1 µ-: NADPH-dependent T3 binding in cytosol 2 3 Satoru Suzuki, Jun-ichirou Mori, Kiyoshi Hashizume 4 5 Department of Aging Medicine and Geriatrics, Institute on Aging and Adaptation, 6 Shinshu University, Graduate School of Medicine, 3-1-1, Asahi, Matsumoto, 7 390-8621, JAPAN, 8 Running title: µ-crystallin binds T3 in cytosol 9 10 key words: CRYM, thyroid hormone, 3,5,3’-triiodo-L-thyronine, CTBP, deafness, 11 crystallin 12 13 Address correspondence to: Satoru Suzuki, M.D., Ph.D., 14 Department of Aging Medicine and Geriatrics, Institute on Aging and Adaptation, 15 Shinshu University, Graduate School of Medicine, 3-1-1, Asahi, Matsumoto, 16 Nagano, 390-8621, Japan 17 TEL: +81-263-37-2686 18 FAX: +81-263-37-2710 E-MAIL: [email protected] 19 Disclosure statement: The authors have nothing to disclose. 20

1 1 Abstract 2 3 Thyroid hormone action is initiated through nuclear thyroid hormone 4 receptors. Prior to the discovery of nuclear receptors, possible major binding 5 sites were thought to be in cytosol because of high binding activity in crude 6 cytosolic fraction. Among several thyroid hormone-binding in cytosol, 7 NADPH-dependent cytosolic 3,5,3’-triiodo-L-thyronine binding protein is identical 8 to µ-crystallin, which was initially cloned as the ortholog of bacterial ornithine 9 cyclodeaminase. The expression is developmentally regulated and cell-type 10 specific. Recently, patients with nonsyndromic deafness were reported to be 11 associated with point mutations in the µ-crystallin . Cytosolic thyroid 12 hormone-binding proteins, especially µ-crystallin, play roles in adaptation to 13 environmental alterations by thyroid hormone and in thyroid hormone action, 14 which may relate to hearing function.

2 1 Introduction 2 3 The thyroid gland produces and secretes two related hormones, 4 thyroxine (T4) and 3,5,3’-triiodo-L-thyronine (T3). These hormones play pivotal 5 roles in cellular differentiation during development and help maintain 6 thermogenic and metabolic homeostasis in the adult, mainly through nuclear 7 receptors that mediate transcriptional activation in the target cells. Following 8 secretion of the hormone from the thyroid gland, T4 binds preferentially to 9 thyroxine-binding globulin in serum. Recent findings showed that thyroid 10 hormones enter mainly through active transporters [1]. Cells have multiple 11 thyroid hormone binding proteins, including nuclear thyroid hormone receptors 12 (TRs). Protein binding analyses using radioisotopes demonstrated that several 13 T3 binding proteins are present in the endoplasmic reticulum [2], mitochondria 14 [3], nuclear envelope [4], and cytoplasm. In this article, we focus on the T3 15 binding proteins in the soluble cytosolic fraction. As there is a great deal of 16 evidence that these proteins are functional, we review and mainly discuss the 17 physiological roles of NADPH-dependent cytosolic T3 binding protein (CTBP), 18 identified as µ-crystallin (CRYM). 19 20 CTBP is identical to CRYM. 21 22 Cytosolic thyroid hormone binding proteins were first described by Tata [5], 23 who presented evidence based on paper chromatography that a specific 24 cytosolic extract from rat skeletal muscle bound [131I]T4. Hamada et al. first 25 demonstrated the possibility that proteins that bind specifically to triiodothyronine 26 are present in the soluble cytosolic fraction [6]. 27 In 1986, it was reported that charcoal treatment of rat renal cytosol 28 abolishes the hormone binding activity, while the addition of NADPH restores 29 this activity [7]. In 1991, NADPH-dependent CTBP was identified and purified 30 from the rat liver. This CTBP has a molecular mass of 76,000 Da and consists of 31 a 38,000-Da peptide dimer with an affinity constant for L-T3 binding of 2.4 32 liter/nmol [8]. Two L-T3 molecules bound to a 76,000-Da unit. In contrast to

3 1 NADPH, NADP suppresses T3 binding activity. Iodothyronine analog-binding 2 specificity is L-T3=D-T3>L-T4>Triac, which differs from that of the nuclear 3 receptors. In 1997, CTBP was identified by protein sequencing analysis of 4 purified human CTBP [9]. The cloned CTBP was CRYM, which had already been 5 cloned as an ortholog of bacterial ornithine cyclodeaminase [10]. CRYM is a 6 taxon-specific protein, which is particularly abundant in kangaroo lens [11]. The 7 protein was also expressed in the human retina, brain, heart, and kidney. 8 9 Physiological function of CRYM as a T3 binder 10 11 Although the T3 binding of CRYM has been demonstrated in vitro, it has 12 not been determined whether the expression of CRYM molecule alters the T3 13 concentration in living cells. To approach this issue, permanent 14 CRYM-expressing GH3 cell lines were established [12]. The expression of 15 CRYM increased cellular uptake of T3 and the efflux rate was also decreased by 16 induction of CRYM. Although these findings indicate that the expression of 17 CRYM increases the concentration of intracellular T3, CRYM expression 18 suppressed the T3-regulated luciferase activity in a series of GH3 cell lines and 19 suppression correlated with the expression level of CRYM. Furthermore, T3 20 induction of rat growth hormone mRNA was lower in cells expressing CRYM 21 than in CRYM-null cells, suggesting that the expression of CRYM suppressed 22 T3-mediated transactivity in CRYM-expressing cells (Fig 1). We further noted 23 that intracellular free T3 concentration is also a crucial factor in peripheral thyroid 24 hormone action. Because of technical problems, it is difficult to discriminate 25 between free and bound fractions of intracellular T3. However, it is speculated 26 that the NADPH-dependency of T3 binding activity on CRYM may affect the free 27 T3 concentration in cytoplasm. 28 To further investigate the in vivo functions of CRYM gene products, we 29 generated mice with targeted disruption of the CRYM gene, which abrogates the 30 production of CRYM [13]. CRYM-knockout mice showed loss of the entire 31 NADPH-dependent T3 binding activity in the cytosol of the brain, kidney, heart, 32 and liver. In the euthyroid state, knockout significantly reduced the serum

4 1 concentrations of T3 and T4 despite normal growth and normal heart rate. 2 Disruption of the gene did not alter the expression of TSHβ mRNA in the pituitary 3 gland. In addition, disruption did not alter the mRNA expressions of glutathione S 4 transferase alpha 2 or deiodinase 1, which are negatively and positively 5 regulated by T3, respectively, in either the liver or kidney. When radiolabeled T3 6 was injected intravenously, labeled T3 entered rapidly into and then escaped 7 from the tissues in CRYM-knockout mice. These observations suggest that, due 8 to rapid T3 turnover, disruption of the CRYM gene decreases T3 concentrations 9 in tissues and serum without alterations of peripheral T3 action in vivo. 10 To date, CRYM-null patients have not been reported. Based on the results 11 of the knockout study, it is anticipated that the concentration of T3 would be low 12 in putative CRYM-null patients. The concentration of thyroid hormone was not 13 remarkable in a patient with CRYM mutation (K314T) (S. Suzuki et al. 14 unpublished), indicating that residual intact CRYM protein expressed in the 15 patient may function in the regulation of thyroid hormone. Pathophysiologically, 16 we speculate that some part of the non-thyroidal illness may be caused by the 17 alteration of CRYM expression in peripheral tissues. 18 Deletion analyses using bacterially expressed mutant proteins indicated 19 that two separate domains are crucial for dimer formation [14]. K314T, which 20 was a mutation at the 3’ end of the amino acids, abolished NADPH-dependent 21 T3 binding [15]. The molecular structure of CRYM has recently been determined 22 by crystallographic analyses. The crystal structure of CRYM demonstrated the 23 presence of an NADPH binding site, dimerization domains, and a putative T3 24 binding site in the molecule [16]. 25 26 CRYM and hearing function 27 28 Microarray analysis demonstrated that CRYM is highly expressed at the 29 mRNA level in the human inner ear [17]. Immunohistochemical analyses showed 30 that the protein is expressed preferentially in type II fibrocytes where Na-K 31 ATPase is enriched [15]. As hearing is one of the most important functions 32 controlled by thyroid hormone, CRYM expression may be related to hearing

5 1 function. 2 There have been reports of two families with hereditary nonsyndromic 3 deafness possessing mutations in CRYM mRNA. One mutation (K314T) 4 eliminated NADPH-dependent T3 binding and patients with K314T showed 5 severe deafness. The other mutation (X315Y) did not alter the binding activity, 6 and the hearing ability in these patients was moderate. These data suggest that 7 T3 binding properties affect the clinical symptoms of deafness [15]. However, 8 auditory-evoked response was normal in CRYM-knockout mice, suggesting that 9 deletion of the gene products does not affect hearing function [13]. The 10 abnormal cellular distribution of mutant protein was demonstrated in a previous 11 article, indicating that expression of abnormal mutant proteins may affect clinical 12 hearing ability [17]. 13 14 Expression of CRYM 15 16 As CRYM may contribute to tissue development through transportation 17 or retention of T3 in the cytoplasm, NADPH-dependent T3 binding activity was 18 assessed in various rat tissues at different developmental stages [18]. The 19 affinity constant was approximately 2×109/M in the tissues studied. While 20 maximal binding capacity was quite low at birth, it increased as development 21 progressed in the kidney, liver, and heart. In the brain, NADPH-dependent T3 22 binding to CRYM, detected in the fetal stage, increased transiently 2 weeks after 23 birth, then increased gradually until maturation. These observations imply that 24 CRYM supplies T3 during the development of the central nervous system. 25 The expression of CRYM mRNA was also assessed. In the central 26 nervous system, high levels of expression were observed in the telencephalon, 27 with low levels in the brainstem and spinal cord [19]. Microarray experiments for 28 serial analysis of gene expression demonstrated that CRYM was also expressed 29 in the nucleus accumbens and medial striatum, but not in the lateral striatum [20]. 30 The heart showed a high level of expression in humans. Moderate expression 31 was detected in the cerebellum and pituitary. The protein expression pattern was 32 similar to the distribution of mRNA expression: i.e., high levels of expression in

6 1 the brain, heart, and kidney. Intriguingly, mosaic expression was observed in 2 granule cells of the human cerebellum. In the human kidney, the renal tubes 3 were stained in contrast to reduced staining in the glomeruli. In the pancreas, 4 islet cells showed positive staining. Thus, the expression of this protein is not 5 only time-specific, but also cell-type specific. 6 CRYM was first demonstrated in the kangaroo lens. Thus, the 7 localization of the protein was also evaluated in the mammalian eye [21]. CRYM 8 was shown to be expressed preferentially in rod cell outer segments of the rat 9 retina. Proteome experiments demonstrated anti-CRYM autoantibody in sera 10 obtained from monkeys with late-onset macular degeneration, indicating that 11 autoimmune responses to CRYM are associated with the progression of macular 12 degeneration in the retina [22]. These observations imply that CRYM may relate 13 to visual function in addition to nuclear T3 receptors as described previously 14 [23]. 15 16 Regulation of CRYM expression 17 18 As expression is both time- and cell-type specific, it is proposed that 19 multiple factors control the NADPH-dependent T3 binding activity, including the 20 level of CRYM expression. NADPH-dependent T3 binding was positively

21 regulated by [24], activated vitamin D3 (1,25 (OH)2D3) [25], 22 insulin [26], and sodium butyrate [27] in cultured cells. In rats, a vitamin 23 D-deficient diet suppressed NADPH-dependent T3 binding. This suppression 24 was reversed by the addition of vitamin D. Streptozotocin-induced diabetic rats 25 show low binding capacity of CRYM in the liver [26], and insulin treatment was 26 shown to promote recovery of the binding capacity. 27 Following the development of microarray techniques, several studies 28 demonstrated the up- and down-regulation of CRYM expression. Clinically, 29 glucocorticoid induced CRYM mRNA in human myeloid cells [28]. The mRNA 30 expression was elevated in the failing heart in patients who died of trauma or 31 cardiomyopathy [29]. The level of expression in the dorsal prefrontal cortex of 32 the brain was low in the majority of subjects with schizophrenia [30].

7 1 As thyroid hormone acts on cellular differentiation, carcinogenesis may 2 be associated with the action of thyroid hormone through CRYM expression. All 3 cancer cell lines examined for CRYM expression showed quite low levels of 4 expression, with the exception of rat hepatoblastoma dRLh-84 cells [25]. CRYM 5 showed a significantly lower mRNA level in hormone-refractory prostate tumors 6 compared with that in primary tumors [31]. CRYM expression was significantly 7 elevated in non-small cell lung carcinoma [32], while the expression level was 8 low in brain tumors [33]. 9 In rodents, several observations have suggested that expression is 10 controlled during the cycle of cellular development. The expression level was 11 shown to be regulated during development of hair follicles in mice [34]. CRYM 12 expression was reduced in ovarian follicles 6 h after FSH treatment relative to 13 that in untreated cells [35]. In situ hybridization studies using randomly cycling 14 adult rats demonstrated that CRYM mRNA was highly expressed only in small 15 growing follicles of the ovary [36]. 16 A recent article demonstrated a high expression of µ-crystallin in the 17 muscle of patients with facioscapulohumeral musclar dystrophy (FSHD) [37]. 18 FSHD is a musclar dystrophy that is thought to be caused by the deletion of the 19 megasatellite repeat on 4q35. The disorder is associated with 20 nonskeletal muscle manifestations include high-frequency hearing loss as well 21 as retinal telangiecatasias. Thus, a high expression of µ-crystallin protein may 22 affect both hearing function and retinal development. 23 Although there is limited evidence at present that alteration of CRYM 24 expression affects physiological or pathological responses in vivo, such research 25 may identify novel molecular mechanisms in the field of thyroid hormone action. 26 27 Conclusions and Perspectives 28 29 In conclusion, CRYM plays pivotal roles in physiological regulation of 30 thyroid hormone action in vivo. The CRYM-mediated retention of T3 affects 31 thyroid hormone concentration in the extranuclear space, resulting in the

8 1 modification of thyroid hormone action in the nuclei. Clinically, although the 2 molecular mechanisms are not well understood, the mutant proteins are known 3 to affect normal development of the inner ear. The binding of pyridine 4 nucleotides to the protein implies that CRYM may possess redundant functions, 5 such as enzyme activity like that of oxidoreductases. These oxidative and 6 reductive mechanisms may alter the free thyroid hormone concentration in the 7 intracellular space, and may also regulate the action of the hormone. The 8 expression of the protein was also regulated in individual cells by multiple factors, 9 indicating that the concentration of intracellular free hormone may also be 10 regulated by numerous factors. However, we propose that the complexity 11 described above may be required in terms of adaptation to environmental 12 alterations by thyroid hormone concentration and other factors that affect thyroid 13 hormone action. 14 Acknowledgement 15 We thank Ms. Izumi Kinoshita for excellent secretarial work.

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13 1 Figure Legend 2 3 Fig. 1 Molecular functions of µ-crystallin on thyroid hormone action. 4 5 T3 moves from outside the plasma membrane into the cytoplasm of the cells. 6 Free T3 enters into the nucleus and initiates the transactivation through binding 7 to nuclear T3 receptors. In the presence of µ-crystallin, the µ-crystallin molecule 8 forms a dimer in the cytoplasm. Each molecule binds one molecule of T3 and 9 NADPH. In the presence of NADPH, the T3-bound form of µ-crystallin increases 10 the T3 concentration in the cytoplasm. Although the mechanisms have not yet 11 been thoroughly elucidated, the expression of µ-crystallin suppresses the 12 transcriptional activity. Regulation of T3 action through µ-crystallin is composed 13 of (1) binding of reduced NADP i.e. NADPH, (2) dimerformation, (3) creation of 14 the T3 binding site, (4) binding of T3, (5) release of T3 by dissociation of the 15 NADPH, (6) induction of the transactivation, and (7) suppression of the 16 transactivation 17 18 CRYM: µ-crystallin, T3: 3,5,3’-triiodo-L-thyronine, TR: thyroid hormone nuclear 19 receptor, RXR: retinoid-x-receptor, NADP: nicotinamide adenine dinucleotide 20 phosphate, NADPH: reduced nicotinamide adenine dinucleotide phosphate 21

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