Distinct post-transcriptional regulation of Igfbp1 gene by hypoxia in lowland mouse and Qinghai-Tibet plateau root vole Microtus oeconomus

Molecular and Cellular Endocrinology 376 (2013) 33–42

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Distinct post-transcriptional regulation of Igfbp1 gene by hypoxia in lowland mouse and Qinghai-Tibet plateau root vole Microtus oeconomus Shengting Zhang a, Yang Zhao a,b,c, Xiaofeng Hu a, Zongyun Liu a,b,c, Xiaocheng Chen d, Xuequn Chen a,b,c,⇑, Jizeng Du a,b,c,⇑ a

Division of Neurobiology and Physiology, Department of Basic Medical Sciences, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Medical Neurobiology of The Ministry of Health, Zhejiang University, Hangzhou, China c Zhejiang Province Key Laboratory of Neurobiology, Zhejiang University, Hangzhou, China d Northwest Plateau Institutes of Biology, the Chinese Academy of Sciences, Xining 810001, Qinghai, China b

a r t i c l e

i n f o

Article history: Received 19 January 2013 Received in revised form 28 May 2013 Accepted 31 May 2013 Available online 6 June 2013 Keywords: Microtus oeconomus Mouse Hypoxia Insulin-like growth factor mRNA stability U-rich element

a b s t r a c t Our previous study revealed the particular expression patterns of insulin-like growth factor 1 (IGF1) and insulin-like growth factor binding protein 1 (IGFBP1) in the Qinghai-Tibet plateau root vole (Microtus oeconomus) under hypoxic challenge. Here we report the molecular mechanisms of Igf gene regulation associated with adaptation to hypoxia. M. oeconomus IGF1 and IGFBP1 were shown to be highly conserved. Hypoxia (8.0% O2, 6 h) did not change the liver-derived Igf1 expression in either M. oeconomus or mouse. Hypoxia significantly upregulated hepatic Igfbp1 gene expression and IGFBP1 levels in the liver and plasma of the mouse, but not in M. oeconomus. A functional U-rich element in the 30 untranslated region was found in mouse Igfbp1 mRNA, which was associated with Igfbp1 mRNA stabilization and upregulation under hypoxia, and this U-rich element was eliminated in the M. oeconomus Igfbp1, resulting in blunted Igfbp1 mRNA upregulation, which might be understood as a sequence variation modified during molecular evolution under hypoxia. Ó 2013 Published by Elsevier Ireland Ltd.

1. Introduction The IGF (insulin-like growth factor) family includes two ligands (IGF1 and IGF2), two cell surface receptors (IGF1R and IGF2R) and six binding proteins (IGFBP1-6). IGF1, a small peptide hormone, is involved in mammalian growth, development and metabolism by regulating cellular proliferation, differentiation and migration (Annunziata et al., 2011). The IGF system is also involved in glucose regulation and the development of diabetes, cardiovascular and immune disease, and cancer (Abbas et al., 2008; Ezzat et al., 2008; Lee et al., 2012; Rajpathak et al., 2009; Smith, 2010). The IGF system can be activated to govern growth, development and cellular protection from injury under hypoxia or ischemia (Kajimura et al., 2005; Liu

⇑ Corresponding authors. Address: Division of Neurobiology and Physiology, Department of Basic Medical Sciences, School of Medicine, Research Building C-507, Zhejiang University, Zijingang Campus, 866 Yuhangtang Road, Hangzhou 310058, China. Tel./fax: +86 571 8820 8182. E-mail addresses: [email protected] (S. Zhang), [email protected] (Y. Zhao), [email protected] (X. Hu), [email protected] (Z. Liu), [email protected] (X. Chen), [email protected] (X. Chen), [email protected] (J. Du). URLs: http://www.cmm.zju.edu.cn/klmn/lab/djz/du.html (X. Chen), http:// www.cmm.zju.edu.cn/klmn/lab/djz/du.html (J. Du). 0303-7207/$ - see front matter Ó 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.mce.2013.05.025

et al., 2011). Furthermore, IGF1 can also activate hypoxia inducible factor 1 (HIF1), which triggers the expression of various target genes and plays a critical role in the cellular hypoxic response (Fukuda et al., 2002; Sutton et al., 2007). IGFBP1 (insulin-like growth factor binding protein 1) is synthesized mainly in hepatocytes and kidney cells, and is involved in the regulation of physiological functions and pathogenesis. IGFBP1 is released from the liver into the circulation and binds to IGF1 with high affinity to govern its bioavailability and action (Rajpathak et al., 2009; Wheatcroft and Kearney, 2009). Furthermore, the igfbp1 gene is known to be modulated through transcriptional regulation by many factors, including insulin, glucocorticoids, cAMP, and hypoxia (Lee et al., 1997; Powell et al., 1995; Sugawara et al., 2000). The root vole (Microtus oeconomus Pallas, 1776) belongs to the species-rich genus Microtus, and is widely distributed in the north of Europe, Asia and North America (Brunhoff et al., 2003), and particularly lives on the Qinghai-Tibet plateau of China, exposed to cold and hypoxia at altitudes of 3000–4500 m. We previously reported that hypoxia mimicked by CoCl2 induces a diversity of HIF1a, IGF1 and IGFBP1 expression in M. oeconomus, the plateau pika (Ochotona curzoniae), and the mouse (Mus musculus) (Chen et al., 2007). This diversity of expression patterns suggests an

34

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

adaptively selective strategy in hypoxic molecular evolution; however, the underlying molecular mechanism for the diversity remains poorly understood. In recent years, understanding how plateau mammals adapt to high-altitude hypoxia has become an increasingly attractive topic. And many species of animals on the Qinghai-Tibet plateau, including yak (Bos grunniens), zokor (Myospalax baileyi), root vole (M. oeconomus), plateau pika (O. curzoniae), and naked carp (Gymnocypris przewalskii) have been used in hypoxia-related studies (Cao et al., 2008, 2009; Chen et al., 2007; Wei et al., 2006; Qiu et al., 2012). In this study, we report the cloning and analysis of the sequence variations of the Igf1 and Igfbp1 genes in M. oeconomus, and a comparison of liver-derived Igf1 and Igfbp1 gene expression in M. oeconomus and mouse under hypoxic challenge. Variation in the U-rich element of Igfbp1 in M. oeconomus was found to lead to blunted upregulation of the igfbp1 gene by hypoxia, which might be correlated with the acclimatization to hypoxia during evolution.

2. Materials and methods 2.1. Animals and hypoxic exposure Adult M. oeconomus (12–21 g) were collected around the Haibei Alpine Meadow Ecosystem Research Station, the Chinese Academy of Sciences, China (37°400 N, 101°230 E, altitude 3200 m). Adult male ICR mice (29–34 g; clean grade) were purchased from the Experimental Animal Center of Zhejiang Province (Hangzhou, Zhejiang, China; approval number: SCXK (Zhe) 2008-0033). All animals were maintained at room temperature and had free access to food and water. They were randomly divided into control and hypoxic exposure groups. For M. oeconomus, hypoxia was mimicked by putting the animals in a chamber ventilated with mixed gas of 8.0% O2 and 92% N2 (an altitude of 7000 m) for 6 h, and the corresponding control group was kept in a laboratory of the Haibei research station (3200 m). For mice, the hypoxia groups were put in a hypobaric hypoxia animal experimental chamber (Guizhou Fenglei Air Ordnance Co., Ltd.) to mimic 8.0%, 10.8% or 16.0% O2 hypoxic conditions (an altitude of 7000 m, 5000 m or 2000 m) for 6 h, and the control group was maintained in a laboratory at Zhejiang University (sea level, 20.9% O2). After hypoxic exposure, all animals were rapidly decapitated. The liver was quickly removed and trunk blood was collected and centrifuged for plasma separation. All tissues and plasma were frozen immediately in liquid nitrogen and stored at 80 °C until use. The experimental procedures followed the National Institutes of Health guide for the care and use of laboratory animals. All protocols concerning animal use were approved by the Institutional Animal Care and Use Committee of the School of Medicine, Zhejiang University.

2.2. Molecular cloning and sequence analysis cDNA and genomic DNA were prepared from the liver of M. oeconomus and mouse, and then used as templates for PCR to amplify the Igf1 and Igfbp1 gene coding regions, and the Igfbp1 gene promoter and intron 1 regions. Primers were designed based on conserved regions of the mouse, rat and human Igf1 or Igfbp1 genes (Supplementary Fig. 1 and Table S1): mouse Igf1 [GenBank: 16000]; mouse Igfbp1 [GenBank: 16006]; rat Igf1 [GenBank: 24482]; rat Igfbp1 [GenBank: 25685]; human IGF1 [GenBank: 3479]; and human IGFBP1 [GenBank: 3484]. The PCR products were TA-cloned and sequenced. Comparisons of the M. oeconomus Igf1 or Igfbp1 gene with the corresponding sequences from other species were generated using Multalin software and the polygenetic trees were constructed by Mega 3.1 software using

the neighbor-joining method based on the ClustalW2 alignment results (Supplementary Fig. 2). 2.3. Plasmid construction and mutagenesis The M. oeconomus and mouse Igfbp1 gene promoter regions (2.8 kb) were amplified by PCR from the sequencing plasmids and fused to pGL2-basic vector (M.O./M.M. Igfbp1 Promoter PGL2-basic) to test the potential hypoxia-induced promoter activation; the M. oeconomus and mouse Igfbp1 gene intron 1 regions (1.4 kb) were also amplified by PCR from the sequencing plasmids and inserted into pGL2-promoter vector which had a minimal SV40 promoter (M.O./M.M. Igfbp1 Intron 1 PGL2-promoter), to test potential hypoxia-induced enhancer activation. For Igfbp1 mRNA stability assessment, a fragment including the 50 -UTR, ORF and 30 -UTR of the Igfbp1 gene was ligated into the pcDNA3.1(+) expression vector (Invitrogen). The coding region of the firefly gene was fused to pcDNA3.1(+) to construct the firefly expression vector for control. For deletion analysis of the 30 -UTR, the indicated gene fragments were amplified by PCR using the full-length Igfbp1 expression vector as the template, and then fused into the pcDNA3.1(+) vector. The mutagenesis of U-rich elements was performed using the Fast mutagenesis system (TransGen Biotech, Beijing, China) following the manufacturer’s instructions. The sequences of primers used in plasmid construction and mutagenesis are shown in the supplementary material (Supplementary Fig. 1 and Table S2). All inserts were confirmed by sequencing before the plasmids were transfected. 2.4. Cell culture, DNA transfection and cellular hypoxic exposure Human hepatoma cells (HepG2) and mouse hepatoma cells (Hepa1–6), obtained from the cell-bank of the Chinese Academy of Sciences (Shanghai, China), were grown in DMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C in a humidified incubator with 21% O2 and 5% CO2. Twenty-four hours after cell plating, transient transfection was performed using Genjet transfection regent (Ver. II) (SignaGen Laboratories, MD, USA). For the dual-luciferase assay, Renilla luciferase reporter plasmid (pRL-SV40, Promega) was co-transfected to control for transfection efficiency. Twenty-four hours after transfection, cellular hypoxia (1% O2) was induced using the Proox Model P110 and ProCO2 Model P120 hypoxia system (BioSpherix, USA), while the control cells were maintained in normal culture conditions. It has been reported that the intracellular pO2 in the hepatic cell is 15 mmHg (1.99% O2) (Jungermann and Kietzmann, 2000). Thus, when an intact rat breathes air with 8% O2, the oxygen level is 1.99% or less in its liver cells (El-Desoky et al., 1999; Seifalian et al., 2001). Therefore, 1% O2 exposure was chosen for the in vitro experiments. Besides, the endogenous hypoxia response pathways in HepG2 cells are triggered by 1% O2 exposure, including HIF1 activation and endogenous Igfbp1 gene upregulation (Kajimura et al., 2006), which makes the potential exogenous hypoxia-responsive promoters can be transcriptionally activated in the luciferase assay. To measure Igfbp1 mRNA decay in Hepa1–6 cells, 100 lM 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB, Sigma) was used to stop transcription, then the DRB-treated cells were divided into hypoxia and control groups, and treated as above. 2.5. Dual-luciferase assay After hypoxia, cells were lyzed to measure luciferase activity by the dual-luciferase reporter assay system (Promega) following the manufacturer’s instructions. The results were normalized to the Renilla luciferase value (pRL-SV40 vector).

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

2.6. Real-time quantitative RT-PCR (qPCR) Total RNA was extracted from tissues and cells, and then reverse-transcribed to cDNA. In the measurement of endogenous Igf1 and Igfbp1 mRNA expression, primers were designed based on the M. oeconomus and mouse Igf1 or Igfbp1 mRNA sequences. The hepatic transcription of the two classes of Igf1 mRNA are controlled by two separate promoters, so in this procedure, qPCR primers were designed to specifically detect the Igf1 class 1 and 2 mRNA levels (Ohtsuki et al., 2005). In measurements of Igfbp1 mRNA stability, qPCR primers were designed to specifically amplify the exogenous Igfbp1 mRNA from plasmids based on the specific sequence of the expression vector. The sequences of primers used in qPCR assays are shown in the supplementary material (Supplementary Fig. 1 and Table S3). The relative quantification of mRNA level was assessed using SYBR Premix Ex TaqTM (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) and analyzed with a PRISM 7900HT real-time PCR system (Applied Biosystems, Foster City, CA). The cycle number at threshold (CT value) was used to calculate the relative amount of mRNA using 18S ribosomal RNA as the reference gene. 2.7. ELISA Liver samples were homogenized and neutral/acid buffer extraction was used for IGF1 measurement in liver as previously described (Goldstein and Phillips, 1991). Protein concentration was determined by the Bradford assay (Beyotime). Acid–ethanol extraction was performed as previously described (Daughaday et al., 1982) for total IGF1 measurement in plasma. The IGF1 concentrations in liver and plasma were measured with an IGF1 ELISA kit (USCN Life Science Inc., E90050Mu: average intra-assay CV is 4.8%, and average inter-assay CV is 5.8%, with a sensitivity of 27 pg/ml), and the IGFBP1 concentrations in liver and plasma were measured with an IGFBP1 ELISA kit (Boster Biotechnology, EK0383: average intra-assay CV is 2.5%, and average inter-assay CV is 7.2%, with a sensitivity of 3 pg/ml), following the instructions of the manufacturer. The IGF1 and IGFBP1 concentrations in liver were normalized to the total protein concentration. 2.8. Statistical analysis All statistical analyses were performed using SPSS version 16.0. Statistical significance compared with the control group was determined with a two-tailed, unpaired Student’s t-test. 3. Results 3.1. Molecular cloning and sequence analysis of Igf1 and Igfbp1 genes of M. oeconomus Due to alternative transcription initiation and mRNA splicing, several Igf1 mRNA isoforms are produced by the Igf1 gene. In this study, the open reading frames (ORFs) of two Igf1 mRNA isoforms ([GenBank: FJ262413.1] and [GenBank: JX987294]) were isolated from M. oeconomus liver cDNA, containing 462 bp and 414 bp respectively (Supplementary Fig. 3A and B). These mRNA isoforms were predicted to be the counterparts of mouse Igf1 mRNA isoform 4 [GenBank: NP_001104745.1] and isoform 5 [GenBank: NP_001104746.1] (also known as IA and IIA). The predicted peptides of the Igf1 mRNA isoforms consisted of 153 and 137 amino-acid residues (Supplementary Fig. 3A and B). The mature M. oeconomus IGF1 peptides predicted from the two Igf1 mRNA isoforms contained the same 70 amino-acid residues, which showed identity with the corresponding sequences in mouse of 98%, in

35

rat 100%, and in human 95%. Four characteristic domains of IGF1 (B, C, A and D) were also predicted in the mature M. oeconomus IGF1 peptide, based on its high degree of conservation with other mammals (Fig. 1A) (Yamada et al., 2004). No special variation associated with IGF1-IGFBP and IGF1-IGFR binding was detected in the mature M. oeconomus IGF1 peptide compared with mouse, rat and human IGF1 (Fig. 1A) (Baxter, 2000; Jansson et al., 1997, 1998). The ORF of M. oeconomus Igfbp1 mRNA [GenBank: JX987293] was cloned from liver cDNA and contained 825 bp (Supplementary Fig. 3C). The predicted M. oeconomus IGFBP1 peptide had 274 amino-acid residues, containing a mature peptide of 249 amino-acid residues (Supplementary Fig. 3C). The mature M. oeconomus IGFBP1 peptide had identity with the corresponding sequences in mouse of 89%, in rat 89%, and in human 73%. Compared with mouse, rat and human, the cysteine-rich N and C domains, which are responsible for IGF binding, were highly conserved in M. oeconomus IGFBP1 (Fig. 1B) (Baxter, 2000; Hwa et al., 1999; Sala et al., 2005; Wheatcroft and Kearney, 2009). The Arg-Gly-Asp (RGD) motif in the C domain responsible for IGF-independent effects was also identified in M. oeconomus IGFBP1 (Fig. 1B) (Jones et al., 1993; Wheatcroft and Kearney, 2009). The phylogenetic tree of the mature IGF1 peptide indicated that M. oeconomus IGF1 was most closely related to rat and mouse IGF1, and this peptide did not evolve convergently with two other hypoxia-adapted mammals, Myospalax baileyi and Ochotona curzoniae, consistent with their taxonomic relationship (Fig. 1C). The phylogenetic tree of the mature IGFBP1 peptide indicated that the M. oeconomus peptide was also most closely related to the rat and mouse IGFBP1 (Fig. 1D). The 3D structure prediction for IGF1, the IGFBP1 N-terminus and the IGFBP1 C-terminus in M. oeconomus and mouse was based on the sequence identity to human by the software (SWISS-MODEL), and it seemed likely that there was no significant difference between them (Supplementary Fig. 4). This suggested that, compared with other mammals, highly-conserved sequences and domains occur in M. oeconomus IGF1 and IGFBP1. 3.2. Effect of hypoxia on Igf1 and Igfbp1 gene expression in M. oeconomus and mouse The liver is the major source of IGF1 and IGFBP1 in the circulation, so we measured hypoxia-induced Igf1 and Igfbp1 mRNA expression in the liver by qPCR assay. Hypoxia (8.0% O2, equal to an altitude of 7000 m, for 6 h) did not influence hepatic Igf1 class 1 or class 2 mRNA expression in either M. oeconomus or mouse (Fig. 2A), while it significantly increased hepatic Igfbp1 mRNA expression in mouse, but not in M. oeconomus (Fig. 2A). The mouse hepatic Igf1 mRNA expression was not affected by graded hypoxic exposure (16.0%, 10.8%, and 8.0% O2), and the Igfbp1 mRNA expression was also unaffected by 16.0% or 10.8% O2, but was significantly upregulated by 8.0% O2 for 6 h (Fig. 2B). Besides, hypobaric hypoxia (8.0% O2, 8 h) in the rat also induced upregulation of the Igfbp1 gene, but did not affect Igf1 gene expression (Supplementary Fig. 5). Consistent with the Igf1 mRNA expression, the IGF1 content in the liver and plasma were not altered by hypoxia (8.0% O2, 6 h) in M. oeconomus and mouse (Fig. 3). The IGFBP1 content in liver and plasma increased significantly under 8.0% O2 exposure for 6 h in mouse, but not in M. oeconomus (Fig. 3). These data revealed differences in hepatic Igfbp1 mRNA expression and IGFBP1 content in the liver and plasma between the plateau M. oeconomus and the lowland mouse under hypoxic challenge. 3.3. Effect of hypoxia on transcriptional regulation of the Igfbp1 gene in M. oeconomus and mouse Hypoxia-induced Igfbp1 upregulation is known to involve the binding of HIF1 to hypoxia response elements (HREs) in zebrafish,

36

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

(A)

(B)

(C)

(D)

Fig. 1. Molecular cloning and sequence analysis of M. oeconomus Igf1 and Igfbp1 genes. The mature peptides of M. oeconomus IGF1 (A) and IGFBP1 (B) were compared with the corresponding sequences in mouse, rat and human. Conserved residues are indicated by (), and gaps are indicated by (-). The four predicted domains (B, C, A and D) of M. oeconomus IGF1 and the three predicted domains (N, Central linker, and C) of M. oeconomus IGFBP1 are also marked. Highly-conserved cysteines in the IGFBP1 N and C domains are indicated by ‘‘⁄’’, and the conserved RGD motif in the C domain is indicated by ‘‘&’’. Phylogenetic trees based on the mature IGF1 (C) and IGFBP1 (D) peptide sequences were constructed using the neighbor-joining method. The analysis was run with 500 bootstrap replications and only bootstrap support values >50% were reported. The M. oeconomus sequences in the alignments and phylogenetic trees are indicated by a solid-line box. In the phylogenetic tree, the IGF1 proteins from another two mammals living on the Qinghai-Tibetan plateau, Myospalax baileyi and Ochotona curzoniae, are indicated by dotted-line boxes.

human and rat (Kajimura et al., 2006; Scharf et al., 2005; Tazuke et al., 1998), however, the transcriptional regulation of the hypoxia-induced Igfbp1 expression in mouse and M. oeconomus remains unclear. In this study, the partial promoter [GenBank: JX987296] and intron 1 regions [GenBank: JX987295] of the M. oeconomus Igfbp1 gene were cloned from the liver genome, and several potential HREs were predicted according to the consensus HRE sequence (A/G)CGTG in these regulatory regions. To investigate the hypoxiainduced transcriptional regulation of the Igfbp1 gene, four recombination plasmids were constructed (M.O./M.M. Igfbp1 Promoter PGL2-basic and M.O./M.M. Igfbp1 Intron 1 PGL2-promoter) and co-transfected with the pRL-SV40 vector into human hepatoma cells (HepG2) (Fig. 4A). After hypoxic exposure (1% O2) for 6 h, the transcriptional activity of the exogenous portions of the Igfbp1 gene was assessed by dual-luciferase assay. The data showed that both the M. oeconomus and mouse Igfbp1 promoter region had basal promoter activity to drive luciferase expression in HepG2 cells. But the transcriptional activity of the M. oeconomus and mouse Igfbp1 promoter was not significantly changed by hypoxia of 1%

O2 for 6 h, compared with the pGL2-basic empty vector (Fig. 4B). And neither the M. oeconomus nor the mouse Igfbp1 intron 1 region was significantly responsive to hypoxia, compared with the pGL2-promoter empty vector (Fig. 4B). The efficiency of cellular hypoxia and the dual-luciferase assay were confirmed by using the hypoxia-responsive zebrafish Igfbp1 promoter pGL2-basic plasmid p1225Luc (a generous gift from Prof. Cunming Duan, University of Michigan), in which the transcriptional activity increased significantly after hypoxia using our procedure, when likewise compared with the pGL2-basic empty vector (Supplementary Fig. 6) (Kajimura et al., 2006). This suggested that these regulatory regions of the M. oeconomus and mouse Igfbp1 gene are not transcriptionally activated by hypoxia (1% O2, 6 h) in HepG2 cells. 3.4. Effect of hypoxia on post-transcriptional regulation of the Igfbp1 gene in M. oeconomus and mouse Since the regulatory regions of the M. oeconomus and mouse Igfbp1 gene were not transcriptionally activated by hypoxia in

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

37

(A)

(B)

Fig. 2. Effect of hypoxia on hepatic Igf1 and Igfbp1 mRNA expression. (A) Effects of acute hypoxia (8.0% O2, 7000 m, 6 h) on hepatic M. oeconomus (M.O.) and mouse (M.M.) Igf1 and Igfbp1 mRNA expression. For M. oeconomus, the control group (Ctrl) was maintained at an altitude of 3200 m, and hypoxia was mimicked by mixed gas of 8.0% O2 and 92% N2 (7000 m) for 6 h. For mouse, the control group (Ctrl) was maintained at sea level, and hypoxia was mimicked using a hypobaric hypoxia animal experimental chamber (7000 m, 8.0% O2, 6 h). (B) Effect of graded hypoxia on mouse hepatic Igf1 and Igfbp1 mRNA expression. The control group (Ctrl) was maintained at sea level, and the hypoxia groups were placed in a hypobaric hypoxia animal experimental chamber to mimic hypoxic conditions of 2000, 5000 and 7000 m (16.0%, 10.8% and 8.0% O2) for 6 h. Igf1 class 1/2 and Igfbp1 mRNA expression in liver was measured by qPCR, using 18S ribosomal RNA as the reference gene. Results are expressed as the fold-change of relative mRNA expression (hypoxia group versus normoxia group). Values are given as mean ± SEM (n = 6–8); P < 0.05, P < 0.01 compared with the control group.

(A)

(B)

Fig. 3. Effects of hypoxia on IGF1 and IGFBP1 protein content in liver and plasma. For M. oeconomus, the control group (Ctrl) was maintained at an altitude of 3200 m, and hypoxia (Hyp) was mimicked by mixed gas of 8.0% O2 and 92% N2 (7000 m) for 6 h. For mouse, the control group (Ctrl) was maintained at sea level, and hypoxia (Hyp) was mimicked using a hypobaric hypoxia animal experimental chamber (7000 m, 8.0% O2, 6 h). (A) Measurement of IGF1 and IGFBP1 protein content in liver by ELISA. Results were normalized to the total protein concentration. (B) Measurement of total IGF1 and IGFBP1 content in plasma by ELISA. Values are given as mean ± SEM (n = 6–8);  P < 0.05, P < 0.01 compared with the control group.

HepG2 cells, we considered that post-transcriptional mRNA stabilization might be involved in the hypoxia-induced Igfbp1 upregulation. The full length of the M. oeconomus and mouse Igfbp1 gene coding region, including the ORF and 50 /30 untranslated region (50 /30 -UTR), were inserted into pcDNA3.1(+) expression vector which contained a CMV promoter to perform constant transcription of Igfbp1 mRNA in hypoxic and control groups of HepG2 cells (Fig. 5A). After hypoxia (1% O2, 6 h), the mRNA expression of plasmids was monitored by qPCR to measure the stabilization of igfbp1

mRNA. To remove the interference of endogenous mRNA expression in HepG2 cells, the qPCR primers were designed to amplify a sequence located downstream of the inserted Igfbp1 gene and upstream of the BGH polyadenylation sequence in pcDNA3.1(+), aiming to specifically detect mRNA expression derived from the recombination plasmids. We found that hypoxia significantly upregulated the plasmid-derived mouse Igfbp1 mRNA, but not that of M. oeconomus, which indicated that cellular hypoxia can stabilize the full-length Igfbp1 mRNA in mouse but not in M. oeconomus

38

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

(A)

(B)

Fig. 4. Transcriptional activity of Igfbp1 promoter and intron 1 regions under hypoxia. (A) Structure of the recombination plasmids. M. oeconomus and mouse Igfbp1 promoters were inserted into promoter-less pGL2-basic vector, and the M. oeconomus and mouse Igfbp1 intron 1 regions were fused into the pGL2-promoter vector. Different regulatory regions are identified and marked according to the mouse Igfbp1 gene. (B) HepG2 cells were co-transfected with recombination plasmids containing different fragments of the M. oeconomus (M.O.) and mouse (M.M.) Igfbp1 genes and Renilla luciferase vector. Dual-luciferase assay was performed after 1% O2 hypoxia for 6 h, and transfection efficiency was normalized to Renilla luciferase activity. Results show the fold-change of relative luciferase activity (hypoxia group versus normoxia group). Values are given as mean ± SEM of three independent transfection assays.

(A)

(C)

(D) (B)

Fig. 5. Effect of hypoxia on Igfbp1 mRNA stability. (A) Structure of the recombination plasmids. M. oeconomus and mouse Igfbp1 gene fragments including the 50 -UTR, ORF and 30 -UTR were inserted into the pcDNA3.1(+) expression vector. Different gene regions are identified and marked according to the mouse Igfbp1 gene. (B) HepG2 cells were transfected with M. oeconomus (M.O.) and mouse (M.M.) Igfbp1 expression plasmids, and Igfbp1 mRNA from plasmids was amplified and analyzed by qPCR using specific primers after hypoxia (1% O2, 6 h). The firefly expression vector was used as control. (C) Endogenous Igfbp1 mRNA of Hepa1-6 cells was analyzed by qPCR after hypoxia (1% O2, 6 h). (D) DRB (100 lM) was applied to Hepa1-6 cells to inhibit transcription before hypoxia (1% O2), and the remaining Igfbp1 mRNA was quantified by qPCR at the indicated time points. These data were fitted to an exponential decay function (Exp) to calculate the half-life of Igfbp1 mRNA. For all qPCR assays, 18S ribosomal RNA was used as the reference gene. Results show the fold-change of relative mRNA expression (hypoxia group (Hyp) versus normoxia group (Ctrl)). Values are given as mean ± SEM (n = 3);  P < 0.01 compared with the control group.

(the firefly expression vector was used as control) (Fig. 5B). Furthermore, a mouse hepatoma cell line, Hepa1–6, in which endogenous Igfbp1 mRNA expression was increased by hypoxia (1% O2, 6 h) (Fig. 5C), was used to investigate the effect of hypoxia on endogenous Igfbp1 mRNA stabilization. DRB (5,6-dichlorobenzimidazole 1-b-D-ribofuranoside) was used to block endogenous

transcription, then the Hepa1–6 cells were exposed to hypoxia (1% O2) and the endogenous Igfbp1 mRNA content (as the remaining Igfbp1 mRNA) was measured by qPCR at different time points. The remaining endogenous Igfbp1 mRNA content in hypoxic (1% O2) Hepa1–6 cells was higher than the control group at 6 h (P < 0.05) and 12 h (P < 0.01). The data collected at different time

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

points were fitted to an exponential decay function, and showed that the half-life of Igfbp1 mRNA in the control group was 3.19 h, while in the hypoxia group it was extended to 5.59 h, indicating that hypoxia significantly attenuated Igfbp1 mRNA decay in Hepa1–6 cells (Fig. 5D). These results suggested that hypoxia stabilizes mouse Igfbp1 mRNA but not that of M. oeconomus and this contributes to the hepatic Igfbp1 mRNA upregulation induced by hypoxia in the mouse. 3.5. U-rich element in the 30 -UTR is involved in hypoxia-induced Igfbp1 mRNA stabilization Previous studies showed that the 30 -UTR of Igfbp1 mRNA is involved in the regulation of its stability (Gay and Babajko, 2000). In this study, the 30 -UTRs in recombination plasmids were deleted to assess their contribution to hypoxia-induced Igfbp1 stabilization. The data showed that hypoxia (1% O2, 6 h) had no effect on the stability of M. oeconomus Igfbp1 mRNA with or without the 30 -UTR. However, the stabilization of mouse Igfbp1 mRNA induced by hypoxia was completely abolished when the 30 -UTR was deleted (Fig. 6A). This result indicated that the 30 -UTR of mouse Igfbp1 mRNA contributes to its stabilization under hypoxia. Elements potentially involved in the regulation of Igfbp1 mRNA stability under hypoxia, like AU-rich (AUUUA) and U-rich (NNUUNNUUU) elements, were predicted in the 30 -UTR of M. oeconomus and mouse Igfbp1 mRNA according to published reports (Dormoy-Raclet et al., 2007; Khabar, 2010). Two potential U-rich elements were located in the mouse Igfbp1 30 -UTR (+4238/+4249 and +4403/+4413), but not in M. oeconomus. Mutations were introduced to the Igfbp1 expression vectors to interchange the two U-rich elements in the mouse Igfbp1 30 -UTR and the corresponding sequences in M. oeconomus respectively (Fig. 6B). The data showed that hypoxia (1% O2, 6 h)-induced mouse Igfbp1 mRNA stabilization was abolished when the first U-rich element (+4238/+4249) was mutated (M.M. MUT1); on the contrary, hypoxia increased the stability of M. oeconomus Igfbp1 mRNA when the first U-rich element of mouse (+4238/+4249) was introduced (M.O. MUT1), while the second U-rich element (+4403/+4413) was not involved in the stabilization (Fig. 6B). This suggested that the first U-rich element (+4238/+4249) in the 30 -UTR contributes to the stabilization of mouse Igfbp1 mRNA induced by hypoxia, and

(A)

39

variations in the corresponding sequence are responsible for the blunting of the stabilization of M. oeconomus Igfbp1 mRNA. 4. Discussion The sequences of the IGF1 and IGFBP1 peptides and the critical domains for IGF-IGFBP and IGF-IGFR binding in M. oeconomus showed high conservation with animals and human (Baxter, 2000; Hwa et al., 1999; Wheatcroft and Kearney, 2009; Duan and Xu, 2005). The phylogenetic trees showed that the mature IGF1 and IGFBP1 peptides in M. oeconomus were closely related to those in the lowland rodents. However, in terms of taxonomic relationship, M. oeconomus belongs to Arvicolinae, Microtus genus, but the mouse (Mus musculus) belongs to Murinae, Mus genus. Brunhoff et al. investigated the species-wide holarctic phylogeography of the root vole (M. oeconomus) and showed that it consists of four main mtDNA phylogenetic lineages, which have a wide allopatric distribution at high latitudes in northern and central Europe, Russia, and northern Canada. They have a distinct phylogeographical structure that was initiated before the latest glaciation. The similarities in the phylogeographical patterns in the root vole and other rodents suggest that geological and climatic events play important roles in structuring biotic communities (Brunhoff et al., 2003). M. oeconomus of the Qinghai-Tibet plateau lives at high altitudes of 3000–4500 m, and has been exposed generation by generation to a hypoxic and cold climate with the raising of Qinghai-Tibet plateau from the collision of the Indian and the Eurasian plates 40–50 million years ago (Harrison et al., 1992). The particular evolutionary sequence variants and special gene expression patterns under hypoxic challenge reflect its adaptive evolution to the plateau environment. IGF1 is a growth factor in the growth and development of vertebrates, and also protects cells against stress. In models of hypoxia or ischemia, IGF1 improves the survival of several types of cell (Eliasz et al., 2010; Gehmert et al., 2008; Liu et al., 2011; Mehrhof et al., 2001), and the availability of IGF1 is regulated by IGFBPs, including IGFBP1. It has been demonstrated that hypoxia-induced Igfbp1 gene upregulation reduces the availability of IGF1 and inhibits the IGFR pathway, contributing to developmental retardation under hypoxia (Kajimura et al., 2005; Murphy, 2000; Sun et al., 2011). In the present study, liver-derived IGF1 remained

(B)

Fig. 6. Mechanism of hypoxia-induced Igfbp1 mRNA stabilization. (A) Deletion analysis of the M. oeconomus (M.O.) and mouse (M.M.) Igfbp1 30 -UTR in hypoxia-induced Igfbp1 mRNA stabilization. Full-length (M.O. and M.M.) and 30 -UTR-deleted (Del 30 M.O. and Del 30 M.M.) Igfbp1 expression plasmids were transfected into HepG2 cells, and Igfbp1 mRNA from plasmids was quantified by qPCR using specific primers after hypoxia (1% O2, 6 h). (B) Mutation analysis of functional U-rich elements in hypoxia-induced Igfbp1 mRNA stabilization. The two U-rich elements (+4238/+4249 and +4403/+4413) in full-length Igfbp1 expression plasmids were each mutated to interchange the U-rich elements in the mouse 30 -UTR and the corresponding sequences in M. oeconomus (M.O. MUT1, M.O. MUT2, M.M. MUT1, M.M. MUT2). The wild-type and mutated recombination plasmids were transfected into HepG2 cells. Igfbp1 mRNA from plasmids was analyzed by qPCR using specific primers after hypoxia (1% O2, 6 h). For all qPCR assays, 18S ribosomal RNA was used as the reference gene. Results show the fold-change of relative mRNA expression (hypoxia group (Hyp) versus normoxia group (Ctrl)). Values are given as mean ± SEM (n = 3); P < 0.05, P < 0.001 compared with the control group.

40

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

unchanged by hypoxic challenge in the lowland mouse and plateau M. oeconomus. In the meantime, sequence variation in the U-rich element of the Igfbp1 mRNA 30 -UTR was found in M. oeconomus to contribute to the blunted IGFBP1 upregulation under hypoxia, which may be a genetic strategy for adaptation to a hypoxic environment. IGF1 and IGFBP1 are synthesized mainly in the liver and released into the circulation, in which they are regulated by developmental and metabolic factors and contribute to growth and development in health as well as in disease (Bonefeld and Moller, 2011; Ohlsson et al., 2009; Wheatcroft and Kearney, 2009). In this study, hypoxia (8.0% O2, 6 h) significantly upregulated hepatic Igfbp1 gene expression and plasma IGFBP1 levels in the lowland mouse, but Igfbp1 expression was unchanged in M. oeconomus, while Igf1 gene expression was unaffected by hypoxia in either species. Hypoxia increased Igfbp1 gene expression in the liver (Fig. 2) and stimulated IGFBP1 release from the liver into the circulation. In addition, it has been reported that hypoxia increases IGFBP1 levels by the induction of IGFBP1 hyperphosphorylation at the posttranslational level to inhibit IGFBP1 proteolysis in the plasma or extracellular matrix (Bunn and Fowlkes, 2003; Gibson et al., 2001; Seferovic et al., 2009), which might markedly increase the IGFBP1 level in mouse plasma (Fig. 3). In the present study, normobaric hypoxia failed to induce an IGF1 response, but a previous study showed that CoCl2 increases the response in M. oeconomus (Chen et al., 2007). This conflict is largely a result of the different hypoxia models and the means by which hypoxia is induced. Hypobaric or normobaric hypoxia depends on reducing the partial pressure of oxygen in air, while CoCl2 is an iron chelator. Hemoglobin binds to Co2+, losing its ability to bind O2, thus leading to a reduced O2 supply for tissues and cells. CoCl2-induced effects are more complex, not only inducing hypoxia but also having other effects. Similarly, hypobaric hypoxia (8.0% O2, 8 h) in the rat (Supplementary Fig. 5) and maternal hypoxia in the mouse (8.0% O2, 6 h) (Ream et al., 2008) and rat (13–14% O2, 7 d) (Tapanainen et al., 1994) all induce upregulation of the Igfbp1 gene, but do not affect Igf1 gene expression, suggesting that the Igfbp1 gene is much more sensitive to hypoxic stress. Transcriptional activity of the IGFBP1 gene can be regulated by cAMP and hypoxia (Sugawara et al., 2000). It is known that transcriptional activation of the Igfbp1 gene under hypoxia involves the binding of HIF1 to the HREs in regulatory regions. Functional HREs have been found in the promoter of the zebrafish (Kajimura et al., 2006) and rat (Scharf et al., 2005) Igfbp1 gene and in the intron 1 region of the human IGFBP1 gene (Tazuke et al., 1998). We have shown previously that HIF1a expression in the liver of M. oeconomus and mouse is upregulated after hypoxia, (Chen et al., 2007). But in HepG2 cells, we did not find that 1% O2 induced any significant change in the transcriptional activity of the Igfbp1 promoter and intron 1 regions. This reminds us to consider the location of HREs or sequence variation of HREs in the Igfbp1 gene of mouse and M. oeconomus that is involved in the blunted response to hypoxia, and further study is needed. Furthermore, in the human IGFBP1 promoter, there is also a cAMP response element that is involved in cAMP-stimulated IGFBP1 expression, and the transcriptional effects of cAMP and hypoxia on IGFBP1 mRNA expression are additive (Sugawara et al., 2000). Our previous study showed that corticotropin-releasing factor (CRF) is activated by hypoxia that triggers the cAMP signaling pathway (Chen and Du, 1996), and CRF receptor 1 (CRFR1) is involved in IGF1 expression in the liver (Chen et al., 2005). In another study, we showed that CRFR1 mediates p53 mRNA activation through cAMP and ERK1/2 signaling (Zhao et al., 2013). This led us to hypothesize, therefore, that this process might contribute to the transcriptional activation of hepatic Igfbp1 expression in mouse, and this is not, however, the case in M. oeconomus, possibly due to its high endurance of hypoxia. While in

HepG2 cells, 1% O2 does not induce transcriptional activation in the Igfbp1 gene of either animal, this might be because there is no CRF release and no cAMP signal activation in this in vitro system. Regarding the post-transcriptional regulation of Igfbp1 mRNA stability, it has been reported that hypoxia significantly increases the half-life of IGFBP1 mRNA in HepG2 cells (Sugawara et al., 2000). IGFBP1 mRNA in HepG2 cells is also stabilized by ethanol treatment for 16 h (Magne et al., 2007). AU-rich elements in the 30 -UTR of human IGFBP1 mRNA are involved in its mRNA decay in human cervical cancer cells (C33) (Gay and Babajko, 2000). In the present study, hypoxia (1% O2, 6 h) significantly stabilized Igfbp1 mRNA in mouse but not in M. oeconomus. In Hepa1-6 cells, endogenous igfbp1 mRNA decay was significantly attenuated by hypoxia, indicating that hypoxia can induce the stabilization of Igfbp1 mRNA in mouse liver. It has been reported that hypoxia activates HuR (ELAVL1), MDM2 (murine double minute 2) and ERBP (erythropoietin mRNA-binding protein) to regulate the mRNA stability of the hypoxia target genes VEGF and EPO (Levy et al., 1998; McGary et al., 1997; Zhou et al., 2011). Previous study showed that the 30 -UTR is important in the regulation of IGFBP1 mRNA stability (Gay and Babajko, 2000). In the present study, 30 -UTR deletion and mutation were used to investigate hypoxia-induced Igfbp1 mRNA stabilization, and showed that the 30 -UTR was essential for mouse Igfbp1 mRNA stabilization. AU-rich and U-rich elements were predicted in the 30 -UTR of Igfbp1 mRNA according to the consensus sequences (Dormoy-Raclet et al., 2007; Khabar, 2010), and one U-rich element (+4238/+4249) was demonstrated by mutation experiments to contribute to the hypoxia-induced mouse Igfbp1 stabilization, revealing a novel mechanism of mouse Igfbp1 gene regulation by hypoxia. In M. oeconomus Igfbp1, the U-rich element was evolutionarily eliminated; when it was reintroduced by site-directed mutations into the 30 -UTR, stabilization by hypoxia occurred again. 5. Conclusions IGF1 and IGFBP1 in the Qinghai-Tibetan plateau root vole M. oeconomus show a high degree of homology and conservation with other mammals. Hypoxic challenge upregulates the Igfbp1 gene in the lowland mouse, but not in M. oeconomus. The stability of mouse Igfbp1 mRNA with a functional U-rich element in its 30 UTR is enhanced by hypoxia, which contributes to the hypoxia-induced Igfbp1 upregulation in mouse liver. Sequence variations in the 30 UTR of M. oeconomus Igfbp1 mRNA eliminate the U-rich element, which leads to a blunting of Igfbp1 upregulation under hypoxia. This special Igfbp1 gene regulation in M. oeconomus may be a genetic adaptation strategy to the hypoxic environment of the plateau. Acknowledgements This work was supported by grants from the Ministry of Science and Technology of China, the National Basic Research Program (973) of China (2012CB518200) and the National Natural Science Foundation of China (31071047; 30870300; 30770807). We are grateful to Prof. Cunming Duan (University of Michigan, Ann Arbor, MI, USA) for kindly giving us the zebrafish igfbp1 promoter pGL2basic plasmid (p1225Luc) to provide a positive control in the dual-luciferase assay. We also thank Prof. IC Bruce (Department of Physiology, School of Medicine, Zhejiang University, China) for editing the manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mce.2013.05.025.

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

References Abbas, A., Grant, P.J., Kearney, M.T., 2008. Role of IGF-1 in glucose regulation and cardiovascular disease. Expert Rev. Cardiovasc. Ther. 6, 1135–1149. Annunziata, M., Granata, R., Ghigo, E., 2011. The IGF system. Acta Diabetol. 48, 1–9. Baxter, R.C., 2000. Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities. Am. J. Physiol. Endocrinol Metab. 278, E967-76. Bonefeld, K., Moller, S., 2011. Insulin-like growth factor-I and the liver. Liver Int. 31, 911–919. Brunhoff, C., Galbreath, K.E., Fedorov, V.B., Cook, J.A., Jaarola, M., 2003. Holarctic phylogeography of the root vole (Microtus oeconomus): implications for late Quaternary biogeography of high latitudes. Mol. Ecol. 12, 957–968. Bunn, R.C., Fowlkes, J.L., 2003. Insulin-like growth factor binding protein proteolysis. Trends Endocrinol. Metab. 14, 176–181. Cao, Y.B., Chen, X.Q., Wang, S., Wang, Y.X., Du, J.Z., 2008. Evolution and regulation of the downstream gene of hypoxia-inducible factor-1alpha in naked carp (Gymnocypris przewalskii) from Lake Qinghai, China. J. Mol. Evol. 67, 570–580. Cao, Y.B., Chen, X.Q., Wang, S., Chen, X.C., Wang, Y.X., Chang, J.P., Du, J.Z., 2009. Growth hormone and insulin-like growth factor of naked carp (Gymnocypris przewalskii) in Lake Qinghai: expression in different water environments. Gen. Comp. Endocrinol. 161, 400–406. Chen, Z., Du, J.Z., 1996. Hypoxia effects on hypothalamic corticotropin-releasing hormone and anterior pituitary cAMP. Zhongguo Yao Li Xue Bao. 17, 489–492. Chen, X.Q., Xu, N.Y., Du, J.Z., Wang, Y., Duan, C., 2005. Corticotropin-releasing factor receptor subtype 1 and somatostatin modulating hypoxia-caused downregulated mRNA of pituitary growth hormone and upregulated mRNA of hepatic insulin-like growth factor-I of rats. Mol. Cell. Endocrinol. 242, 50–58. Chen, X.Q., Wang, S.J., Du, J.Z., Chen, X.C., 2007. Diversities in hepatic HIF-1, IGF-I/ IGFBP-1, LDH/ICD, and their mRNA expressions induced by CoCl(2) in QinghaiTibetan plateau mammals and sea level mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R516-26. Daughaday, W.H., Parker, K.A., Borowsky, S., Trivedi, B., Kapadia, M., 1982. Measurement of somatomedin-related peptides in fetal, neonatal, and maternal rat serum by insulin-like growth factor (IGF) I radioimmunoassay, IGF-II radioreceptor assay (RRA), and multiplication-stimulating activity RRA after acid-ethanol extraction. Endocrinology 110, 575–581. Dormoy-Raclet, V., Menard, I., Clair, E., Kurban, G., Mazroui, R., Di Marco, S., von Roretz, C., Pause, A., Gallouzi, I.E., 2007. The RNA-binding protein HuR promotes cell migration and cell invasion by stabilizing the beta-actin mRNA in a U-richelement-dependent manner. Mol. Cell. Biol. 27, 5365–5380. Duan, C., Xu, Q., 2005. Roles of insulin-like growth factor (IGF) binding proteins in regulating IGF actions. Gen. Comp. Endocrinol. 142, 44–52. El-Desoky, A.E., Seifalian, A.M., Davidson, B.R., 1999. Effect of graded hypoxia on hepatic tissue oxygenation measured by near infrared spectroscopy. J. Hepatol. 31, 71–76. Eliasz, S., Liang, S., Chen, Y., De Marco, M.A., Machek, O., Skucha, S., Miele, L., Bocchetta, M., 2010. Notch-1 stimulates survival of lung adenocarcinoma cells during hypoxia by activating the IGF-1R pathway. Oncogene 29, 2488–2498. Ezzat, V.A., Duncan, E.R., Wheatcroft, S.B., Kearney, M.T., 2008. The role of IGF-I and its binding proteins in the development of type 2 diabetes and cardiovascular disease. Diabetes Obes. Metab. 10, 198–211. Fukuda, R., Hirota, K., Fan, F., Jung, Y.D., Ellis, L.M., Semenza, G.L., 2002. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J. Biol. Chem. 277, 38205–38211. Gay, E., Babajko, S., 2000. AUUUA sequences compromise human insulin-like growth factor binding protein-1 mRNA stability. Biochem. Biophys. Res. Commun. 267, 509–515. Gehmert, S., Sadat, S., Song, Y.H., Yan, Y., Alt, E., 2008. The anti-apoptotic effect of IGF-1 on tissue resident stem cells is mediated via PI3-kinase dependent secreted frizzled related protein 2 (Sfrp2) release. Biochem. Biophys. Res. Commun. 371, 752–755. Gibson, J.M., Aplin, J.D., White, A., Westwood, M., 2001. Regulation of IGF bioavailability in pregnancy. Mol. Hum. Reprod. 7, 79–87. Goldstein, S., Phillips, L.S., 1991. Extraction and nutritional/hormonal regulation of tissue insulin-like growth factor 1 activity. J. Biol. Chem. 266, 14725–14731. Harrison, T.M., Copeland, P., Kidd, W.S., Yin, A., 1992. Raising tibet. Science 255, 1663–1670. Hwa, V., Oh, Y., Rosenfeld, R.G., 1999. The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr. Rev. 20, 761–787. Jansson, M., Uhlen, M., Nilsson, B., 1997. Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor. Biochemistry 36, 4108–4117. Jansson, M., Andersson, G., Uhlen, M., Nilsson, B., Kordel, J., 1998. The insulin-like growth factor (IGF) binding protein 1 binding epitope on IGF-I probed by heteronuclear NMR spectroscopy and mutational analysis. J. Biol. Chem. 273, 24701–24707. Jones, J.I., Gockerman, A., Busby Jr., W.H., Wright, G., Clemmons, D.R., 1993. Insulinlike growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence. Proc. Natl. Acad. Sci. USA 90, 10553–10557. Jungermann, K., Kietzmann, T., 2000. Oxygen: modulator of metabolic zonation and disease of the liver. Hepatology 31, 255–260.

41

Kajimura, S., Aida, K., Duan, C., 2005. Insulin-like growth factor-binding protein-1 (IGFBP-1) mediates hypoxia-induced embryonic growth and developmental retardation. Proc. Natl. Acad. Sci. USA 102, 1240–1245. Kajimura, S., Aida, K., Duan, C., 2006. Understanding hypoxia-induced gene expression in early development: in vitro and in vivo analysis of hypoxiainducible factor 1-regulated zebra fish insulin-like growth factor binding protein 1 gene expression. Mol. Cell. Biol. 26, 1142–1155. Khabar, K.S., 2010. Post-transcriptional control during chronic inflammation and cancer: a focus on AU-rich elements. Cell. Mol. Life Sci. 67, 2937–2955. Lee, P.D., Giudice, L.C., Conover, C.A., Powell, D.R., 1997. Insulin-like growth factor binding protein-1: recent findings and new directions. Proc. Soc. Exp. Biol. Med. 216, 319–357. Lee, C., Raffaghello, L., Longo, V.D., 2012. Starvation, detoxification, and multidrug resistance in cancer therapy. Drug Resist. Update 15, 114–122. Levy, N.S., Chung, S., Furneaux, H., Levy, A.P., 1998. Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J. Biol. Chem. 273, 6417–6423. Liu, W., D’Ercole, J.A., Ye, P., 2011. Blunting type 1 insulin-like growth factor receptor expression exacerbates neuronal apoptosis following hypoxic/ ischemic injury. BMC Neurosci. 12, 64. Magne, L., Blanc, E., Marchand, A., Fafournoux, P., Barouki, R., Rouach, H., Garlatti, M., 2007. Stabilization of IGFBP-1 mRNA by ethanol in hepatoma cells involves the JNK pathway. J. Hepatol. 47, 691–698. McGary, E.C., Rondon, I.J., Beckman, B.S., 1997. Post-transcriptional regulation of erythropoietin mRNA stability by erythropoietin mRNA-binding protein. J. Biol. Chem. 272, 8628–8634. Mehrhof, F.B., Muller, F.U., Bergmann, M.W., Li, P., Wang, Y., Schmitz, W., Dietz, R., von Harsdorf, R., 2001. In cardiomyocyte hypoxia, insulin-like growth factor-Iinduced antiapoptotic signaling requires phosphatidylinositol-3-OH-kinasedependent and mitogen-activated protein kinase-dependent activation of the transcription factor cAMP response element-binding protein. Circulation 104, 2088–2094. Murphy, L.J., 2000. Overexpression of insulin-like growth factor binding protein-1 in transgenic mice. Pediatr. Nephrol. 14, 567–571. Ohlsson, C., Mohan, S., Sjogren, K., Tivesten, A., Isgaard, J., Isaksson, O., Jansson, J.O., Svensson, J., 2009. The role of liver-derived insulin-like growth factor-I. Endocr. Rev. 30, 494–535. Ohtsuki, T., Otsuki, M., Murakami, Y., Maekawa, T., Yamamoto, T., Akasaka, K., Takeuchi, S., Takahashi, S., 2005. Organ-specific and age-dependent expression of insulin-like growth factor-I (IGF-I) mRNA variants: IGF-IA and IB mRNAs in the mouse. Zool. Sci. 22, 1011–1021. Powell, D.R., Allander, S.V., Scheimann, A.O., Wasserman, R.M., Durham, S.K., Suwanichkul, A., 1995. Multiple proteins bind the insulin response element in the human IGFBP-1 promoter. Prog. Growth Factor Res. 6, 93–101. Qiu, Q., Zhang, G., Ma, T., Qian, W., Wang, J., Ye, Z., Cao, C., Hu, Q., Kim, J., Larkin, D.M., Auvil, L., Capitanu, B., Ma, J., Lewin, H.A., Qian, X., Lang, Y., Zhou, R., Wang, L., Wang, K., Xia, J., Liao, S., Pan, S., Lu, X., Hou, H., Wang, Y., Zang, X., Yin, Y., Ma, H., Zhang, J., Wang, Z., Zhang, Y., Zhang, D., Yonezawa, T., Hasegawa, M., Zhong, Y., Liu, W., Huang, Z., Zhang, S., Long, R., Yang, H., Lenstra, J.A., Cooper, D.N., Wu, Y., Shi, P., Liu, J., 2012. The yak genome and adaptation to life at high altitude. Nat. Genet. 44, 946–949. Rajpathak, S.N., Gunter, M.J., Wylie-Rosett, J., Ho, G.Y., Kaplan, R.C., Muzumdar, R., Rohan, T.E., Strickler, H.D., 2009. The role of insulin-like growth factor-I and its binding proteins in glucose homeostasis and type 2 diabetes. Diabetes Metab. Res. Rev. 25, 3–12. Ream, M., Ray, A.M., Chandra, R., Chikaraishi, D.M., 2008. Early fetal hypoxia leads to growth restriction and myocardial thinning. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R583-95. Sala, A., Capaldi, S., Campagnoli, M., Faggion, B., Labo, S., Perduca, M., Romano, A., Carrizo, M.E., Valli, M., Visai, L., Minchiotti, L., Galliano, M., Monaco, H.L., 2005. Structure and properties of the C-terminal domain of insulin-like growth factorbinding protein-1 isolated from human amniotic fluid. J. Biol. Chem. 280, 29812–29819. Scharf, J.G., Unterman, T.G., Kietzmann, T., 2005. Oxygen-dependent modulation of insulin-like growth factor binding protein biosynthesis in primary cultures of rat hepatocytes. Endocrinology 146, 5433–5443. Seferovic, M.D., Ali, R., Kamei, H., Liu, S., Khosravi, J.M., Nazarian, S., Han, V.K., Duan, C., Gupta, M.B., 2009. Hypoxia and leucine deprivation induce human insulinlike growth factor binding protein-1 hyperphosphorylation and increase its biological activity. Endocrinology 150, 220–231. Seifalian, A.M., El-Desoky, H., Delpy, D.T., Davidson, B.R., 2001. Effect of graded hypoxia on the rat hepatic tissue oxygenation and energy metabolism monitored by near-infrared and 31P nuclear magnetic resonance spectroscopy. FASEB J. 15, 2642–2648. Smith, T.J., 2010. Insulin-like growth factor-I regulation of immune function: a potential therapeutic target in autoimmune diseases? Pharmacol. Rev. 62, 199– 236. Sugawara, J., Tazuke, S.I., Suen, L.F., Powell, D.R., Kaper, F., Giaccia, A.J., Giudice, L.C., 2000. Regulation of insulin-like growth factor-binding protein 1 by hypoxia and 30 ,50 -cyclic adenosine monophosphate is additive in HepG2 cells. J. Clin. Endocrinol. Metab. 85, 3821–3827. Sun, C.F., Tao, Y., Jiang, X.Y., Zou, S.M., 2011. IGF binding protein 1 is correlated with hypoxia-induced growth reduce and developmental defects in grass carp (Ctenopharyngodon idellus) embryos. Gen. Comp. Endocrinol. 172, 409–415. Sutton, K.M., Hayat, S., Chau, N.M., Cook, S., Pouyssegur, J., Ahmed, A., Perusinghe, N., Le Floch, R., Yang, J., Ashcroft, M., 2007. Selective inhibition of MEK1/2

42

S. Zhang et al. / Molecular and Cellular Endocrinology 376 (2013) 33–42

reveals a differential requirement for ERK1/2 signalling in the regulation of HIF1 in response to hypoxia and IGF-1. Oncogene 26, 3920–3929. Tapanainen, P.J., Bang, P., Wilson, K., Unterman, T.G., Vreman, H.J., Rosenfeld, R.G., 1994. Maternal hypoxia as a model for intrauterine growth retardation: effects on insulin-like growth factors and their binding proteins. Pediatr. Res. 36, 152– 158. Tazuke, S.I., Mazure, N.M., Sugawara, J., Carland, G., Faessen, G.H., Suen, L.F., Irwin, J.C., Powell, D.R., Giaccia, A.J., Giudice, L.C., 1998. Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia. Proc. Natl. Acad. Sci. USA 95, 10188–10193. Wei, D.B., Wei, L., Zhang, J.M., Yu, H.Y., 2006. Blood-gas properties of plateau zokor (Myospalax baileyi). Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 145, 372– 375.

Wheatcroft, S.B., Kearney, M.T., 2009. IGF-dependent and IGF-independent actions of IGF-binding protein-1 and -2: implications for metabolic homeostasis. Trends Endocrinol. Metab. 20, 153–162. Yamada, N., Yanai, R., Nakamura, M., Inui, M., Nishida, T., 2004. Role of the C domain of IGFs in synergistic promotion, with a substance P-derived peptide, of rabbit corneal epithelial wound healing. Invest. Ophthalmol. Vis. Sci. 45, 1125–1131. Zhao, Y., Wang, M.Y., Hao, K., Chen, X.Q., Du, J.Z., 2013. CRHR1 mediates p53 transcription induced by high altitude hypoxia through ERK 1/2 signaling in rat hepatic cells. Peptides. Zhou, S., Gu, L., He, J., Zhang, H., Zhou, M., 2011. MDM2 regulates vascular endothelial growth factor mRNA stabilization in hypoxia. Mol. Cell. Biol. 31, 4928–4937.