Transthyretin Blocks Retinol Uptake and Cell Signaling by the Holo-Retinol-Binding Protein Receptor STRA6

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Transthyretin blocks retinol uptake and cell signalling by the holo-retinol-binding protein 1

receptor STRA6 2

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Daniel C. Berry1,2, Colleen M. Croniger2, Norbert B. Ghyselinck3, and Noa Noy1,2* 6

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1Departments of Pharmacology and 2Nutrition, Case Western Reserve University School of 8

Medicine, Cleveland, OH 44106, USA, 3Institut de Génétique et de Biologie Moléculaire et 9

Cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS UMR7104), Institut 10

National de la Santé et de la Recherche Médicale (INSERM) U964, Université de Strasbourg, 11

Illkirch, France 12

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Running Title: TTR inhibits STRA6 activities 15

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*Address correspondence to this author at Department of Pharmacology, Case Western Reserve 18

University School of Medicine, 10900 Euclid Ave. W333, Cleveland, OH 44106. Tel: 216-368-19

0302, Fax: 216-368-1300, E. mail: [email protected] 20

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.00775-12 MCB Accepts, published online ahead of print on 23 July 2012

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Abstract 23

Vitamin A is secreted from cellular stores and circulates in blood bound to retinol binding 24

protein (RBP). In turn, holo-RBP associates in plasma with transthyretin (TTR) to form a ternary 25

RBP-retinol-TTR complex. It is believed that binding to TTR prevents the loss of RBP by 26

filtration in the kidney. At target cells, holo-RBP is recognized by STRA6, a plasma membrane 27

protein that serves a dual role: it mediates uptake of retinol from extracellular RBP into cells and 28

it functions as a cytokine receptor that, upon binding holo-RBP, triggers a JAK/STAT signalling 29

cascade. We previously showed that STRA6-mediated signalling underlies the ability of RBP to 30

induce insulin resistance. However, the role that TTR, the binding partner of holo-RBP in blood, 31

in STRA6-mediated activities remained unknown. Here we show that TTR blocks the ability of 32

holo-RBP to associate with STRA6 and thereby effectively suppresses both STRA6-mediated 33

retinol uptake and STRA6-initiated cell signalling. Consequently, TTR protects mice from RBP-34

induced insulin resistance reflected by reduced phosphorylation of insulin receptor and glucose 35

tolerance tests. The data indicate that STRA6 functions only under circumstances where plasma 36

RBP levels exceeds that of TTR and demonstrate that, in addition to preventing the loss of RBP, 37

TTR plays a central role in regulating holo-RBP/STRA6 signalling. 38

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Introduction 41

Vitamin A (retinol, ROH) plays critical roles both in the embryo and in the adult where it 42

regulates multiple cellular processes and is essential for embryonic development, reproduction, 43

immune function and vision (29, 32, 33). The vitamin exerts many of its biological activities by 44

giving rise to active metabolites: the visual chromophore 11-cis-retinaldehyde and retinoic acid 45

(RA), which regulates gene transcription by activating specific nuclear receptors (11, 27). ROH 46

is stored in various tissues, including white adipose tissue (WAT), lung, and retinal pigment 47

epithelium in the eye but its main storage site is the liver. ROH is secreted from storage into the 48

circulation bound to retinol-binding protein (RBP), a 21 KDa polypeptide that contains one 49

binding site for ROH. In most mammals, ROH-bound RBP (holo-RBP) does not circulate alone 50

but is associated with another protein called transthyretin (TTR), a 56 KDa homotetramer that, in 51

addition to associating with RBP, functions as a carrier for thyroid hormones (23, 24). ROH thus 52

reaches target tissues bound in a holo-RBP-TTR complex that, under normal circumstances, 53

displays a 1:1 molar stoichiometry. It is believed that binding of RBP to TTR serves to prevent 54

the loss of the smaller protein from blood by filtration in the glomeruli. The concentration of the 55

holo-RBP-TTR complex in plasma is kept constant at 1-2 µM except in extreme cases of vitamin 56

A deficiency or in disease states. Notably, RBP levels are markedly elevated in blood of obese 57

mice and humans and it was reported that, under these circumstances, the protein induces insulin 58

resistance (35). 59

Association with the TTR-RBP complex allows the poorly soluble ROH to circulate in 60

blood but the vitamin dissociates from RBP prior to entering cells. It was proposed that, due to 61

its hydrophobic nature, ROH can readily move from extracellular RBP into cells by diffusion 62

across the plasma membranes at fluxes that are dictated by its extra-to-intra-cellular 63


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concentration gradient (10, 14, 20, 21). However, it has also been suggested that uptake of ROH 64

from circulating holo-RBP is mediated by a cell-surface receptor (13, 28). Indeed, a plasma 65

membrane protein termed STRA6 (Stimulated by Retinoic Acid 6) was found to bind holo-RBP 66

and transport ROH into cells (15). Our recent studies revealed that, in addition to its function as 67

an ROH transporter, STRA6 is a cytokine receptor. We thus found that binding of holo-RBP 68

triggers phosphorylation of a tyrosine residue in the cytosolic domain of STRA6, resulting in 69

recruitment and activation of the Janus kinas JAK2 and, in a cell-dependent manner, the 70

transcription factors STAT3 or STAT5. Holo-RBP thus activates STRA6-mediated signalling 71

that culminates in upregulation of STAT target genes (2, 4). As STAT target genes in white 72

adipose tissue (WAT) and muscle include Suppressor of cytokine signalling 3 (Socs3), a potent 73

inhibitor of insulin signalling (8), these findings suggested a rationale for understanding how 74

elevated serum levels of RBP in obese animals induce insulin resistance (35). Additional studies 75

showed that activation of STRA6 is triggered not simply by binding of holo-RBP but by a 76

STRA6-mediated translocation of ROH from extracellular holo-RBP to an intracellular acceptor, 77

the retinol-binding protein CRBP-I. Importantly, this movement was found to be critically linked 78

to the intracellular metabolism of ROH (5). The data further established that ROH uptake and 79

signalling by STRA6 are interdependent, i.e. that activation of a JAK2/STAT cascade by the 80

receptor requires ROH uptake and vice versus that phosphorylation of STRA6 is essential for 81

enabling ROH transport to proceed (5). 82

While these recent studies provided surprising new insights into the involvement of 83

STRA6 in vitamin A biology, the role that TTR, the binding partner of holo-RBP in blood, may 84

play in STRA6-mediated functions remained unknown. Here, we show that TTR blocks the 85

ability of holo-RBP to associate with STRA6 and thereby effectively suppresses both STRA6-86


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mediated ROH uptake and STRA6-initiated cell signalling. We show further that, consequently, 87

TTR protects mice from RBP-induced insulin resistance. The data indicate that, in addition to 88

preventing the loss of RBP by filtration in the kidney, TTR plays a central role in regulating 89

holo-RBP/STRA6 signalling. 90

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Materials and Methods 92

Reagents. Human STRA6 was cloned as N-terminal hexahisitidine tagged protein in a 93

pReciever-M01 vector (Genecopoeia). Point mutations of RBP were generated using the 94

Stratagene Quikchange II mutagenesis kit. Lentiviruses harboring shRNA for STRA6, were 95

purchased from Openbiosystems. Antibodies against actin and hexahistidine were from Santa 96

Cruz biotechnology. Antibodies against JAK2, pJAK2, pAKT, pIR, IR, pSTAT5, STAT5 and 97

phospho-tyrosine were from Cell Signaling. RBP antibodies were purchased from Dako. 98

Retinol and bovine insulin were purchased from Sigma Chemical Co. and retinol was purchased 99

from Calbiochem. 100

Immunoblots and immunoprecipitations were performed as previously described (2). 101

Mouse Studies. Mice with a mixed C57BL/6-129/Sv (50%–50%) genetic background were 102

maintained on a 12 h light and dark cycle on a normal chow diet. Mice were housed according to 103

ARC protocol. The breeding diets (Diet #5P76 from LabDiet) contained 25,000-29,000 IU of 104

vitamin A per kg. The mice had access to water and diet ad libitum. 105

Cells. HepG2 and NIH3T3 cells were cultured in DMEM supplemented with 10% FBS and 10% 106

calf serum, respectively. NIH3T3-L1 preadipocytes were induced to differentiate and 107

differentiation was verified as previously described (3). 108


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Analysis of mRNA. RNA was extracted using TRIZOL (Molecular Research Center). cDNA 109

was generated using GeneAmp RNA PCR (Applied Biosystems). Q-PCR was carried out using 110

TaqMan chemistry and Assays-on-Demand probes (Applied Biosystems) for hSTRA6 111

(Hs00223621_m1), hSOCS3 (Hs02330328_g1), mSOCS3 (Mm01249143), mPPARγ 112

(Mm00440945_m1) and 18s rRNA (4352930). RNA was extracted from lipid tissues and 113

skeletal muscle using RNeasy lipid tissue minikit (Qiagen cat#74804) and RNeasy fibrous tissue 114

minikit (cat#74704). 115

Recombinant proteins. Bacterial expression vectors for histidine-tagged hRBP and hTTR were 116

respectively provided by Lawreen Connors, Boston University School of Medicine and Silke 117

Vogel, Columbia University. RBP and TTR were expressed in E. coli and purified as previously 118

described (see (34) for RBP and (16) for TTR. Purified proteins were dialyzed against 300 mM 119

NaCl, 100 mM Tris pH 7.4, and 5% glycerol. For RBP, the method generates holo-RBP, i.e. an 120

RBP-ROH complex at a 1:1 mole ratio. 121

Vitamin A uptake by cultured cells. Holo-RBP was incubated with 3H-retinol (~5000 122

cpm/nmole) for 2 h on ice. In experiments including TTR, 3H-labeled holo-RBP was 123

precomplexed with TTR prior to experimentation. Cultured cells were placed in serum free 124

media for 12 h prior to experimentation. Cells were then washed three times with phosphate-125

buffered saline (PBS), placed in serum free media and treated with 1 µM 3[H]-RBP-ROH or 126

3[H]-RBP-ROH-TTR for three minutes. Cells were washed twice with PBS and then placed in 127

100% ethanol for ten minutes. The ethanol phase was immediately counted using a scintillation 128

counter (Beckman Coulter LS6500). Protein content of each well was measured using the 129

Bradford assay and used for data normalization. 130


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Uptake of ROH from holo-RBP in vivo. Mice fed a regular chow diet were intraperitoneally 131

injected with 0.2 mg holo-RBP or holo-RBP-TTR containing 0.02 mCi 3H-ROH-RBP (total 132

volume 60 μl). Two hours post injection, a blood sample was collected and hearts were perfused 133

with 0.9% saline to remove blood containing labeled ROH. Tissues were isolated, weighed and 134

stored in liquid nitrogen. Tissues were homogenized in PBS, and homogenates placed in 135

scintillation fluid and counted (Beckman Coulter). Counts were normalized per gram tissue. 136

Glucose tolerance tests (GTT). Mice were fasted for 12 hours and injected intraperitoneally with 137

glucose (2 g kg-1 body weight). Blood was sampled from the tail vein and at 0, 15, 30, 60, and 138

120 minutes using an UltraTouch or Accu-Chek Performa glucometer. 139

Flurorescence titrations. Binding of ROH to RBP or its mutant was monitored by fluorescence 140

titrations as described (7). Prior to titrations, ROH was extracted from purified holo-RBP as 141

described (7). Ligand-binding was monitored by following the increase in ROH fluorescence 142

(λex=325; λem=480nm) that accompanies binding to the protein. Titration curves were 143

analyzed by fitting the data to an equation derived from simple binding theory (19) using the 144

software Origin (MicroCal). 145

Fluorescence anisotropy titrations. Association between holo-RBP and TTR was monitored by 146

by following the increase in fluorescence anisotropy of RBP-bound ROH (λex=325; 147

λem=480nm) that accompanies complex formation. Measurements were carried out using a 148

Photon Technology International Quantamaster spectrofluorometer equipped with Glan-149

Thompson polarizers. 150

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RESULTS 152


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TTR inhibits STRA6-mediated uptake of ROH from holo-RBP. In most mammals, holo-RBP 153

circulates in blood in complex with transthyretin (TTR). To begin to examine the effect of TTR 154

on STRA6 function, hepatocarcinoma HepG2 cells, which endogenously express STRA6, were 155

used to compare cellular uptake of ROH from holo-RBP and from TTR-bound holo-RBP. 156

Recombinant RBP and TTR were expressed in E. coli and purified (see Materials and Methods). 157

HepG2 cells were treated with RBP complexed with 3H-retinol at a 1 μM concentration, similar 158

to the serum RBP level, or with 1 μM 3H-retinol-labeled RBP complexed with TTR at a 1:1 159

molar stoichiometry, similar to that found in blood (24). Media were removed, cells washed, 160

organic compounds extracted from the cells into ethanol, and the amount of 3H-retinol taken up 161

within the incubation period measured by scintillation counting. The rates of uptake of retinol 162

under the assay conditions were constant during the initial 5 min. (Fig. 1a) and subsequent 163

experiments were carried out with a single 3 min. time point, well within the initial linear rate. 164

The rate of ROH uptake from the holo-RBP-TTR complex was slower than uptake from holo-165

RBP alone (Fig. 1a). Moreover, increasing the TTR/RBP ratio by increasing the concentration of 166

TTR inhibited ROH uptake in a dose dependent manner (Fig. 1b). The dose response of the 167

initial rate of ROH transport from holo-RBP showed a two phase behavior comprised of an 168

initial saturable component, likely attributable to STRA6-mediated uptake, followed by a non-169

saturable phase, reflecting passive diffusion of ROH across the plasma membranes (Fig. 1c). In 170

contrast, uptake of ROH from the holo-RBP-TTR complex displayed a single, non-saturable 171

phase (Fig. 1c). These observations suggest that TTR does not impede the ability of ROH to 172

enter cells by passive diffusion but that it effectively blocks ROH transport mediated by STRA6. 173

In agreement with this notion, increasing the expression level of STRA6 in HepG2 cells (Fig. 1d) 174

facilitated ROH uptake from holo-RBP in a dose-responsive manner but had no effect on 175


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transport of ROH from TTR-bound holo-RBP (Fig. 1e). Also in agreement, decreasing the 176

expression of STRA6 in HepG2 cells (Fig. 1f) or in NIH3T3-L1 adipocytes (Fig. 1g) reduced the 177

rate of ROH uptake from holo-RBP but did not affect uptake from TTR-bound holo-RBP. The 178

observation that, in both cell lines, rates of uptake from the holo-RBP-TTR complex were similar 179

to those observed in the absence of STRA6 supports the conclusion that TTR specifically inhibits 180

STRA6-mediated transport. 181

The effect of TTR on ROH uptake from holo-RBP was then examined in vivo using our 182

newly generated STRA6-null mice (26). Twelve week old WT and STRA6-null male mice were 183

injected intraperitoneally with 3H-ROH-labeled holo-RBP or with holo-RBP complexed with 184

TTR, and ROH uptake into tissues was assessed 2 h later. Uptake of ROH into the STRA6-185

expressing tissues WAT, skeletal muscle, and the eye was modestly but significantly lower in 186

STRA6-null vs. WT mice (Fig. 1h), reflecting that the contribution of STRA6 to overall vitamin 187

A uptake by tissues in vivo is small. ROH uptake from TTR-bound holo-RBP was all but 188

identical to that observed in STRA6-/- animals (Fig. 1h). Neither ablation of STRA6 nor the 189

presence of TTR affected ROH uptake by the liver, an organ that does not express STRA6 (Fig. 190

1h). Hence, TTR specifically inhibits STRA6-mediated uptake of ROH in vivo. 191

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TTR inhibits the association of holo-RBP with STRA6. STRA6 may bind the ternary RBP-193

ROH-TTR complex or, alternatively, it may recognize only free holo-RBP. To dissect between 194

these possibilities, we considered that, unlike in most mammals, holo-RBP in zebrafish (Danio 195

rerio) does not associate with TTR. Thus, presumably, zebrafish STRA6 does not contain a 196

TTR-binding region and, while ROH uptake by the mammalian STRA6 may involve recognition 197

of TTR, ROH uptake by zebrafish STRA6 (dSTRA6) will not. In these experiments, NIH3T3 198


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fibroblasts, which do not endogenously express STRA6, were used. We previously showed that 199

ROH metabolism is essential both for STRA6-mediated ROH transport and for holo-RBP-200

induced STRA6 signalling (5). Hence, to enable STRA6 action in these cells, an NIH3T3 line in 201

which ROH metabolism is enhanced by stably over-expressing lecithin:ROH-acyltransferase 202

(LRAT), which catalyzes ROH esterification, was generated. Ectopic over-expression of either 203

hSTRA6 or dSTRA6 in LRAT-expressing NIH3T3 fibroblasts enhanced ROH uptake from holo-204

RBP to a similar extent and introduction of TTR similarly decreased the rate of uptake (Fig. 2a, 205

2b). The similarity of the response of dSTRA6, which is unlikely to contain a TTR-binding 206

capability, to that of hSTRA6 suggests that STRA6 in both species recognizes only free and not 207

TTR-bound holo-RBP. 208

The question of whether STRA6 binds free or TTR-bound holo-RBP was then directly 209

assessed. Recombinant holo-RBP was incubated alone or in the presence of TTR with the 210

chemical cross-linker Bis[sulfosuccinimidyl] suberate (0.5 mM, 14 h.), resulting in efficient 211

cross-linking of the holo-RBP-TTR complex (Fig. 2c). The mixtures and additional cross-linker 212

were added to NIH3T3 cells ectopically over-expressing histidine-tagged STRA6. STRA6 was 213

immunoprecipitated, precipitated proteins were resolved by SDS-PAGE, and immunoblotted for 214

RBP-containing complexes (Fig. 2d). Cross-linking of cells with holo-RBP resulted in the 215

appearance of a band with a molecular weight of ~100 KDa, corresponding to that of an RBP-216

bound STRA6. No such band was observed in cells cross-linked with the RBP-ROH-TTR 217

complex and no bands which may correspond to a STRA6-RBP-TTR (~150 KDa) appeared. The 218

data thus indicate that STRA6 associates only with free holo-RBP and that the presence of TTR 219

prevents the association. 220


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To further examine whether TTR inhibits STRA6-mediated ROH uptake by preventing 221

holo-RBP from binding to the receptor, an RBP mutant defective in its ability to bind TTR was 222

generated. The reported three dimensional crystal structure of the holo-RBP-TTR complex 223

suggests that the interactions between the two proteins are mediated by several residues 224

including Phe96 and Leu97 (18). An RBP mutant in which these residues were replaced with 225

alanines (RBP-F96A/L97A) was thus generated. The mutations did not alter the affinity of RBP 226

for retinol (Fig. 2e), indicating that the overall fold of the mutant is intact. As expected, the 227

F96A/L97A mutations disrupted the association of RBP with TTR (Fig. 2f). Measurements of 228

ROH uptake showed that, in contrast with its inhibitory activity on ROH uptake from WT-RBP, 229

TTR had no effect on ROH uptake from RBP-F96A/L97A (Fig. 2g). These observations further 230

establish that TTR inhibits STRA6-mediated ROH uptake by sequestering holo-RBP and not by 231

direct association with the receptor. 232

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TTR inhibits holo-RBP-induced STRA6 signalling. The effect of TTR of RBP-induced 234

STRA6 signalling was then examined using NIH3T3-L1 adipocytes. We previously showed that 235

in, in these cells, activation of STRA6 by holo-RBP triggers a JAK2/STAT5 cascade to induce 236

the STAT target genes SOCS3 and PPARγ and inhibit insulin responses (2). Preadipocytes 237

NIH3T3-L1 cells were grown two days past confluence and induced to differentiate using a 238

standard hormone mix (10 µg/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 239

mM dexamethasone). Three days later, media were replaced and cells grown for four days. 240

Differentiation was verified by monitoring lipid accumulation and by examining the expression 241

of the adipocyte marker FABP4 (3). As expected, treatment of differentiated adipocytes with 242

holo-RBP (R-R) increased the phosphorylation levels of JAK2 and STAT5 (Fig. 3a). In contrast, 243


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the holo-RBP-TTR complex did not alter the phosphorylation status of these proteins (Fig. 3a). 244

Accordingly, TTR-bound holo-RBP failed to induce the expression of SOCS3 and PPARγ (Fig. 245

3b). To examine the effect of TTR on the ability of holo-RBP to suppress insulin responses, 246

cells were pre-treated with holo-RBP or holo-RBP-TTR for 8 h., treated with insulin for 15 min., 247

and the levels of phosphorylation of the insulin receptor (IR) and its downstream effector AKT 248

were monitored. The data show that inhibition of insulin-induced phosphorylation of IR and 249

AKT by holo-RBP was blunted in the presence of TTR (Fig. 3c). TTR also inhibited the ability 250

of holo-RBP, but not of holo-RBP-F96A/L97A, defective in TTR binding, to trigger STAT5 251

phosphorylation (Fig. 3d) or to induce the expression of SOCS3 in NIH3T3-L1 adipocytes (Fig. 252

3e) or in HepG2 cells (Fig. 3f). 253

The effect of TTR on signaling by the zebrafish STRA6 was then examined. Notably, the 254

phosphotyrosine in the cytosolic domain of STRA6, the STAT recruitment site of the receptor, is 255

present in the dSTRA6, suggesting evolutionary conservation of STRA6 signalling (Fig. 3g). In 256

these experiments, NIH3T3 fibroblasts that ectopically over-express LRAT were transfected 257

with expression vectors for either hSTRA6 or dSTRA6. Treatment of cells expressing either 258

hSTRA6 or dSTRA6 with holo-RBP-induced phosphorylation of STAT3, the preferred STRA6-259

activated STAT in these cells (Fig. 3h) and upregulation of SOCS3 (Fig. 3i). TTR suppressed the 260

ability of holo-RBP to induce STAT3 phosphorylation and to upregulate SOCS3 expression in 261

cells expressing either hSTRA6 or dSTRA6 (Fig. 3h, 3i). 262

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TTR inhibits the ability of holo-RBP to suppress insulin responses in vivo. The effect of TTR 264

on the ability of holo-RBP to promote insulin resistance in vivo was then investigated. Eight 265

week old mice were injected with recombinant holo-RBP, TTR, or holo-RBP-TTR. Mice were 266


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injected three times at 2 h. intervals and sacrificed an hour after the last injection. The 267

treatments resulted in respective elevation of serum levels of RBP, TTR, or both (Fig. 4a, 4b). 268

As expected, treatment of mice with holo-RBP reduced the phosphorylation levels of the insulin 269

receptor and AKT and induced the expression of SOCS3 and PPARγ in WAT (Fig. 4c, 4f) and 270

skeletal muscle (Fig. 4d, 4g) but not in liver (Fig. 4e, 4h). In contrast, treatment with RBP-ROH-271

TTR did not affect the phosphorylation of IR and AKT or the expression levels of the STAT 272

target genes (Fig. 4c-4h). 273

The observations that only free and not TTR-bound holo-RBP activates STRA6 suggest 274

that the serum RBP/TTR ratio is key for regulating STRA6 signalling. In agreement with the 275

report that expression of RBP in adipose tissue increases in obese rodents and humans, resulting 276

in elevation of serum RBP levels (35), feeding mice a high fat high sucrose (HFHS) diet for 10 277

weeks resulted in upregulation of the expression of RBP in WAT but not in liver (Fig. 5a). In 278

contrast, TTR expression in these organs was not affected by the diet (Fig. 5b). Accordingly, 279

serum level of RBP was markedly elevated while serum level of TTR remained unchanged in 280

obese mice (Fig. 5c). Hence, the RBP/TTR ratio is significantly higher in blood of obese vs. lean 281

mice. 282

To directly determine if TTR prevents holo-RBP- induced insulin resistance, mice were 283

treated with holo-RBP or holo-RBP-TTR for three weeks prior to carrying out glucose tolerance 284

tests (GTT). Mice were treated by implanting alzet osmotic pumps containing the appropriate 285

proteins (1 μM) thereby delivering constant amounts of proteins over the three week period. 286

Similarly to the short-term treatments (Fig. 4), three week treatment of mice with holo-RBP 287

induced phosphorylation of STAT5, reduced the activation level of IR and upregulated SOCS3 288

and PPARγ in WAT (Fig. 5e, 5g) and muscle (Fig. 5f, 5h) but not in the liver (Fig. 5i). In 289


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contrast, treatment with TTR-bound holo-RBP had no effect on the phosphorylation of STAT5 290

or IR and did not alter the expression levels of the STAT target genes (Fig. 5e-5i). Accordingly, 291

while holo-RBP treatment resulted in a sluggish response in GTT, reflecting the development of 292

insulin resistance, treatment with the holo-RBP-TTR complex did not alter the insulin responses 293

of the mice (Fig. 5j). Hence, association with TTR suppresses the ability of holo-RBP to 294

interfere with insulin signaling. 295

296

Discussion 297

298

Upon binding of extracellular holo-RBP, STRA6 transports ROH into cells and it activates a 299

signalling cascade culminating in induction of STAT target genes (4, 5). The observations 300

described here reveal that the binding partner of RBP in blood, TTR, effectively blocks 301

association of holo-RBP with STRA6. Consequently, STRA6 mediates cellular ROH uptake 302

only from free and not from TTR-bound holo-RBP. The data further show that, even in the 303

presence of free holo-RBP, STRA6-mediated ROH uptake by tissues comprises only a small 304

fraction of total uptake by target tissues in vivo (Fig. 1h). The observations thus support the 305

previously proposed model that supply of ROH from circulating holo-RBP or holo-RBP-TTR to 306

cells occurs primarily by diffusion through the plasma membranes (10, 14, 20, 21). Taken 307

together with the observations that ROH transport by STRA6 is critical for enabling activation of 308

STRA6 signalling (5), the data indicate that, with the exception of the eye (26), the main role of 309

ROH transport by STRA6 is not to provide the vitamin to cells but to couple sensing of 310

circulating free holo-ROH levels to cell signalling. It is worth noting that even in the eye, 311


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morphological changes and reduction in visual function in Stra6-null mice are mild, indicating 312

that STRA6 is not the only pathway by which ROH enters the retinal pigment epithelium (26). 313

The data reveal that, in addition to its function in preventing filtration of the 21 KDa RBP 314

in the kidney, TTR plays an important role in protecting cells from holo-RBP-induced signalling 315

mediated by STRA6. The observations that STRA6 “senses” only free and not TTR-bound RBP 316

establish that the receptor functions only under circumstances in which serum RBP level exceeds 317

that of TTR. Such circumstances are encountered, for example, in obese animals in which serum 318

level of RBP is elevated while TTR level is not (Fig. 5c). The circumstances in which the plasma 319

RBP concentration exceeds that of TTR in healthy lean animals remain to be clarified. It is 320

interesting to note in regard to this that it has long been known that insulin responsiveness varies 321

in a circadian fashion (17, 31). The molecular basis for these diurnal variations is incompletely 322

understood but the data presented here raise the intriguing possibility that they may arise from 323

diurnal variations in plasma RBP/TTR ratio. 324

The RBP/TTR ratio in blood may be altered by changes in the expression level of RBP, 325

or TTR, or both. TTR is expressed in the central nervous system and in the liver with the latter 326

serving as the main source for the protein in serum (9). Expression of hepatic TTR is 327

downregulated and, consequently, serum TTR level dramatically decreases during acute phase 328

response (APR), a process characterized by rapid reprogramming of gene expression and 329

metabolism in response to inflammatory cytokine signaling (1, 22). The low serum level of TTR 330

associated with APR may release holo-RBP thereby activating STRA6. Hence, STRA6 331

signalling may play a role in APR. It has also been reported that hepatic TTR expression is 332

regulated by sex hormones (12) and is directly controlled by hepatocyte nuclear factor 4α (HNF-333

4α) (30). The expression of RBP in brown adipose tissue and liver was reported to be regulated 334


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by cAMP-mediated pathways and by the nuclear receptors PPARα and PPARγ (6, 25). Whether, 335

by controlling TTR or RBP expression, these factors regulate the RBP-TTR ratio in blood and 336

thus STRA6 signalling remains to be clarified. 337

Notably, as free holo-RBP is rapidly excreted by glomeruli filtration, its lifetime in serum 338

is short. Holo-RBP thus functions like a classical cytokine: its availability to its membrane 339

receptor is tightly regulated and its signalling activities are constrained by a short half-life in the 340

circulation. These characteristics of the signalling activities of holo-RBP strikingly differ from 341

the characteristics of its role as a shuttling protein which mobilizes ROH from liver stores. 342

Unlike in the former capacity where holo-RBP functions on its own, delivery of ROH to target 343

tissues is mediated by the holo-RBP-TTR complex. The plasma level of this complex is under 344

tight homeostatic control and it provides ROH to target cells to support tissue requirement for 345

vitamin A without the need for a specialized receptor. 346

347

Acknowledgments 348

We are grateful to Michele Mumaw for help in early stages of this work. We thank Lawreen 349

Connors, Boston University School of Medicine, and Silke Vogel, Columbia University School 350

of Physicians and Surgeons, for the TTR and RBP expression constructs. This work was 351

supported by NIH grants DK060684 and CA107013 to N.N. The Mouse Metabolic Phenotyping 352

Center (MMPC) of Case Western Reserve University is supported by NIH grant DK59630. The 353

Stra6-null mouse line was established at the Mouse Clinical Institute (http://www-mci.u-354

strasbg.fr/) in the Genetic Engineering and Model Validation Department with Inserm and FRM 355

(DEQ20071210544) grants to NBG. 356

357


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References 358

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451

Figure legends 452

453

Figure. 1. TTR inhibits STRA6-mediated uptake of ROH from holo-RBP. a) Uptake of 3H-454

ROH by HepG2 cells treated with RBP-3H-ROH or RBP-3H-ROH-TTR (1 µM) for denoted 455

times. b) Uptake of 3H-ROH by HepG2 cells treated with the denoted concentrations of RBP-3H-456

ROH or RBP-3H-ROH-TTR for 3 min. c) Uptake of 3H-ROH by HepG2 cells following a 3 min. 457

incubation with 1 µM RBP-3H-ROH in the presence of denoted concentrations of TTR. d) 458

Levels of STRA6 mRNA in HepG2 cells transfected with varying amounts of STRA6 cDNA. e) 459

Effect of increasing the expression level of STRA6 in HepG2 cells on uptake of 3H-ROH from 460

RBP-3H-ROH or RBP-3H-ROH-TTR (1 µM, 3 min.). f) Top: expression level of STRA6 in 461

HepG2 cells transfected with an empty vector (e.v.) or vector harboring STRA6shRNA. Bottom: 462

Effect of decreasing the expression level of STRA6 in HepG2 cells on uptake of 3H-ROH from 463

RBP-3H-ROH or from RBP-3H-ROH-TTR (1 µM, 3 min.). g) Top: expression level of STRA6 in 464

NIH3T3-L1 cells transfected with an empty vector (e.v.) or vector harboring STRA6shRNA. 465

Effect of decreasing the expression level of STRA6 in NIH3T3-L1 adipocytes on uptake of 3H-466

ROH from RBP-3H-ROH or from RBP-3H-ROH-TTR (1 µM, 3 min.). h) 12 week old WT and 467

STRA6-null male mice were injected intraperitoneally with RBP-3H-ROH (100 μl, 0.1 mCi, 1 468

µM). 2 h later, tissues were isolated, weighed, homogenized, and 3H-ROH counted. Data are 469

means±SEM, *p<0.01 RBP-ROH- vs. RBP-ROH-TTR-treated groups. All p values were 470

calculated using a two-tailed student t-test. 471

Figure 2. STRA6 does not bind the holo-RBP-TTR complex. a) NIH3T3 cells stably over-472

expressing LRAT were transfected with an e.v, or with expression vectors encoding human 473


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(hSTRA6) or zebrafish (dSTRA6) STRA6, resulting in similar levels of mRNAs. b) Uptake of 474

3H-ROH from RBP-3H-ROH or RBP-3H-ROH-TTR (1 µM, 3 min.) by cells expressing hSTRA6 475

or dSTRA6. c) RBP-ROH or RBP-ROH-TTR (1 μM) was incubated with the chemical cross-476

linker Bis[sulfosuccinimidyl] suberate (0.5 mM) for 14 h. Proteins were resolved by SDS-PAGE 477

and visualized by Coomasie-blue staining. d) Cross-linked complexes and additional cross-linker 478

(0.5 mM) were added to HepG2 cells transfected with an e.v. or with a vector encoding histidine-479

tagged STRA6. Following a 15 min. incubation, his-STRA6 was immunoprecipitated using 480

antibodies against the tag and precipitated RBP and STRA6 visualized by immunoblots. e) 481

Fluorescence titrations of RBP and its F96A/L97A mutant (1 µM) with ROH. Progress of 482

titrations was monitored by following the increase in ROH fluorescence upon binding to the 483

protein (λex - 330 nm; λem - 460 nm). f) Fluorescence anisotropy titrations of holo-RBP and 484

holo-RBP-F96A/L97A (3 µM) with TTR. Progress of titrations was monitored by measuring the 485

fluorescence anisotropy of bound ROH (λex - 330 nm; λem - 460 nm). g) Uptake of 3H-ROH 486

from holo-RBP-F96A/L97A (1 µM, 3 min.) in the presence or absence of TTR. Data are 487

means±SEM, *p<0.01 vs. cells transfected with an empty vector. **p=0.01 vs. cells transfected 488

with an empty vector and treated with RBP-ROH. All p values were calculated using a two-489

tailed student t-test. 490

491

Figure 3. TTR blocks activation of STRA6 signaling by holo-RBP. a) NIH3T3-L1 adipocytes 492

were treated with 1 µM RBP-ROH, TTR, or RBP-ROH-TTR for 15 min. Cell were lyzed and 493

phosphorylated JAK2 (pJAK2) and STAT5 (pSTAT5) visualized by immunoblots. b) NIH3T3-494

L1 adipocytes cells were treated with 1 µM RBP-ROH, TTR, or RBP-ROH-TTR for 4 h and 495

levels of SOCS3 and PPARγ mRNA assessed by Q-PCR. Data are means±SEM. *p<0.001 vs. 496


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non-treated cells. c) NIH3T3-L1 adipocytes were pre-treated with 1 µM RBP-ROH, TTR or 497

RBP-ROH-TTR for 8 h and then treated with insulin (25 nM, 15 min.). Phosphorylated IR (pIR) 498

and AKT (pAKT) were visualized by immunoblots. Bottom panel: quantitation of band 499

intensities. Mean of two independent experiments. d) NIH3T3-L1 adipocytes were treated with 500

RBP-ROH or RBP-F96A/L97A-ROH (RBP96/97-R) in the presence or absence of TTR (1 µM 501

each, 15 min). Lysates were immunoblotted for pSTAT5. e) NIH3T3-L1 adipocytes were 502

treated with RBP-ROH or RBP-F96A/L97A-ROH in the presence or absence of TTR (1 µM 503

each, 4 h). Levels of SOCS3 mRNA were assessed by Q-PCR. Data are means±SEM. *p<0.001 504

vs. non-treated cells, **p<0.001 vs. R-R-TTR-treated cells. f) HepG2 cells were treated with 505

RBP-ROH in the presence or absence of TTR (1 µM each, 4 h). Levels of SOCS3 mRNA were 506

assessed by Q-PCR. Data are means±SEM. *p<0.001 vs. non-treated cells, g) The 507

phosphotyrosine motif in mouse, human and zebrafish STRA6 (mSTRA6, hSTRA6, dSTRA6). 508

h) NIH3T3 fibroblasts stably expressing LRAT were transfected with zebrafish and human 509

STRA6 and treated with 1 µM RBP-ROH or RBP-ROH-TTR for 15 min and lysates were 510

immunoblotted for pSTAT3. i) NIH3T3 fibroblasts stably over-expressing LRAT were 511

transfected with dSTRA6 or hSTRA6 and treated with 1 µM RBP-ROH or RBP-ROH-TTR for 4 512

h. Levels of SOCS3 mRNA were assessed by Q-PCR. Data are means±SEM. *p<0.001 vs. non-513

treated cells. All p values were calculated using a two-tailed student t-test. 514

515

Figure 4. TTR suppress activation of STRA6 by holo-RBP in vivo. Mice were injected three 516

times with 0.1 μmole RBP-ROH or 0.1 μmole RBP-ROH complexed with TTR and sacrificed 1 517

h after the last injection. a, b) Immunoblots of RBP (a) and TTR (b) in serum following 518

respective injections. Blots from 2 mice of each group are shown. c-e) Immunoblots of 519


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phosphorylated insulin receptor (pIR), AKT (pAKT) and STAT5 (pSTAT5) in WAT (c), skeletal 520

muscle (d) and liver (e) of mice treated as denoted. Total IR served as a loading control. f-h) 521

Levels of mRNA of SOCS3 and PPARγ in WAT (f), skeletal muscle (g) and liver (h) of treated 522

mice. Data are means ±SEM *p<0.001 buffer-treated vs. RBP-ROH treated mice. 523

524

Figure 5. TTR is protectives against holo-RBP-induced insulin resistance. a-b) Levels of 525

mRNA of RBP (a) and TTR (b) in WAT and liver of lean mice and of mice fed a HFHS diet for 526

10 weeks (obese). c) Immunoblots of RBP and TTR in serum of mice fed a HFHS diet for 0, 3, 527

6 and 10 weeks. d-j) Mice were implanted with an Alzet pump that contained buffer, 0.1 μM 528

holo-RBP or 0.1 μM holo-RBP complexed with TTR. Implants were replaced once a week for 3 529

weeks. d) Immunoblots of RBP (top) and TTR (bottom) in serum following 3 weeks of denoted 530

treatments. e, f) Immunoblots of pIR and pSTAT5 in WAT (e) and skeletal muscle (f) of mice 531

treated as denoted. g-i) Levels of SOCS3 mRNA in WAT (g), skeletal muscle (h) and liver (i) of 532

mice treated as denoted. j). Glucose tolerance tests carried out following 3 weeks of denoted 533

treatments. Data are means ±SEM. *p<0.001 lean vs. obese mice; **p<0.001 buffer-treated vs. 534

RBP-ROH-treated mice. All p values were calculated using a two-tailed student t-test. 535


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Fig. 1a

0.20

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chow 3 wk 6 wk 10 wkc25

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2

3

4

fold

mR

NA

/18 PPARγ

0 00.20.40.60.81.0

fold

mR

NA

/18 γ

****

0 20 40 60 80 100 120100150

seru

time (min.)

RBP TTRbuffer R-R R-R-TTR

0buffer R-R R-RTTR

0buffer R-R R-R-TTR

0.0


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Dow

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http://mcb.asm.org/

Transthyretin Blocks Retinol Uptake and Cell Signaling by the Holo-Retinol-Binding Protein Receptor STRA6

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