.
The impact of exogenous thyroid hormone on the developmental curve of ferritin H mRNA is shown in Fig. 3. Exogenous T4 elevated ferritin H mRNA abundance during development as compared to age-matched controls. A trend toward this increase was first seen on postnatal day 2 Ž24 h after injection.. By postnatal day 7 brain ferritin H mRNA
Fig. 2. Serum thyroxine ŽT4 , mgrml. during the postnatal period Ž ns 3. in ŽA. animals treated with exogenous thyroxine Ž2 mgrg body weight. on postnatal day 1 and ŽB. pups born to dams treated with PTU from gestational day 7 to induce hypothyroidism.
107
Fig. 3. The effect of exogenous thyroxine ŽT4 . on ferritin H mRNA. Pups were injected with a vehicle or T4 Ž2 mgrg body weight. on postnatal day 1. Ferritin H mRNA was measured by northern analysis during the first three postnatal weeks and normalized to the abundance of 28S rRNA. Bars represent mean"S.D. Ž ns 3.. U Significantly different from age-matched controls at pF 0.05. UU Significantly different from agematched controls at pF 0.001.
abundance was 2.4-fold higher in thyroxine-treated pups Ž p F 0.001.. In contrast, ferritin L mRNA was reduced by exogenous thyroid hormone during development ŽFig. 4.. On postnatal day 2 ferritin L mRNA in PTU treated pups was reduced to 60% of control Ž p F 0.01.. By day 14 ferritin L mRNA was 40% of age-matched controls Ž p F 0.05., and by day 21 it was approximately 20% of control Ž p F 0.01.. Fig. 5 shows the effect of gestational hypothyroidism on ferritin H mRNA abundance in control and PTU-treated male rat pups. PTU treatment did not result in significant change in ferritin H mRNA abundance during the first week of postnatal development, but did significantly lower ferritin H mRNA abundance by postnatal day 14 Ž p F 0.05, Fig. 5.. Likewise, ferritin L mRNA was not altered during the first week, but was increased by 15% compared to
Fig. 4. The effect of exogenous thyroxine ŽT4 . on ferritin L mRNA. Pups were injected with a vehicle or T4 Ž2 mgrg body weight. on postnatal day 1. Ferritin L mRNA was measured by northern analysis during the first three postnatal weeks and normalized to the abundance of 28S rRNA. Bars represent mean"S.D. Ž ns 3.. U Significantly different from age-matched controls at pF 0.05. UU Significantly different from agematched controls at pF 0.01.
108
C.W. LeÕenson, C.A. Fitch r DeÕelopmental Brain Research 119 (2000) 105–109
Fig. 5. Effect of hypothyroidism on the developmental pattern of brain ferritin H mRNA abundance. Hypothyroid pups ŽPTU. were born to dams treated with PTU Ž0.05% in drinking water. from gestational day 7. Ferritin H mRNA was measured by northern analysis and normalized to the abundance of 28S rRNA during the first two weeks after birth. Bars represent mean"S.D. Ž ns 3.. Northern inset show representative autoradiogram of ferritin H mRNA in control and hypothyroid ŽPTU. pups at postnatal day 14. U Significantly different from age-matched controls at pF 0.05.
age-matched controls on postnatal day 14 and increased by 63% at day 21 Ž p F 0.05..
4. Discussion While the developmental regulation of hepatic and intestinal ferritin subunit gene expression has been studied w35x, there has not been work examining the regulation of ferritin H and L subunit mRNA in the developing brain. The pattern of mRNA abundance for ferritin H, but not ferritin L, during development is consistent with the pattern of total ferritin in the brain previously reported w16,19x. Ferritin H mRNA, like total ferritin protein, declines from birth levels for approximately 2 weeks, but increases later during the developmental period, while ferritin L mRNA exhibits a steady decline after birth. The fact that reported levels of total brain ferritin during development follow the same pattern as ferritin H mRNA is consistent with the observation that ferritin H is the predominant subunit of brain ferritin w17x. However, it should be noted that the distribution of the ferritin subunits varies with cell type w17,18x. Microglia contain ferritin that predominately consists of the L subunit, while neurons synthesize the H subunit. Ferritin H has also been localized to neuronal nuclei w11x. Only oligodendrocytes have consistently been shown to contain high abundances of both H and L w18x. Alterations in thyroid hormone status are known to regulate ferritin. Hepatic synthesis of ferritin was reduced by 36% in hypothyroid rats and increased by a similar amount in hyperthyroid animals w21x. While this increase is due in part to post-transcriptional regulation of ferritin w26,27x, thyroid hormone may also act at the level of transcription to regulate ferritin H. Hyperthyroidism increased and hypothyroidism decreased rat kidney ferritin H
mRNA w25x. In brain, the only work reported to date is in cultured C6 glioma cells where T3 increased the transcription rate of ferritin H without altering ferritin H mRNA stability w25x. Thus, it is possible that the effects of hyperthyroidism and hypothyroidism during brain development observed here are due to a combination of transcriptional and post-transcriptional events. The data reported here are also consistent with the fact that ferritin abundance is regulated during the process of cellular differentiation and development w33x. Both ferritin L and H genes have been shown to be regulated during the differentiation of the human promyelocytic cell line HL-60 w13x and Friend erythroleukemic cells w6,14x. It is interesting to note that in each of these studies the H and L subunits were regulated coordinately, while in rat brain the developmental pattern of mRNA abundance was different for H and L. This suggests a higher level of ferritin regulation in the brain and, given the different functions of H and L, the increased ability of the brain to regulate stored vs. utilizable iron during development. The suggestion that the differentiation state of the brain is a primary factor in determining the abundance of ferritin mRNA is supported by the finding that a single injection of T4 on postnatal day 1 and a transient increase in serum T4 was sufficient to increase ferritin H and decrease ferritin L mRNA throughout the first three postnatal weeks. However, it should be noted that the effect of T4-treatment on day 1 did not increase ferritin H mRNA until postnatal day 7, suggesting that the effect of T4 on ferritin H mRNA may be the result of the role of T4 on brain development, rather than directly on ferritin H gene expression. Whether the increase in ferritin H mRNA at day 21 represents a secondary peak in development with T4 treatment is not known at this time. However, it is possible that the regulation of ferritin H mRNA is related to the ability of thyroid hormone to accelerate oligodendrocyte development and differentiation w3,4x, and a subsequent increase in ferritin H expression in the differentiated oligodendrocytes w32x. The possibility that hyperthyroidism or hypothyroidism, and the accompanying changes in ferritin subunit expression, during the critical period of brain development may have long-term consequences needs to be considered. For example, the precocious differentiation of oligodendrocytes induced by neonatal hyperthyroidism can result in myelin deficits in adulthood w29x. Furthermore, alterations in ferritin have been reported in the brains of Parkinson’s patients w22x and Alzheimer’s diseased brains w20x. Thus, the role of ferritin in the long-term disruption of myelin by thyroid hormone and its possible role in the development of other disease states associated with development and aging needs to be explored. Acknowledgements The authors would like to thank Dr. Elizabeth Leibold, University of Utah, for her generous contribution of the
C.W. LeÕenson, C.A. Fitch r DeÕelopmental Brain Research 119 (2000) 105–109
ferritin L cDNA and Dr. Marc Freeman, Program in Neuroscience, Florida State University, for his contribution of neonatal animals and helpful discussions. This work was supported by NIH grant DK50472 from the National Institute of Diabetes and Digestive and Kidney Diseases ŽNIDDK..
References w1x P. Arosio, T.G. Adelman, J.W. Drysdale, On ferritin heterogeneity. Further evidence for heteropolymers, J. Biol. Chem. 253 Ž1978. 4451–4458. w2x P. Arosio, S. Levi, P. Santambrogio, A. Cozzi, A. Luzzago, G. Cesareni, A. Albertini, Structural and functional studies of human ferritin H and L chains, Curr. Stud. Hematol. Blood Transfus., 1999, pp. 127–131. w3x D. Baas, D. Bourbeau, L.L. Sarlieve, ` M.E. Ittel, J.H. Dussault, J. Puymirat, Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone, Glia 19 Ž1997. 324–332. w4x B.A. Barres, M.A. Lazar, M.C. Raff, A novel role for thyroid hormone, glucocorticoids and retinoic acid in timing oligodendrocyte development, Development 120 Ž1994. 1097–1108. w5x J.L. Beard, J.R. Connor, B.C. Jones, Iron in the brain, Nutr. Rev. 51 Ž1993. 157–170. w6x C. Beaumont, S.K. Jain, M. Bogard, Y. Nordmann, J. Drysdale, Ferritin synthesis in differentiating Friend erythroleukemic cells, J. Biol. Chem. 262 Ž1987. 10619–10623. w7x G. Blissman, S. Menzies, J. Beard, C. Palmer, J. Connor, The expression of ferritin subunits and iron in oligodendrocytes in neonatal porcine brains, Dev. Neurosci. 18 Ž1996. 274–281. w8x A. Bomford, C. Conlon-Hollingshead, H.N. Munro, Adaptive responses of rat tissue isoferritins to iron administration. Changes in subunit synthesis, isoferritin abundance, and capacity for iron storage, J. Biol. Chem. 256 Ž1981. 948–955. w9x G. Cairo, L. Bardella, L. Schiaffonati, P. Arosio, S. Levi, A. Bernelli-Zazzera, Multiple mechanisms of iron-induced ferritin synthesis in HeLa cells, Biochem. Biophys. Res. Commun. 133 Ž1985. 314–321. w10x G. Cairo, E. Rappocciolo, L. Tacchini, L. Schiaffonati, Expression of the genes for the ferritin H and L subunits in rat liver and heart. Evidence for tissue-specific regulations at pre- and post-translational levels, Biochem. J. 275 Ž1991. 813–816. w11x P. Cheepsunthorn, C. Palmer, J.R. Connor, Cellular distribution of ferritin subunits in postnatal rat brain, J. Comp. Neurol. 400 Ž1998. 73–86. w12x P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. w13x C.C. Chou, R.A. Gatti, M.L. Fuller, P. Concannon, A. Wong, S. Chada, R.C. Davis, W.A. Salser, Structure and expression of ferritin genes in a human promyelocytic cell line that differentiates in vitro, Mol. Cell. Biol. 6 Ž1986. 566–573. w14x E.M. Coccia, E. Stellacci, R. Orsatti, U. Testa, A. Battistini, Regulation of ferritin H-chain expression in differentiating Friend leukemia cells, Blood 86 Ž1995. 1570–1579. w15x J.R. Connor, Iron acquisition and expression of iron regulatory proteins in the developing brain: manipulation by ethanol exposure, iron deprivation and cellular dysfunction, Dev. Neurosci. 16 Ž1994. 233–247.
109
w16x J.R. Connor, Iron regulation in the brain at the cell and molecular level, Adv. Exp. Med. Biol. 356 Ž1994. 229–238. w17x J.R. Connor, K.L. Boeshore, S.A. Benkovic, S.L. Menzies, Isoforms of ferritin have a specific cellular distribution in the brain, J. Neurosci. Res. 37 Ž1994. 461–465. w18x J.R. Connor, S.L. Menzies, Cellular management of iron in the brain, J. Neurol. Sci. 134 Ž1995. 33–44, Suppl. w19x J.R. Connor, S.L. Menzies, Relationship of iron to oligodendrocytes and myelination, Glia 17 Ž1996. 83–93. w20x J.R. Connor, B.S. Snyder, P. Arosio, D.A. Loeffler, P. LeWitt, A quantitative analysis of isoferritins in select regions of aged, parkinsonian, and Alzheimer’s diseased brains, J. Neurochem. 65 Ž1995. 717–724. w21x U.R. Deshpande, G.D. Nadkarni, Relation between thyroid status and ferritin metabolism in rats, Thyroidology 4 Ž1992. 93–97. w22x D.T. Dexter, A. Carayon, M. Vidailhet, M. Ruberg, F. Agid, Y. Agid, A.J. Lees, F.R. Wells, P. Jenner, C.D. Marsden, Decreased ferritin levels in brain in Parkinson’s disease, J. Neurochem. 55 Ž1990. 16–20. w23x J.H. Dussault, J. Ruel, Thyroid hormones and brain development, Annu. Rev. Physiol. 49 Ž1987. 321–334. w24x C.A. Fitch, Y. Song, C.W. Levenson, Developmental regulation of hepatic ceruloplasmin mRNA and serum activity by exogenous thyroxine and dexamethasone, Proc. Soc. Exp. Biol. Med. 221 Ž1999. 27–31. w25x Y. Iwasa, K. Aida, N. Yokomori, M. Inoue, T. Onaya, Transcriptional regulation of ferritin heavy chain messenger RNA expression by thyroid hormone, Biochem. Biophys. Res. Commun. 167 Ž1990. 1279–1285. w26x D.B. Jump, C.N. Mariash, J.H. Oppenheimer, Evidence for posttranscriptional effects of T3 on hepatic ferritin synthesis, Biochem. Biophys. Res. Commun. 104 Ž1982. 701–707. w27x P.J. Leedman, A.R. Stein, W.W. Chin, J.T. Rogers, Thyroid hormone modulates the interaction between iron regulatory proteins and the ferritin mRNA iron-responsive element, J. Biol. Chem. 271 Ž1996. 12017–12023. w28x E.A. Leibold, H.N. Munro, Characterization and evolution of the expressed rat ferritin light subunit gene and its pseudogene family. Conservation of sequences within noncoding regions of ferritin genes, J. Biol. Chem. 262 Ž1987. 7335–7341. w29x C.B. Marta, A.M. Adamo, E.F. Soto, J.M. Pasquini, Sustained neonatal hyperthyroidism in the rat affects myelination in the central nervous system, J. Neurosci. Res. 53 Ž1998. 251–259. w30x H. Ozawa, A. Nishida, T. Mito, S. Takashima, Development of ferritin-containing cells in the pons and cerebellum of the human brain, Brain Dev. 16 Ž1994. 92–95. w31x A.J.I. Roskams, J.R. Connor, Iron, transferrin, and ferritin in the rat brain during development and aging, J. Neurochem. 63 Ž1994. 709–716. w32x B. Sanyal, P.E. Polak, S. Szuchet, Differential expression of the heavy-chain ferritin gene in non-adhered and adhered oligodendrocytes, J. Neurosci. Res. 46 Ž1996. 187–197. w33x E.C. Theil, Ferritin mRNA translation, structure, and gene transcription during development of animals and plants, Enzyme 44 Ž1990. 68–82. w34x S.N. Walters, P. Morell, Effects of altered thyroid states on myelogenesis, J. Neurochem. 36 Ž1981. 1792–1801. w35x K.Y. Yeh, X. Alvarez-Hernandez, J. Glass, M. Yeh, Rat intestinal and hepatic ferritin subunit expression during development and after dietary iron feeding, Am. J. Physiol. 270 Ž1996. G498–505.
Research report
Effect of altered thyroid hormone status on rat brain ferritin H and ferritin L mRNA during postnatal development Cathy W. Levenson ) , Cheryl A. Fitch Program in Neuroscience and Department of Nutrition, Food and Exercise Sciences, Florida State UniÕersity, Tallahassee, FL 32306-4340, USA Accepted 12 October 1999
Abstract The iron binding protein ferritin is a heterogeneous mix of 24 heavy ŽH. and light ŽL. subunits. The H subunit is associated with iron utilization, while the L subunit is responsible for iron storage. Examination of the developmental pattern of mRNA abundance in rat brain revealed that ferritin L mRNA is highest at birth and declines during the first postnatal week. A similar decline was seen in ferritin H mRNA, but was followed by an increase in ferritin H mRNA in the second postnatal week which continued through postnatal day 21. The pattern of H mRNA regulation is similar to that in previous reports of total ferritin protein in the developing rat brain and is consistent with the fact that brain ferritin is predominately ferritin H. The effect of thyroid hormone on the developmental regulation of ferritin mRNAs was examined by the subcutaneous injection of a single dose of exogenous thyroxine ŽT4 ; 2 mgrg. on postnatal day 1. Hypothyroidism was induced in pregnant dams with propylthiouracil ŽPTU; 0.05% in drinking water. from gestational day 7. Northern analysis from postnatal days 2–21 showed that T4 increased ferritin H mRNA throughout development, while ferritin L mRNA was decreased compared to age-matched controls. PTU treatment decreased ferritin H and increased L mRNA in the later stages Ždays 14–21. of development. Given the distinct functions of ferritin H and L this suggests a role for thyroid hormone in the ability of the brain to regulate stored vs. utilizable iron during critical periods of development. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Thyroxine; Hyperthyroidism; Hypothyroidism; Iron
1. Introduction The iron-binding protein ferritin is a heterogenous assembly of 24 heavy ŽH. and light ŽL. subunits w1x that are transcribed from distinct genes on separate chromosomes w9x. The heavier of the two subunits ŽH. has an approximate molecular weight of 21 kDa, and the lighter subunit ŽL. is around 19 kDa w1,11x. These two subunits appear to have different functions. The L subunit is closely associated with iron storage w8x, while the H subunit is associated with cellular iron utilization w2x. In fact, the functions of the two subunits are so distinct that the abundance of one subunit in a particular cell type or organ is a good indicator of the iron utilization and storage pattern w7x. For example, the L subunit is predominant in liver and spleen which are organs known to participate significantly in body iron storage w10x. In ) Corresponding author. Florida State University, 237 Biomedical Research Facility, Tallahassee, FL 32306-4340, USA. Fax: q1-850-6440989; e-mail: [email protected]
contrast, organs with high iron utilization requirements, such as brain, synthesize ferritin that is largely composed of H subunits w10,17x. Brain iron utilization is important for normal brain development, myelination, electron transport, and the activity of a number of enzymes responsible for neurotransmitter synthesis, such as tyrosine hydroxylase and tryptophan hydroxylase w5x. Brain development is accompanied by changes in ferritin protein abundance in both humans w30x and rodentsw15,19x. Brain ferritin is high at birth w16,31x and declines over the first two postnatal weeks w31x. After postnatal week 3 there is an increase in ferritin that continues throughout adulthood w16,31x. The distribution of ferritin in the brain also appears to be altered during development. In the early postnatal period brain ferritin is predominately localized to the microglia. By postnatal day 30 ferritin is found most abundantly in oligodendrocytes w11x. Thyroid hormone has been shown to be a significant regulator of brain development w23x. For example, hyperthyroidism accelerates myelin deposition in the central
0165-3806r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 1 6 3 - 7
106
C.W. LeÕenson, C.A. Fitch r DeÕelopmental Brain Research 119 (2000) 105–109
nervous system ŽCNS. w29x, while hypothyroidism delays the developmental pattern of myelination w29,34x and the expression of a number of myelin-associated proteins such as myelin basic protein and 2X ,3X-cyclic nucleotide phosphodiesterase w34x. Given the unique developmental pattern of brain ferritin protein that is characterized by an initial decrease followed by an increase to adult levels w11,16,31x, the current study was designed to explore the developmental regulation of the mRNA for the individual ferritin subunits, ferritin H and ferritin L. Furthermore, given the role of thyroid hormone in brain development, we hypothesized that thyroid hormone may play a role in the developmental regulation of the ferritin subunits, particularly with respect to mRNA abundance. Thus, we tested whether alterations in thyroid hormone Žhyperthyroidism and hypothyroidism. during the early neonatal period would alter the developmental pattern of H andror L gene expression in the rat.
2. Materials and methods 2.1. Animals Sprague–Dawley rats ŽCharles River Labs, Raleigh, NC. were used for all experiments. Pregnant dams were housed individually in standard cages with bedding and maintained on a 12 hr12 h lightrdark cycle and fed a standard rodent diet Ž5001; Ralston-Purina, St. Louis, MO. and distilled water ad libitum. The Institutional Animal Care and Use Committee ŽIACUC. approved all experiments. Treatment groups were similar to those described previously w24x, with each group consisting of at least three animals. Neonatal hyperthyroidism was induced by administration of thyroxine ŽT4 ; Sigma, St. Louis, MO. to pups on postnatal day 1 Žthe day of birth was considered day 0.. The T4 was dissolved in normal saline with 0.05 mM NaOH and administered subcutaneously Ž2 mgrg body weight.. Vehicle-treated pups served as controls and were injected with the same volume of salinerNaOH as T4treated pups. Hypothyroidism was induced in developing rats using propylthiouracil ŽPTU; Sigma. administered in the drinking water Ž0.05% wrv. to pregnant dams from day 7 of gestation. After parturition, dams remained on PTU water to insure that pups would develop hypothyroidism. Pups from dams given distilled water served as controls. Whole brain and trunk blood were collected for isolation of total cellular RNA and serum, respectively. 2.2. Serum T4 measurement Serum T4 was measured by radioimmunoassay ŽRIA. using a commercially prepared kit ŽDiagnostic Systems Labs, Webster, TX.. Briefly, 10 ml serum, 25 ml standards, and 200 ml I 125-labeled T4 were added to antibody-
coated tubes and incubated at room temperature for 1 h. After decanting, tubes were rinsed and left to dry for several minutes. Tubes were then counted on a gamma counter ŽICN Micromedic Systems, Costa Mesa, CA.. The sensitivity of the assay was 10 ngrml, with an intra-assay coefficient of variation of - 5%. 2.3. Northern analysis of ferritin mRNA Total cellular RNA was isolated from whole brain using the guanidinium – isothiocyanate – phenol– chloroform method w12x. RNA Ž20 mg. from each pup was subjected to agarose gel Ž1% agarose with 0.66 M formaldehyde. electrophoresis and transferred to a nylon membrane ŽGeneScreen, NENrDupont, Boston, MA.. Northern blots were hybridized to 32 P-labeled cDNA probes specific for rat ferritin H, ferritin L w28x and 28S rRNA ŽRandom Primer-Labeling System, Gibco BRL, Gaithersburg, MD. overnight at 658C. After exposure of hybridized blots to X-ray film at y808C, densitometry was used to measure relative amounts of bound cDNA probes. Blots were stripped by boiling Ž1% SDS, 15 mM NaCl, 1 mM NaH 2 PO4 , 0.1 mM EDTA. between hybridizations. Complete removal of the ferritin H probe was confirmed before hybridization with the ferritin L probe. Relative ferritin mRNA abundances Žboth H and L. were determined by computer evaluated densitometry ŽQuantity One Quantification. created by Protein and DNA Imaging ŽPDI, Boston, MA. and expressed as a function of 28S rRNA Žto control for loading.. Results are reported as mean " S.D. Ž n s 3.. Time points with fewer than three animals were eliminated. ANOVA and a post hoc t-test were used to determine significance between treatment groups. Differences were considered statistically significant at p F 0.05.
3. Results Northern analysis of normal ferritin H and L mRNA during postnatal development revealed that ferritin H mRNA abundance decreased between day 1 and day 7, after which time there was a gradual increase through the end of the developmental study, day 21 ŽFig. 1.. Ferritin L mRNA abundance declined from peak levels at day 1 during the first postnatal week. Low levels of ferritin L mRNA were maintained in the brain after the end of the first week. ŽFig. 1.. Early neonatal administration of T4 resulted in a transient, but highly significant increase in serum T4 levels. Serum T4 levels rose after injection and remained elevated Ž p - 0.001. through postnatal day 5 ŽFig. 2.. Administration of water containing PTU to pregnant dams resulted in significantly reduced Ž p - 0.05. serum T4 levels compared to age-matched control pups. Pups remained hypothyroid throughout the developmental period ŽFig. 2..
C.W. LeÕenson, C.A. Fitch r DeÕelopmental Brain Research 119 (2000) 105–109
Fig. 1. Developmental regulation of rat brain ferritin H and ferritin L mRNA. Total brain RNA Ž15 mg. was collected from brains of normal rat pups Ž ns 3. on postnatal days 2–21 and separated by agarose gel electrophoresis, transferred to a nylon membrane and hybridized to 32 P-labeled cDNA probes for ferritin H, ferritin L and 28S rRNA Žloading control.. Data are expressed as a function of 28S rRNA abundance. Inset shows representative autoradiograph of ferritin H and L mRNA as well as 28S rRNA control from postnatal days 2–21.
The impact of exogenous thyroid hormone on the developmental curve of ferritin H mRNA is shown in Fig. 3. Exogenous T4 elevated ferritin H mRNA abundance during development as compared to age-matched controls. A trend toward this increase was first seen on postnatal day 2 Ž24 h after injection.. By postnatal day 7 brain ferritin H mRNA
Fig. 2. Serum thyroxine ŽT4 , mgrml. during the postnatal period Ž ns 3. in ŽA. animals treated with exogenous thyroxine Ž2 mgrg body weight. on postnatal day 1 and ŽB. pups born to dams treated with PTU from gestational day 7 to induce hypothyroidism.
107
Fig. 3. The effect of exogenous thyroxine ŽT4 . on ferritin H mRNA. Pups were injected with a vehicle or T4 Ž2 mgrg body weight. on postnatal day 1. Ferritin H mRNA was measured by northern analysis during the first three postnatal weeks and normalized to the abundance of 28S rRNA. Bars represent mean"S.D. Ž ns 3.. U Significantly different from age-matched controls at pF 0.05. UU Significantly different from agematched controls at pF 0.001.
abundance was 2.4-fold higher in thyroxine-treated pups Ž p F 0.001.. In contrast, ferritin L mRNA was reduced by exogenous thyroid hormone during development ŽFig. 4.. On postnatal day 2 ferritin L mRNA in PTU treated pups was reduced to 60% of control Ž p F 0.01.. By day 14 ferritin L mRNA was 40% of age-matched controls Ž p F 0.05., and by day 21 it was approximately 20% of control Ž p F 0.01.. Fig. 5 shows the effect of gestational hypothyroidism on ferritin H mRNA abundance in control and PTU-treated male rat pups. PTU treatment did not result in significant change in ferritin H mRNA abundance during the first week of postnatal development, but did significantly lower ferritin H mRNA abundance by postnatal day 14 Ž p F 0.05, Fig. 5.. Likewise, ferritin L mRNA was not altered during the first week, but was increased by 15% compared to
Fig. 4. The effect of exogenous thyroxine ŽT4 . on ferritin L mRNA. Pups were injected with a vehicle or T4 Ž2 mgrg body weight. on postnatal day 1. Ferritin L mRNA was measured by northern analysis during the first three postnatal weeks and normalized to the abundance of 28S rRNA. Bars represent mean"S.D. Ž ns 3.. U Significantly different from age-matched controls at pF 0.05. UU Significantly different from agematched controls at pF 0.01.
108
C.W. LeÕenson, C.A. Fitch r DeÕelopmental Brain Research 119 (2000) 105–109
Fig. 5. Effect of hypothyroidism on the developmental pattern of brain ferritin H mRNA abundance. Hypothyroid pups ŽPTU. were born to dams treated with PTU Ž0.05% in drinking water. from gestational day 7. Ferritin H mRNA was measured by northern analysis and normalized to the abundance of 28S rRNA during the first two weeks after birth. Bars represent mean"S.D. Ž ns 3.. Northern inset show representative autoradiogram of ferritin H mRNA in control and hypothyroid ŽPTU. pups at postnatal day 14. U Significantly different from age-matched controls at pF 0.05.
age-matched controls on postnatal day 14 and increased by 63% at day 21 Ž p F 0.05..
4. Discussion While the developmental regulation of hepatic and intestinal ferritin subunit gene expression has been studied w35x, there has not been work examining the regulation of ferritin H and L subunit mRNA in the developing brain. The pattern of mRNA abundance for ferritin H, but not ferritin L, during development is consistent with the pattern of total ferritin in the brain previously reported w16,19x. Ferritin H mRNA, like total ferritin protein, declines from birth levels for approximately 2 weeks, but increases later during the developmental period, while ferritin L mRNA exhibits a steady decline after birth. The fact that reported levels of total brain ferritin during development follow the same pattern as ferritin H mRNA is consistent with the observation that ferritin H is the predominant subunit of brain ferritin w17x. However, it should be noted that the distribution of the ferritin subunits varies with cell type w17,18x. Microglia contain ferritin that predominately consists of the L subunit, while neurons synthesize the H subunit. Ferritin H has also been localized to neuronal nuclei w11x. Only oligodendrocytes have consistently been shown to contain high abundances of both H and L w18x. Alterations in thyroid hormone status are known to regulate ferritin. Hepatic synthesis of ferritin was reduced by 36% in hypothyroid rats and increased by a similar amount in hyperthyroid animals w21x. While this increase is due in part to post-transcriptional regulation of ferritin w26,27x, thyroid hormone may also act at the level of transcription to regulate ferritin H. Hyperthyroidism increased and hypothyroidism decreased rat kidney ferritin H
mRNA w25x. In brain, the only work reported to date is in cultured C6 glioma cells where T3 increased the transcription rate of ferritin H without altering ferritin H mRNA stability w25x. Thus, it is possible that the effects of hyperthyroidism and hypothyroidism during brain development observed here are due to a combination of transcriptional and post-transcriptional events. The data reported here are also consistent with the fact that ferritin abundance is regulated during the process of cellular differentiation and development w33x. Both ferritin L and H genes have been shown to be regulated during the differentiation of the human promyelocytic cell line HL-60 w13x and Friend erythroleukemic cells w6,14x. It is interesting to note that in each of these studies the H and L subunits were regulated coordinately, while in rat brain the developmental pattern of mRNA abundance was different for H and L. This suggests a higher level of ferritin regulation in the brain and, given the different functions of H and L, the increased ability of the brain to regulate stored vs. utilizable iron during development. The suggestion that the differentiation state of the brain is a primary factor in determining the abundance of ferritin mRNA is supported by the finding that a single injection of T4 on postnatal day 1 and a transient increase in serum T4 was sufficient to increase ferritin H and decrease ferritin L mRNA throughout the first three postnatal weeks. However, it should be noted that the effect of T4-treatment on day 1 did not increase ferritin H mRNA until postnatal day 7, suggesting that the effect of T4 on ferritin H mRNA may be the result of the role of T4 on brain development, rather than directly on ferritin H gene expression. Whether the increase in ferritin H mRNA at day 21 represents a secondary peak in development with T4 treatment is not known at this time. However, it is possible that the regulation of ferritin H mRNA is related to the ability of thyroid hormone to accelerate oligodendrocyte development and differentiation w3,4x, and a subsequent increase in ferritin H expression in the differentiated oligodendrocytes w32x. The possibility that hyperthyroidism or hypothyroidism, and the accompanying changes in ferritin subunit expression, during the critical period of brain development may have long-term consequences needs to be considered. For example, the precocious differentiation of oligodendrocytes induced by neonatal hyperthyroidism can result in myelin deficits in adulthood w29x. Furthermore, alterations in ferritin have been reported in the brains of Parkinson’s patients w22x and Alzheimer’s diseased brains w20x. Thus, the role of ferritin in the long-term disruption of myelin by thyroid hormone and its possible role in the development of other disease states associated with development and aging needs to be explored. Acknowledgements The authors would like to thank Dr. Elizabeth Leibold, University of Utah, for her generous contribution of the
C.W. LeÕenson, C.A. Fitch r DeÕelopmental Brain Research 119 (2000) 105–109
ferritin L cDNA and Dr. Marc Freeman, Program in Neuroscience, Florida State University, for his contribution of neonatal animals and helpful discussions. This work was supported by NIH grant DK50472 from the National Institute of Diabetes and Digestive and Kidney Diseases ŽNIDDK..
References w1x P. Arosio, T.G. Adelman, J.W. Drysdale, On ferritin heterogeneity. Further evidence for heteropolymers, J. Biol. Chem. 253 Ž1978. 4451–4458. w2x P. Arosio, S. Levi, P. Santambrogio, A. Cozzi, A. Luzzago, G. Cesareni, A. Albertini, Structural and functional studies of human ferritin H and L chains, Curr. Stud. Hematol. Blood Transfus., 1999, pp. 127–131. w3x D. Baas, D. Bourbeau, L.L. Sarlieve, ` M.E. Ittel, J.H. Dussault, J. Puymirat, Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone, Glia 19 Ž1997. 324–332. w4x B.A. Barres, M.A. Lazar, M.C. Raff, A novel role for thyroid hormone, glucocorticoids and retinoic acid in timing oligodendrocyte development, Development 120 Ž1994. 1097–1108. w5x J.L. Beard, J.R. Connor, B.C. Jones, Iron in the brain, Nutr. Rev. 51 Ž1993. 157–170. w6x C. Beaumont, S.K. Jain, M. Bogard, Y. Nordmann, J. Drysdale, Ferritin synthesis in differentiating Friend erythroleukemic cells, J. Biol. Chem. 262 Ž1987. 10619–10623. w7x G. Blissman, S. Menzies, J. Beard, C. Palmer, J. Connor, The expression of ferritin subunits and iron in oligodendrocytes in neonatal porcine brains, Dev. Neurosci. 18 Ž1996. 274–281. w8x A. Bomford, C. Conlon-Hollingshead, H.N. Munro, Adaptive responses of rat tissue isoferritins to iron administration. Changes in subunit synthesis, isoferritin abundance, and capacity for iron storage, J. Biol. Chem. 256 Ž1981. 948–955. w9x G. Cairo, L. Bardella, L. Schiaffonati, P. Arosio, S. Levi, A. Bernelli-Zazzera, Multiple mechanisms of iron-induced ferritin synthesis in HeLa cells, Biochem. Biophys. Res. Commun. 133 Ž1985. 314–321. w10x G. Cairo, E. Rappocciolo, L. Tacchini, L. Schiaffonati, Expression of the genes for the ferritin H and L subunits in rat liver and heart. Evidence for tissue-specific regulations at pre- and post-translational levels, Biochem. J. 275 Ž1991. 813–816. w11x P. Cheepsunthorn, C. Palmer, J.R. Connor, Cellular distribution of ferritin subunits in postnatal rat brain, J. Comp. Neurol. 400 Ž1998. 73–86. w12x P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. w13x C.C. Chou, R.A. Gatti, M.L. Fuller, P. Concannon, A. Wong, S. Chada, R.C. Davis, W.A. Salser, Structure and expression of ferritin genes in a human promyelocytic cell line that differentiates in vitro, Mol. Cell. Biol. 6 Ž1986. 566–573. w14x E.M. Coccia, E. Stellacci, R. Orsatti, U. Testa, A. Battistini, Regulation of ferritin H-chain expression in differentiating Friend leukemia cells, Blood 86 Ž1995. 1570–1579. w15x J.R. Connor, Iron acquisition and expression of iron regulatory proteins in the developing brain: manipulation by ethanol exposure, iron deprivation and cellular dysfunction, Dev. Neurosci. 16 Ž1994. 233–247.
109
w16x J.R. Connor, Iron regulation in the brain at the cell and molecular level, Adv. Exp. Med. Biol. 356 Ž1994. 229–238. w17x J.R. Connor, K.L. Boeshore, S.A. Benkovic, S.L. Menzies, Isoforms of ferritin have a specific cellular distribution in the brain, J. Neurosci. Res. 37 Ž1994. 461–465. w18x J.R. Connor, S.L. Menzies, Cellular management of iron in the brain, J. Neurol. Sci. 134 Ž1995. 33–44, Suppl. w19x J.R. Connor, S.L. Menzies, Relationship of iron to oligodendrocytes and myelination, Glia 17 Ž1996. 83–93. w20x J.R. Connor, B.S. Snyder, P. Arosio, D.A. Loeffler, P. LeWitt, A quantitative analysis of isoferritins in select regions of aged, parkinsonian, and Alzheimer’s diseased brains, J. Neurochem. 65 Ž1995. 717–724. w21x U.R. Deshpande, G.D. Nadkarni, Relation between thyroid status and ferritin metabolism in rats, Thyroidology 4 Ž1992. 93–97. w22x D.T. Dexter, A. Carayon, M. Vidailhet, M. Ruberg, F. Agid, Y. Agid, A.J. Lees, F.R. Wells, P. Jenner, C.D. Marsden, Decreased ferritin levels in brain in Parkinson’s disease, J. Neurochem. 55 Ž1990. 16–20. w23x J.H. Dussault, J. Ruel, Thyroid hormones and brain development, Annu. Rev. Physiol. 49 Ž1987. 321–334. w24x C.A. Fitch, Y. Song, C.W. Levenson, Developmental regulation of hepatic ceruloplasmin mRNA and serum activity by exogenous thyroxine and dexamethasone, Proc. Soc. Exp. Biol. Med. 221 Ž1999. 27–31. w25x Y. Iwasa, K. Aida, N. Yokomori, M. Inoue, T. Onaya, Transcriptional regulation of ferritin heavy chain messenger RNA expression by thyroid hormone, Biochem. Biophys. Res. Commun. 167 Ž1990. 1279–1285. w26x D.B. Jump, C.N. Mariash, J.H. Oppenheimer, Evidence for posttranscriptional effects of T3 on hepatic ferritin synthesis, Biochem. Biophys. Res. Commun. 104 Ž1982. 701–707. w27x P.J. Leedman, A.R. Stein, W.W. Chin, J.T. Rogers, Thyroid hormone modulates the interaction between iron regulatory proteins and the ferritin mRNA iron-responsive element, J. Biol. Chem. 271 Ž1996. 12017–12023. w28x E.A. Leibold, H.N. Munro, Characterization and evolution of the expressed rat ferritin light subunit gene and its pseudogene family. Conservation of sequences within noncoding regions of ferritin genes, J. Biol. Chem. 262 Ž1987. 7335–7341. w29x C.B. Marta, A.M. Adamo, E.F. Soto, J.M. Pasquini, Sustained neonatal hyperthyroidism in the rat affects myelination in the central nervous system, J. Neurosci. Res. 53 Ž1998. 251–259. w30x H. Ozawa, A. Nishida, T. Mito, S. Takashima, Development of ferritin-containing cells in the pons and cerebellum of the human brain, Brain Dev. 16 Ž1994. 92–95. w31x A.J.I. Roskams, J.R. Connor, Iron, transferrin, and ferritin in the rat brain during development and aging, J. Neurochem. 63 Ž1994. 709–716. w32x B. Sanyal, P.E. Polak, S. Szuchet, Differential expression of the heavy-chain ferritin gene in non-adhered and adhered oligodendrocytes, J. Neurosci. Res. 46 Ž1996. 187–197. w33x E.C. Theil, Ferritin mRNA translation, structure, and gene transcription during development of animals and plants, Enzyme 44 Ž1990. 68–82. w34x S.N. Walters, P. Morell, Effects of altered thyroid states on myelogenesis, J. Neurochem. 36 Ž1981. 1792–1801. w35x K.Y. Yeh, X. Alvarez-Hernandez, J. Glass, M. Yeh, Rat intestinal and hepatic ferritin subunit expression during development and after dietary iron feeding, Am. J. Physiol. 270 Ž1996. G498–505.