Association of estradiol on expression of melanocortin receptors and their accessory proteins in the liver of chicken (Gallus gallus)

Accepted Manuscript Association of Estradiol on Expression of Melanocortin Receptors and Their Accessory Proteins in the Liver of Chicken (Gallus gallus) Junxiao Ren, Yanmin Li, Naiyi Xu, Hong Li, Cuicui Li, Ruili Han, Yanbin Wang, Zhuanjian Li, Xiangtao Kang, Xiaojun Liu, Yadong Tian PII: DOI: Reference:

S0016-6480(16)30337-9 http://dx.doi.org/10.1016/j.ygcen.2016.10.012 YGCEN 12516

To appear in:

General and Comparative Endocrinology

Received Date: Revised Date: Accepted Date:

4 April 2016 18 October 2016 24 October 2016

Please cite this article as: Ren, J., Li, Y., Xu, N., Li, H., Li, C., Han, R., Wang, Y., Li, Z., Kang, X., Liu, X., Tian, Y., Association of Estradiol on Expression of Melanocortin Receptors and Their Accessory Proteins in the Liver of Chicken (Gallus gallus), General and Comparative Endocrinology (2016), doi: http://dx.doi.org/10.1016/j.ygcen. 2016.10.012

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1

Association of Estradiol on Expression of Melanocortin Receptors and

2

Their Accessory Proteins in the Liver of Chicken (Gallus gallus)

3

Junxiao Ren1†, Yanmin Li1†, Naiyi Xu 1, Hong Li1, Cuicui Li1, Ruili Han1, 2, 3, Yanbin

4

Wang1, 2, 3, Zhuanjian Li1, 2, 3, Xiangtao Kang1, 2, 3, Xiaojun Liu 1, 2, 3*, Yadong Tian1, 2, 3

5

*

6 7

1

8

Zhengzhou 450002, China

9 10 11 12

2

College of Animal Science and Veterinary Medicine, Henan Agricultural University,

Henan Innovative Engineering Research Center of Poultry Germplasm Resource,

Henan Agricultural University, Zhengzhou 450002, China 3

International Joint Research Laboratory for Poultry Breeding of Henan, Henan

Agricultural University, Zhengzhou 450002, China

13 14

†

These authors contributed equally to this work.

15 16

*Corresponding authors

17

Yadong Tan: [email protected]

18

Xiaojun Liu: [email protected]

19 20 21 22

23

Abstract

24

The melanocortin receptor accessory proteins (MRAP and MRAP2) are small

25

single-pass transmembrane proteins that regulate the biological functions of the

26

melanocortin receptor (MCR) family. MCRs comprise five receptors (MC1R–MC5R)

27

with diverse physiological roles in mammals. Five MCR members and two MRAPs

28

were also predicted in the chicken (Gallus gallus) genome. However, little is known

29

about their expression, regulation and biological functions. In this study, we cloned

30

the MRAP and MRAP2 genes. Sequencing analysis revealed that the functional

31

domains of MRAP and MRAP2 were conserved among species, suggesting that the

32

physiological roles of chicken MRAP and MRAP2 could be similar to their

33

mammalian counterparts. Tissue expression analysis demonstrated that MRAP was

34

expressed in the adrenal gland, liver, spleen, glandular stomach and lungs, while

35

MRAP2 is predominantly expressed in the adrenal gland. All five MCRs were present

36

in the adrenal gland, but showed different expression patterns in other tissues. The

37

MC5R was the only MCR member that was expressed in the chicken liver. The

38

expression levels of MRAP in chicken liver were significantly increased at sexual

39

maturity stage, and were significantly up-regulated (P < 0.05) when chickens and

40

chicken primary hepatocytes were treated with 17β-estradiol in vivo and in vitro,

41

respectively; however, expression levels of PPARγ were down-regulated, and no

42

effect on MC5R was observed. Our results suggested that estrogen could stimulate the

43

expression of MRAP in the liver of chicken through inhibiting the expression of

44

transcription regulation factor PPARγ, and MRAP might play its biological role in a

45

different way rather than forming an MRAP/MC2R complex in chicken liver during

46

the egg-laying period.

47 48 49

Keywords: Chicken; melanocortin receptors; melanocortin receptor accessory proteins; 17β-estradiol

50

1. Introduction

51

G-protein-coupled receptors (GPCRs) play important roles in GPCR signal

52

transduction. Many accessory proteins interact with GPCRs to alter either the GPCRs’

53

ligand binding mechanism or their functional responses (Metherell et al., 2005; Rana,

54

2003). The melanocortin receptor (MCR) accessory proteins (MRAPs) are GPCR

55

accessory proteins that function in the melanocortin system. There are two subtypes

56

of MRAPs, MRAP and MRAP2, which have been identified in many species (Hinkle

57

and Sebag, 2009; Webb and Clark, 2010). Comprehensive evolutionary analysis of the

58

MRAP and MRAP2 homologs revealed that the two subtypes possibly originated from

59

a single ancestor (Valsalan et al., 2013).

60

Both MRAP and MRAP2 are small proteins containing a single transmembrane

61

domain with no signal peptide. The MRAP gene in humans has six exons and is

62

located on chromosome 21q22.1. Exons 3 to 5 encode the 172-amino acid MRAP

63

protein. Comparative protein sequence studies of MRAP found that the N-terminal

64

and transmembrane regions are highly conserved, while the C-terminal regions vary

65

greatly in length and show little sequence homology between species. However,

66

MRAP possesses only a small amount of overall conservation in gene structure or

67

protein sequence among species (Valsalan et al., 2013; Webb and Clark, 2010). The

68

human MRAP2 gene comprises four exons that encode a 205-amino acid protein.

69

Molecular modeling also predicted the presence of a transmembrane domain that is

70

fully conserved among MRAP2 sequences (Agulleiro et al., 2010). Similar to MRAP,

71

the C-terminal domain is not conserved. However, the IPNFV domain is fully

72

conserved in all MRAP2 sequences (Cerdá-Reverter et al., 2013). The MRAP and

73

MRAP2 often form highly unusual antiparallel homodimers, which bind directly with

74

the MCRs and form a stable immunoprecipitable complex, which is essential for

75

trafficking of MCRs from the endoplasmic reticulum to the plasma membrane, where

76

they mediate their pharmacological effects (Metherell et al., 2005; Roy et al., 2007;

77

Sebag and Hinkle, 2007).

78

Five subtypes (MC1R, MC2R, MC3R, MC4R, and MC5R) have been identified

79

in the MCR family in many species, including chicken (Gallus gallus) (Cone et al.,

80

1993; Dores, 2013; Klovins et al., 2004; Ling et al., 2004; Mountjoy et al., 1992; Roy

81

et al., 2007), and they are distributed in various tissues (Cone, 2006; Davis et al., 2013;

82

Dores, 2013). All of the accumulated data suggested the co-expression and physical

83

interaction of MC2R with MRAP. MC2R，also referred to as the ACTH receptor, is

84

expressed primarily in the adrenal cortex and spleen (Cone et al., 1993; Mountjoy et

85

al., 1992). In the mammalian adrenal gland, MRAP facilitates MC2R expression and

86

trafficking from the endoplasmic reticulum to the plasma membrane (Chung et al.,

87

2008). It also plays an essential and independent role in influencing the

88

three-dimensional conformation of MC2R, facilitating ACTH binding and activating

89

the MC2R/MRAP complex on the plasma membrane, leading to receptor signaling

90

(Hinkle and Sebag, 2009; Webb and Clark, 2010). MRAP is also expressed outside

91

the adrenal gland, this suggested that MRAP plays a wider physiological purpose

92

beyond MC2R-mediated adrenal steroidogenesis (Novoselova et al., 2013). The

93

MRAP2 is reported to be required for growth and development, as well as metabolism,

94

across species via melanocortin receptor signaling (Asai et al., 2013; Cone, 2006;

95

Sebag et al., 2013). It is predominantly expressed in the brain and interacts directly

96

with MC4R to enhance MC4R-mediated generation of the second messenger, cyclic

97

AMP (Asai et al., 2013). In zebrafish, MC1R physically interacts with the MRAP2

98

system; however, this interaction does not result in any modification of the studied

99

pharmacological profile (Cortés et al., 2014). More interestingly, MRAP2 can interact

100

with MC2R to aid MC2R trafficking to the membrane. However, data on functional

101

activation by ACTH are controversial (Agulleiro et al., 2010; Sebag and Hinkle,

102

2009). Both MRAP and MRAP2 reduce the expression level of MC4R and MC5R but

103

not of MC1R and MC3R in the plasma membrane. The MC5R homodimerizes in the

104

absence of MRAP, as does MC2R, but the co-expression of MRAP inhibits MC5R

105

homodimerization (Sebag and Hinkle, 2009).

106

It was reported that the transcriptional activation of MRAP gene was regulated by

107

peroxisome proliferator-activated receptor γ (PPARγ), which is one members of the

108

nuclear hormone receptor superfamily of transcription factors (Mangelsdorf et al.,

109

1995). Previous studies demonstrated that there were peroxisome proliferator

110

response element (PPRE) sites in the MRAP promoter, and PPARγ could bind to the

111

PPRE in the MRAP promoter to regulate the transcriptional activation of MRAP in

112

3T3-L1 cells (Kim et al., 2013). In addition, PPARγ can be negatively regulated by

113

estradiol treatment in human abdominal adipose tissue (Lundholm et al., 2008).

114

Both the MRAP and MRAP2 genes were identified in chicken genome. In

115

addition, there were two transcript variants of MRAP2 genes in the chicken genome,

116

MRAP2α and MRAP2β. The isoform in this study refers to MRAP2α. MRAP and

117

MRAP2 are located on chromosomes 1 and 3, and encode the deduced proteins of 120

118

and 206 amino acid residues, respectively. Five MCR family member genes were also

119

found in the chicken genome, among which MC1R and MC3R reside on chromosome

120

11 and 20, respectively, and MC2R, MC4R and MC5R reside on chromosome 2.

121

However, the expression patterns and regulatory mechanism of these genes are poorly

122

understood. To gain insights into the biological functions of these genes, we cloned

123

the MRAP and MRAP2, and investigated their tissue distribution and regulatory

124

mechanism. Our results demonstrated that MRAP was present not only in the adrenal

125

gland, but also in the liver, spleen, glandular stomach, lungs and brain, while MRAP2

126

is predominantly expressed in the adrenal gland and brain. All of the five MCRs were

127

present in the adrenal gland, but showed varied expression patterns in other tissues.

128

Only MC5R was present in the liver. Moreover, MRAP was significantly upregulated

129

by estradiol in chicken liver and primary hepatocytes, however MC5R expression

130

showed no changes. Our studies, for the first time, systematically investigated the

131

expression and regulation of melanocortin receptors and their accessory proteins in

132

chicken, and suggested that MRAP might play a role alone rather than forming the

133

MRAP/MC5R complex in the chicken liver during the egg-laying period.

134

2. Materials and methods

135

2.1 Animals, treatment and sampling

136

All experimental designs and procedures were performed in accordance with the

137

protocol approved by the Institutional Animal Care and Use Committee of Henan

138

Agricultural University. All animals used in the experiments were female Lushi

139

Green-shell-egg chickens obtained from the Animal Center of Henan Agricultural

140

University. They were raised in the same environmental conditions with food and

141

water ad libitum.

142

To clone the MRAP, MRAP2 and related genes, and determine their expression

143

profiles, eight female birds at different ages were humanely killed. The heart, liver,

144

pectoral, kidney, adrenal gland, spleen, abdominal fat, duodenum, glandular stomach,

145

pancreas and lung were quickly removed, snap-frozen in liquid nitrogen and stored at

146

−80°C in a freezer until use.

147

To investigate the effect of estradiol on the expression of MRAP, 40 birds at 10

148

weeks old were divided randomly into four groups of 10 birds. The birds in groups 1,

149

2 and 3 were injected intramuscularly with 0.5 mg, 2.0 mg and 8.0 mg of

150

17β-estradiol (Sigma, St Louis, MO, USA) (dissolved in olive oil)/kg of body weight,

151

respectively. The birds in group 4 served as the controls and were injected

152

intramuscularly with solvent olive oil only. All the birds were killed after treatment

153

for 12 hours. Their livers were collected and stored as mentioned above.

154

2.2 Primary hepatocyte culture and treatment

155

Hepatocytes were isolated from chicken embryonic livers according to the

156

method of Fischer and Marks (Fischer and Marks, 1976), with some modifications. In

157

brief, livers were removed from 18-day-old female embryonic chickens and washed

158

with D-Hanks solution (Solarbio, Beijing, China). The livers were manually minced,

159

washed with D-Hanks solution, and digested using collagenase type II with gentle

160

shaking at 37 °C for 0.5 hours. Dispersed cells were filtered through a 200-mesh sieve

161

and a 500-mesh sieve. After washing and centrifugation, unadulterated hepatocytes

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were obtained using non-continuous density Percoll gradient centrifugation. Then the

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cells were resuspended in DMEM medium (Sigma) supplemented with 5% fetal calf

164

serum, 100 mg/mL streptomycin and 100 U/mL penicillin, and counted using a Luna

165

automated cell counter (Biosystems L10001, Korea). The cell density was adjusted to

166

5×105 cells/mL. The cells with viability greater than 90%, as assessed using the

167

TrypanBlue exclusion assay, were plated on 6-well plates at 2 mL per well. Cells were

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incubated at 38.5 °C under a water-saturated atmosphere containing 95% air humidity

169

and 5% CO2. When the cells reached 90% confluence (after approximately 24 hours),

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the cell culture medium was replaced by serum-free DMEM medium with 100 mg/mL

171

streptomycin and 100 U/mL penicillin, and incubated for 12 hours.

172

To assess the effect of estradiol on expression of the MRAP gene in hepatocytes,

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the cells were divided into four groups, with four repeats in each group, and treated

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with 25 nM, 50 nM and 100 nM of 17β-estradiol, respectively. The control group

175

received solvent ethanol alone at a final concentration of 0.1%. After 12 hours, the

176

cells were washed with fresh D-Hanks solution, collected by the Trizol reagent

177

(Takara, Kyoto, Japan), and stored at −80°C until use.

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2.3 RNA extraction and reverse transcription

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Total RNA from primary hepatocytes and chicken tissues was extracted with

180

Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA,

181

USA). The quantity and quality of the RNA samples were assessed using a NanoDrop

182

2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and standard

183

denaturing agarose gel electrophoresis. The RNA samples with OD260/280 ratios

184

above 1.8 were used for further study. One microgram of RNA from each sample was

185

treated with DNase I (Invitrogen #18068015) to remove trace amounts of DNA

186

according to the manufacturer’s instructions. Each of the RNA samples was treated

187

with DNase I (Invitrogen #18068015) to remove trace amounts of genomic DNA

188

according to the manufacturer’s instructions. The treated RNA samples were checked

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by PCR with primer pairs for amplification of internal control β-actin and GAPDH

190

genes (Table s1). Only the samples showed no amplification from RNA without

191

reverse transcription were used for the next study. One microgram of the DNase I

192

treated RNA was then reverse transcribed into cDNA using random hexamer primers

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with the Thermo Scientific™ Revert Aid First Strand cDNA Synthesis kit (Thermo

194

Scientific # K1621) according to the manufacturer’s instructions. The cDNA was

195

stored at −20°C until use.

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2.4 Gene cloning and sequencing analysis

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According to the predicted chicken MRAP and MRAP2 sequences in GenBank

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(accession numbers XR_001470382 and XM_015284715, respectively), two pairs of

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specific PCR primers (Supplemental Table 1) for cloning the coding sequences (CDS)

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of the genes were designed, using the software primer 5.0. The PCR was performed in

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a 12.5-µL reaction volume containing 0.5 µL of first-strand cDNA, 6.25 µL of 2×PCR

202

Mix (Takara), 0.5 µL each of the forward and reverse primers (10 µM), and 4.75 µL

203

RNase-free water. The reactions were amplified using the following conditions: 95°C

204

for 5 minutes; 30 cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 30

205

seconds; followed by 72°C for 10 minutes. The PCR products were sequenced by

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Sangon Biotech Co. Ltd. (Shanghai, China). Each sequence was confirmed by

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sequencing twice and on both strands.

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Database searches were performed at NCBI (http://www.ncbi.nlm.nih.gov/)

209

using BLASTn and BLASTx under default settings. Peptide translations were made

210

using SDSC Biology Workbench (http://workbench.sdsc.edu/). Signal peptide and

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transmembrane helices analyses were performed using the Signal P Server 4.1 and

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TMHMM Server v. 2.0 available at http://www.cbs.dtu.dk/services/SignalP/ and

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http://www.cbs.dtu.dk/services/TMHMM/, respectively. N-linked glycosylation sites

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were

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http://www.cbs.dtu.dk/services/NetNGlyc/. Amino acid alignments were generated

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using Clustal V in the Molecular Evolutionary Genetics Analysis version 6.0 (MEGA

217

6).

218

2.5 Tissue distribution of MRAP, MRAP2 and MCR genes

predicted

using

NetNGlyc

1.0

servers

at

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The expression profiles of MRAP, MRAP2 and MCR genes in the 11 tissues

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including heart, liver, pectoral, kidney, adrenal gland, spleen, abdominal fat,

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duodenum, glandular stomach, pancreas and lung were analyzed by RT-PCR. First,

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the same amount of cDNA sample from each tissue of eight individuals was mixed.

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The mixed cDNA sample was used to generate amplification products by PCR using

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primer pairs for the MRAP, MRAP2 and MCR genes (Supplemental Table 1). To

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confirm the effectiveness of reverse transcription, PCR was also carried out to

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amplify a cDNA fragment of the chicken β-actin gene using the specific primer pair

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shown in Supplemental Table 1. The PCR was carried out in a 12.5-µL reaction

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volume with the same amplification conditions mentioned in section 2.4.

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Amplification products were separated by electrophoresis on a 2% agarose gel and

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stained with DNA Green (LaiFeng, HangZhou, China).

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2.6 Quantitative real-time PCR (qPCR)

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The expression levels of the genes were quantified using SYBR Green method in

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a Roche Lightcycle R96 instrument according to the method of Nolan (Nolan et al.,

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2006). The β-actin was used as the endogenous control. Each reaction contained 5 µl

235

of SYBR Green PCR Master Mix (Takara), 3.5 µl of RNase-free water, 0.5 µl each of

236

forward and reverse primers (Supplemental Table 1) and 0.5 µl of extracted cDNA.

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The reaction conditions were as follows: denaturation at 95°C for 5 minutes; followed

238

by 40 PCR cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 20

239

seconds; followed by a further 10-minute extension at 72°C. All reactions were

240

performed in triplicate. The expression levels were measured in terms of the cycle

241

threshold (Ct), and then normalized by β-actin using the 2 -△△Ct method.

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2.7 Western blotting analysis

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Total protein was extracted from liver tissue using the RIPA lysis buffer (R00zO,

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Solarbio, China). The protein concentration was determined using a Bradford assay

245

(Bradford, 1976). A total of 50 µg protein from each sample was separated on 12%

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SDS-polyacrylamide gel. Separated protein was then transferred to polyvinylidene

247

difluoride (PVDF) membranes (IPVH00010, Millipore, USA) and membranes were

248

then blocked with nonfat milk for 1 hour. Then the membranes were washed with

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PBST three times and incubated with the MRAP primary antibodies (MRAP BK-730,

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Broad-ocean Bio-science, China) at 4°C for overnight. The membranes were then

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washed three times using PBST, and incubated with secondary antibody conjugated

252

with HRP (ZB-2305, ZSGB-BIO, China) for one hour at room temperature. Images

253

were captured and analyzed by UVP GDS-8000 System (Thermo Scientific). β-actin

254

was used as an internal control.

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2.8 Statistical analysis

256

Statistical analyses of the qPCR results were carried out using SPSS version 20.0.

257

One-way ANOVA and repeated measures ANOVA were used for statistical analysis of

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relative expression levels, followed by Dunnett’s test. Graphics were drawn using

259

Graphpad Prism 5 (Graphpad Software, San Diego, CA, USA), and differences were

260

considered significant at P ≤ 0.05. Data was presented as the mean ± SD.

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3. Results

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3.1 Cloning and sequence analysis of chicken MRAP and MRAP2

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The full-length coding sequences for the chicken MRAP and MRAP2 were

264

obtained by PCR amplification using chicken adrenal gland cDNA as the template.

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Sequencing analysis indicated that the coding sequences of MRAP and MRAP2 were

266

entirely identical to the predicted coding sequences of the two genes in GenBank

267

(accession number XR_001470382 and XM_015284715, respectively). The chicken

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MRAP gene comprised of three exons and encoded a 120-amino acid MRAP protein

269

(Fig. 1A). The cDNA sequences and deduced amino acid sequences are shown in

270

Fig.1B. Bioinformatics analyses results revealed that there was no signal peptide but a

271

possible transmembrane domain from amino acid 38 to 60 in the MRAP protein

272

sequence with N-terminus inside the cell and C-terminus outside (Supplemental Fig.

273

l). In addition, there were two potential N-linked glycosylation (Asn-X-Ser/Thr) sites

274

at positions 3 and 6 in the N-terminal intracellular domain and one at amino acid 91 in

275

the extracellular domain. The MRAP2 gene of chicken also comprised of three exons

276

and encoded a 206- amino acid MRAP2α protein (Fig. 2A). The cDNA sequences and

277

deduced amino acid sequences are shown in Fig. 2B. Similar to MRAP, MRAP2 had

278

no signal peptide but a possible transmembrane domain between amino acid 45 and

279

67 (Supplemental Fig.2).

280

Multiple alignments of MRAP and MRAP2 amino acid sequences among

281

different species including chicken and 10 other species from mammal, avian，reptile，

282

batrachians, and fish are obtained by Clustal V (Fig. 3A and Fig. 3B). Amino acid

283

sequence alignment analysis among species manifested that chicken MRAP and

284

MRAP2 shared low identifies with other species. The most conserved parts were in

285

the N-terminus and transmembrane regions. Amino acid sequence alignment analysis

286

between chicken MRAP and MRAP2 indicated that MRAP was only 16.02% identical

287

to MRAP2 (Fig. 3C). The most conserved parts were in the N-terminal and

288

transmembrane regions. The N-terminal were 41.67% identical. The molecular

289

evolution and the relationships between the homologs were investigated using

290

phylogenetic methods. The evolution analysis result showed that the phylogeny tree

291

was divided into two phylogenetic groups by MRAP and MRAP2 (Fig. 4). The avian

292

MRAP and MRAP2 clusters separate from mammalian and reptile MRAP and

293

MRAP2.

294

3.2 Tissue distribution of MRAP, MRAP2 and MCRs

295

To detect the tissue specific expression patterns of MRAP, MRAP2 and MCRs

296

mRNA in chicken, tissues including heart, liver, pectoralis, kidney, adrenal, spleen,

297

abdominal fat, duodenum, glandular stomach, pancreas lungs and hypothalamus were

298

studied using semi-quantitative PCR in 30-week-old laying chickens (Fig. 5). MRAP2

299

can be detected in the adrenal gland and brain tissues, and MC2R only can be detected

300

in the adrenal gland. Only MRAP and MC5R can be detected in the liver tissue of

301

chicken. Other genes were extensively expressed in multiple tissues.

302

3.3 Differential expression of MRAP, MC5R and PPARγ in the liver at the different

303

developmental stages of chicken

304

To further understand the biological functions of MRAP in the chicken liver, the

305

mRNA expression levels of MRAP, MC5R and PPARγ in the liver during the different

306

developmental stages of chicken were studied using qPCR. In addition, the protein

307

levels of MRAP were also detected by Western Blotting. The relative expression

308

levels of MRAP mRNA (Fig. 6A) and protein (Fig. 6B) in 30-week-old chickens were

309

significantly higher than those in 10- and 20-week-old (P<0.01). The relative

310

expression levels of PPARγ mRNA were significantly lower (P<0.01), while MC5R

311

mRNA was not changed (Fig. 6C).

312

3.4 Effect of 17β-estradiol on MRAP, MC5R and PPARγ expression in vitro and in

313

vivo

314

To sight into the effect of 17β-estradiol on the expression of MRAP, MC5R and

315

PPARγin vivo, the mRNA expression levels of the three genes were investigated in the

316

livers of chickens treated with 17β-estradiol. Meantime, the protein levels of MRAP

317

were also analyzed by Western Blotting. The results showed that the expression levels

318

of MRAP mRNA (Fig. 7A) and protein (Fig. 7B) (P<0.05) were significantly

319

increased in the livers of 17β-estradiol treated chickens. By contrast, the PPARγ

320

mRNA expression levels were significantly decreased by varying degrees after the

321

chickens treated with different doses of 17β-estradiol for 12 hours. No alteration was

322

observed to the expression levels of MC5R mRNA when chickens treated with

323

17β-estradiol (Fig. 7C).

324

To further verify whether the expressions of MRAP, MC5R and PPARγ genes

325

were regulated by sex steroids in vitro, chicken primary hepatocytes were treated with

326

different concentrations of 17β-estradiol. The results showed that the expression level

327

of MRAP mRNA presented a dose dependent increase, and the PPARγ appeared a

328

corresponding decrease; the MC5R mRNA expression level was unaffected by

329

17β-estradiol (Fig. 8).

330

4. DISCUSSION

331

In this study, we cloned the whole coding sequences of MRAP and MRAP2 in

332

chicken for the first time. Sequence analysis indicated that chicken MRAP is a small

333

protein of 120 amino acids with no signal peptide, which has a tyrosine-rich domain

334

in the N-terminal region. The chicken MRAP shares relatively low amino acid

335

sequence identities with MRAPs of mammalian species (approximately 40% identity).

336

Three functionally distinct domains of MRAP are highly conserved in chickens (Fig.

337

3A) (Webb and Clark, 2010). These observations suggested that the physiological

338

roles of chicken MRAP might be similar to its mammalian counterparts. The chicken

339

MRAP2 shared relatively low amino acid sequence identities with chicken MRAP and

340

other mammalian MRAP2s. The functional domains of MRAP2 are varied (Agulleiro

341

et al., 2010; Cerdá-Reverter et al., 2013); therefore, its biological functions need to be

342

explored further.

343

The importance of MRAP for the functional expression of the MC2R/ACTH

344

receptor and MRAP/MC2R for physiological function of adrenal gland has been

345

extensively studied since MCRs and MRAP were first discovered and cloned about 25

346

years and 40 years ago, respectively (Mountjoy et al., 1992; Schwyzer, 1977). It has

347

been well documented that MRAP interacts with MC2R and trafficking MC2R from

348

the endoplasmic reticulum to the cell surface. However, there are still some confusing

349

facets of the system. One study reported that MC2R was expressed on cell surface in

350

the absence of MRAP (Roy et al., 2007). Very recently, the MC2R orthologs of the

351

stingray, Dasyatis akajei, and the elephant shark, Callorhinchus milii, were reported

352

also functionally expressed in CHO cells in the absence of co-transfection of an

353

exogenous MRAP cDNA (Dores, 2016; Reinick et al., 2012). In addition, studies

354

showed that human MRAP was expressed in a wide range of tissues including the

355

hippocampus, prefrontal cortex, cerebellum, and spinal cord, among other tissues

356

(Gardiner et al., 2002), but MC2R was only detectable in the adrenal, bone, adipose

357

tissue, ovaries, testes, skin, and the pituitary (Metherell et al., 2005). MRAP

358

expression clearly extends beyond MC2R expression (Jackson et al., 2015). Up to

359

now, the observation of MRAP expression outside the adrenal gland suggested a

360

special physiological purpose

361

steroidogenesis. However, few studies looked into the functional roles of MRAP in

362

the absence of MC2R.

beyond

MC2R

or

MC5R-mediated

adrenal

363

Our results showed that the MRAP and MC5R were expressed in the adrenal

364

gland and liver; however, MC2R was only detected in the adrenal gland in chickens. A

365

previous study reported that MC2R was found in the adrenal gland and spleen in

366

chicken (Takeuchi et al., 1998). The possible reason that made the difference might be

367

due to the chickens used for the studies were in the different developmental stages.

368

With the absence of MC2R in chicken liver and other tissues, the question arises as to

369

whether any other MCR family members, instead of MC2R, interact with MRAP in

370

the chicken liver or whether MRAP is involved in other biological processes besides

371

the above-mentioned functions. Previous studies in mammals have showed that

372

MRAPs have no effect on the trafficking of MC1R and MC3R but reduce surface

373

expression of MC4R and MC5R (Chan et al., 2009; Sebag and Hinkle, 2009). Another

374

study has also shown a significant reduction in MC5R signaling in the presence of

375

MRAPs (Sebag and Hinkle, 2010). However, our data showed that expression of

376

MRAP was up-regulated, but MC5R maintained no change when the individuals and

377

hepatocytes were treated by estradiol. The different mechanism might be in part due

378

to differences in species, cell-lines, tissue or be dependent on the ortholog studied.

379

Hence, there is still a considerable amount of work need to be done to clarify the

380

physiological roles of the MRAP.

381

The significant physiological difference between hens at 20 weeks and those at

382

30 weeks lies in their sexual maturity and egg laying. Estrogen is vitally important for

383

sexual maturity and the development of the female reproductive system (Hess et al.,

384

2011; Sato et al., 1996). The plasma estrogen in female chicken reaches peak level

385

just before the onset of the first egg production (Tanabe et al., 1981; Williams and

386

Harvey, 1986), then goes down gradually, but still maintains with a relative higher

387

level than that in the immature pullets for certain period (Williams and Harvey, 1986).

388

Moreover, for chicken, the liver is one of the main target organs for estrogen.

389

Estrogens initiate the transcription of estrogen-dependent genes and enhance the

390

stability of their transcripts (Flouriot et al., 1996). The MRAP was up-regulated by

391

estrogen, which suggested that estrogen might be served as an activator of MRAP.

392

Nuclear hormone receptors are ligand-activated transcription factors that regulate

393

gene expression by interacting with specific DNA sequences in the upstream region

394

(promoter) of their target genes. Through sequence analysis, PPREs was found in the

395

chicken MRAP promoter region. PPARγ, a key regulator of adipogenesis, is involved

396

in the regulation of lipid metabolism (Luquet et al., 2004; Watanabe et al., 2003). It

397

has been reported to increase the expression of certain genes involved in adipogenesis,

398

for instance the genes encoding stearoyl–CoA desaturase-1 (SCD-1) (Ikeda et al.,

399

2015), disulfide-bond A oxidoreductase-like protein (DsbA-L) (Jin et al., 2015),

400

insulin-dependent glucose transporter 4 (GLUT4) (Wu et al., 1998), adipocyte fatty

401

acid binding protein (aP2)(Tontonoz et al., 1994), lipoprotein lipase (LPL)

402

(Schoonjans et al., 1996) and the fatty acid translocase (CD-36/FAT) (Sato and Akiba,

403

2002). In addition, PPARγ can be negatively regulated by estradiol treatment in human

404

abdominal adipose tissue (Lundholm et al., 2008). Previous study demonstrated that

405

PPARγ could bind to the transcriptional regulation factor PPRE of MRAP to regulate

406

the transcriptional activation of MRAP in 3T3-L1 cells (Kim et al., 2013). As our in

407

vitro and in vivo experiments indicated, estradiol exhibited an inhibition role on the

408

expression of PPARγ, which likely in turn relieves the transcriptional suppression role

409

to MRAP, leading to the increase of MRAP expression in mRNA and protein level.

410

In summary, given the above findings, it was implied that estradiol might

411

contribute to the expression of MRAP through inhibiting the expression of

412

transcription regulation factor PPARγ in chicken liver. MRAP might play its

413

biological role in a different way rather than forming an MRAP/MC2R complex

414

under estradiol induction in chicken. However, further studies need to be carried out

415

to confirm the functions of MRAP in chicken liver.

416 417

Figure legends

418

Fig. 1 Cloning and sequence analysis of chicken MRAP. (A) The gene structure

419

of chicken MRAP. Exons are indicated by boxes. The gray regions of boxes represent

420

the coding sequence, and the white regions represent the regions that were cut off in

421

the transcribed mRNA. The introns are regions within the broken lines which

422

indicated the cut off in the mRNA. (B) cDNA and deduced amino acid sequence for

423

chicken MRAP. Nucleotides are shown in lower case letters and amino acids in capital

424

letters above the first nucleotide in each codon triplet. The predicted start (atg) and

425

stop (tga) codons, are shown in bold text with underlining. Predicted glycosylated

426

amino acids (N) are indicated by gray shading. The predicted transmembrane is

427

shown as black boxes with white text. The numbers on left hand side indicated the

428

number of nucleotides counting continuously from the first one.

429 430

Fig. 2 Cloning and sequence analysis of chicken MRAP2. Descriptions for Fig. 2A and 2B are the same as described in Fig. 1A and 1B, respectively.

431

Fig. 3 Multiple alignments of MRAP and MRAP2 amino acid sequences among

432

different species by Clustal V. 50%-70% percentage identity sites are indicated by

433

gray shading and more than 70% percentage identity sites are indicated by black

434

shading. (A) Multiple alignment of MRAP amino acid sequences. The amino acid

435

sequences name and NCBI accession numbers used in the multiple alignment were as

436

follows: hs MRAPα, Homo sapiens (human), NM_178817.3; hs MRAPβ, Homo

437

sapiens

438

NM_001285394.1; mm MRAP, Mus musculus (house mouse), NM_029844.3; gg

439

MRAP, Gallus gallus (chicken), XR_001470382.1 ; mg MRAP, Meleagris gallopavo

(human),

NM_206898.1;

hs

MRAPγ,

Homo

sapiens

(human),

440

(turkey), XM_003202927.2; mu MRAP, Melopsittacus undulates (budgerigar),

441

XM_005151786.1; ap MRAP, Anas platyrhynchos (mallard), XM_005013839.2; cp

442

MRAP, Chrysemys picta (painted turtle), XM_005283913.1; ps MRAP, Pelodiscus

443

sinensis (Chinese soft-shelled turtle), XM_006126058.1; xt MRAP, Xenopus

444

(Silurana) tropicalis (western clawed frog), XM_002938443.3; dr MRAP, Danio rerio

445

(zebrafish), Ensembl version: ENSDART00000148193.2. The position of ligand

446

binding domain (1), antiparallel dimerization domain (2), and the transmembrane

447

domain (3) are shown by black lines. (B) Multiple alignment of MRAP2 amino acid

448

sequences. The amino acid sequence names were described as above, the NCBI

449

accession numbers were as follows: hs MRAP2, NM_138409.2; mm MRAP2,

450

NM_001101482.2; gg MRAP2α, NM_001320907.1; gg MRAP2β, XM_015284715.1;

451

mg MRAP2, XM_010707659.1; mu MRAP2, XM_005153140.1; ap MRAP2,

452

XM_013093506.1; ps MRAP2α, XM_006133346.2; ps MRAP2β, XM_006133347.2;

453

xt MRAP2, XM_002933917.2; dr MRAP2, XM_001342887.5. (C) Amino acid

454

sequences alignment of chicken MRAP and MRAP2α. The asterisk represents the

455

same amino acid.

456

Fig. 4 Phylogenetic relationship between representative MRAP and MRAP2

457

homologs. The amino acid sequence alignments were conducted using Clustal V

458

based on a BLOSUM protein weight matrix. The bootstrap consensus tree was

459

inferred from 1000 replicates. The evolutionary distances were computed using the

460

Poisson correction method. The fragmented sequences were removed for obtaining a

461

stable topology. Evolutionary analyses were conducted in MEGA6. Branch lengths

462

reflect evolutionary divergence.

463

Fig. 5 Tissue distribution of chicken MRAP, MRAP2 and MCRs mRNAs using

464

RT-PCR. Distribution is shown of amplification products separated by electrophoresis

465

on a 2% agarose gel.

466

Fig. 6 Expression patterns of MRAP, MC5R and PPARγ in liver in different

467

developmental stages. (A) The MRAP mRNA levels were normalized to the mRNA

468

levels of β-actin. (B) Bottom: Western blotting of MRAP using equal amounts of

469

protein extracted from livers of chickens with different ages. Top: Corresponding

470

MRAP densitometry values were normalized to β-actin, respectively. (C) The MC5R

471

and PPARγ mRNA levels were normalized to the mRNA levels of β-actin. Graphed

472

data represent the mean ± SD, n = 6. Values with different superscripts indicate

473

statistical difference (p<0.05).

474

Fig. 7 Effects of 17β-Estradiol on the expression of MRAP, MC5R and PPARγ in

475

chicken liver. (A) The MRAP mRNA levels of the genes were normalized to the

476

mRNA levels of β-actin. (B) Bottom: Western blotting of MRAP using equal amounts

477

of protein extracted from livers treated with increasing concentrations of 17β-estradiol.

478

Top: Corresponding MRAP densitometry values were normalized to β-actin,

479

respectively. (C) The MC5R and PPARγ mRNA levels were normalized to the mRNA

480

levels of β-actin. Graphed data represent the mean ± SD, n = 6. Values with

481

different superscripts indicate statistical difference (p<0.05).

482

Fig. 8 Effects of 17β-Estradiol on the expression of MRAP, MC5R and PPARγ in

483

primary hepatocytes. The mRNA levels of the genes were normalized to the mRNA

484

levels of β-actin. Each data point represents the mean ± SD, n = 6. Values with

485

different superscripts indicate statistical difference (P<0.05).

486 487

Supporting Information

488

Supplemental Table.1 List of PCR and real-time PCR primers used. All primers were

489

designed by primer 5.0 and synthases by Sangon Biotech (Shanghai, China). Products

490

have been sequenced and alignment with the reference sequence.

491

Supplemental Fig.1 The output of MRAP transmembrane domain performed by

492

TMHMM Server v. 2.0.

493

Supplemental Fig.2 The output of MRAP2 transmembrane domain performed by

494

TMHMM Server v. 2.0.

495

Author contributions

496

RJ and LY performed the experiments and wrote the manuscript. LH, XN and LC

497

participated in the management of the experimental animals and the sample collection.

498

WY and LZ contributed to the cell culture and qRT-PCR analyses. KX and HR

499

participated in critical discussion. TY and LX conceived the study, participated in the

500

experiment design and critical discussion. All authors read and approved the final

501

manuscript.

502

Disclosure statement

503 504

The authors have no conflicts of interest to disclose. Acknowledgments

505

This work was supported by the Henan International Cooperative Research

506

Project (162102410030), Earmarked Fund for Modern Agro-Industry Technology

507

Research System (CARS-41-K04), Key Science and Technology Research Project of

508

Henan Province (112101110800).

509

References

510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538

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Primers name

Gene

Primer sequence (5'-3')

Accession

location

number

(5'-3') F:

MRAP

Primer

R:

oduct size

426-445

XR_00147038 CTCAGCAGTCCATTCCCAGA 2.1

Pr

636-617

211

GTCAGGTTTGGTCTTCGCTG F: MRAP2

24-43

NM_00132090 TAACAGAACCTCCCAGCAGG 7.1

R:

217-198

194

GTGCTCCTGTCTTTGTCAGC F: MC1R

273-292

NM_00103146 CAAGACGCTCTTCATGCTGC 2.1

R:

398-379

126

AGGAAGGAGAGGGAGGACAC F: MC2R

812-831

NM_00103151 ACTGTGCCTGCTACATGTCC 5.1

R:

915-896

104

CCGTAATTCTGGGCTTCGGA F: RT-PCR primer

MC3R

XM_00494723 CCGTTCCACCGTTCACCTAA 6.2

R: CTTACTGCTGGCTGTTGGGA F:

MC4R

3322-334 1 3482-346 3 475-494

NM_00103151 ATCATGACGGTCAAGCGTGT 4.1

R:

161

774-755

300

GGCCCAGCACACAACAAAAA F: MC5R

691-710

NM_00103101 AGAACCAGCATGAAGGGAGC 5.1

R:

959-940

269

CCACAGACCATTCTCACGCT F:GAACATCATCCCAGCGTC GAPDH

NM_204305.1

663-682

CA R:

795-776

133

ACGGCAGGTCAGGTCAACAA F: β-actin

NM_205518.1

415-434

GAGAGAAGATGACACAGATC R:

530-511

116

GTCCATCACAATACCAGTGG q-PCR primer

MRAP

XR_00147038 2.1

F:

426-445

CTCAGCAGTCCATTCCCAGA R:

211 636-617

GTCAGGTTTGGTCTTCGCTG F: MC5R

66-85

NM_00103101 TGTGCCTACTGTCAAGAGCA 5.1

R:

276-257

211

CTCCCAAGCATTAGACACGC F: β-actin

NM_205518.1

GAGAGAAGATGACACAGATC R: GTCCATCACAATACCAGTGG

633 634

415-434

530-511

116

Fig. 1A

Fig. 1B

Fig. 2A

Fig. 2B

Fig. 3A

Fig. 3B

Fig. 3C

Fig. 4

Fig. 5

Fig. 6A

Fig. 6B

Fig. 6C

Fig. 7A

Fig. 7B

Fig. 7C

Fig. 8

635 636 637

Highlights

638

MRAP and MRAP2 were conserved between species.

639

MRAP and MCRs were expressed in adrenal gland, and also in other tissues in

640

chicken.

641

Only MRAP and MC5R expressed in chicken liver.

642

The expression of MRAP was regulated by estrogen in vivo and in vitro.

643

MRAP might play its biological role in a different way rather than forming an

644

MRAP/MC2R complex

645 646