ORIGINAL
RESEARCH
Esterase 1 is a Novel Transcriptional Repressor of Growth Hormone Receptor Gene Expression: A Unique Noncatalytic Role for a Carboxyesterase Protein Jinhong Sun,* P. Anil Kumar,* Jamuna Thimmarayappa, Natinder Saini, Pooja Goel, Travis Maures, Chunxia Lu, and Ram K. Menon Pediatrics and Communicable Diseases (J.S., P.A.K., J.T., N.S., P.G., C.L., R.K.M.) and Molecular and Integrative Physiology (R.K.M.), University of Michigan, Ann Arbor, Michigan 48109-5718; and Stanford University (T.M.), Palo Alto, California 94305-4060
The pleiotropic actions of GH result from its engagement with the GH receptor (GHR). GHR expression is regulated by free fatty acids (FFA). A cDNA phage expression library was screened to identify a phage clone expressing esterase 1 (ES1) binding to the FFA-response element (FARE), L2-D1, in the murine GHR promoter. Ectopically expressed ES1 inhibited GHR promoter activity via effects at two FARE, L2-D1 and L2-A2. Chromatin immunoprecipitation experiments demonstrated specific association of ES1 with the FARE. Catalytically inactive ES1 retained inhibitory activity on the GHR promoter and excluded the possibility that the effect on the GHR promoter was an indirect effect secondary to ES1’s actions on the intracellular metabolism of FFA. Ectopically expressed ES1 inhibited the endogenous GHR mRNA and protein expression in 3T3-F442A preadipocytes. Subcellular fractionation and confocal microscopy established that ES1 localizes both to the cytoplasm and the nucleus. Experiments demonstrated chromosome region maintenance 1-dependent nuclear export and the presence of a functional nuclear export signal in ES1. The domain of ES1 responsible for the effect on the GHR promoter was localized to the C-terminal portion of the protein. The in vivo significance of ES1’s effect on GHR expression was suggested by decreased liver GHR mRNA expression in mice on a high-fat diet correlating with increased steadystate abundance of liver ES1 mRNA. Our results identify and characterize ES1 as a novel transcriptional regulator of GHR gene expression, thereby establishing a unique nonenzymatic role for a carboxyesterase and expanding the potential biological roles of this protein superfamily. (Molecular Endocrinology 25: 1351–1363, 2011)
ituitary GH is essential for postnatal linear growth and regulation of metabolism of fat, carbohydrates, and proteins (1). These somatogenic and metabolic actions of GH are mediated by GH’s engagement of a specific cell-surface protein, the GH receptor (GHR). In addition to the direct effect of GH on target cells, GH also exerts its biological activity via stimulation of IGF-I. The biological activity of GH is also modulated by GH binding protein (GHBP) (2). GHBP is generated by alternative splicing of the GHR pre-mRNA in rodents and by posttranslational proteolytic shedding of the extracellular do-
P
ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/me.2011-0097 Received February 28, 2011. Accepted May 12, 2011. First Published Online June 9, 2011
main of the GHR protein in other mammals. Dysregulation of the GH/GHR axis is implicated in the etiology of short stature (3); in the pathogenesis of abnormalities of glucose homeostasis, including hypoglycemia (4) and poor glycemic control in diabetes mellitus (1, 5); in mechanisms underlying the chronic microvascular complications of diabetes mellitus, such as nephropathy and reti* J.S. and P.A.K. contributed equally to this work. Abbreviations: ChIP, Chromatin immunoprecipitation; CoA, coenzyme A; CRM1, chromosome region maintenance 1; DAPI, 4⬘,6-diamidino-2-phenylindole; ECL, enhanced chemiluminescence; ES1, esterase 1; F, forward; FARE, FFA-response element; FFA, free fatty acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GHBP, GH binding protein; GHR, GH receptor; HFD, high-fat diet; mAadac, murine arylacetemide deacetylase; NASH, nonalcoholic steatohepatitis; ND, normal chow diet; NES, nuclear export signal; NLS, nuclear localization signal; PARP, poly(ADP-ribose) polymerase; R, reverse; RT-qPCR, real-time quantitative RT-PCR; SDS, sodium dodecyl sulfate; 5⬘UTR, 5⬘-untranslated region; ZBP-89, 89-kDa zinc finger binding protein.
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nopathy (6, 7); and in morbidity associated with trauma, sepsis, and surgery (8). Because engagement of the GHR is essential for GH’s actions, control of GHR/GHBP gene expression is a nodal point in the regulation of the GHR/GH axis. A feature common to GHR transcripts from different species is the heterogeneity in the 5⬘-untranslated region (5⬘UTR) with disparate first exons being spliced to exon 2, resulting in mRNA with alternative 5⬘UTR controlled by distinct promoters (9). In the mouse, three 5⬘UTR (termed L1, L2, and L5) have been identified (10 –15). L1 is exclusively expressed in the liver and only during pregnancy (13, 14). In postnatal life, L2 is the widely expressed and dominant transcript, constituting 50 – 80% of the hepatic GHR transcripts in the nonpregnant adult animal (16). In a prior study, we reported that free fatty acids (FFA) directly inhibit GHR expression, and we identified and characterized FFA-response elements (FARE) in the L2GHR promoter. Our studies led to the discovery that the Kruppel-type zinc finger transcription factor [89-kDa zinc finger binding protein (ZBP-89)] was one of the cognate transcription factors that interacts with FARE to mediate FFA-dependent regulation of GHR expression (17). In the course of that investigation, we obtained evidence to suggest that additional factors interact with the FARE to mediate the effects of FFA on GHR gene expression. In the current report, we detail studies that establish a novel role for esterase 1 (ES1), a member of the carboxylesterase family of proteins (18), as a repressor of GHR gene transcription resulting from interaction with FARE of the L2-GHR promoter.
Results Mouse liver cDNA phage expression screening identifies ES1 protein binding to the FARE in the L2-GHR promoter We screened a mouse liver phage expression library with a DNA probe (FARE1) (Fig. 1A) from the L2-D1 FARE in the L2-GHR promoter (17), to identify a phage clone that bound to FARE1. Specificity of the DNA binding of the identified clone was confirmed by lack of binding of the identified phage clone to a mutated FARE1 sequence. DNA sequence analysis of the phage clone revealed that it coded for ES1 (Ces1c; Ces-N; Es1; BC028907; NM_007954; MGI 95420), a member of the carboxylesterase family of proteins. Although a recent report has proposed the use of the name Ces1c for this gene (19), in the current manuscript, we refer to this protein as ES1, the name most commonly used in published reports concerning this protein.
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ES1 decreases L2 promoter activity To investigate the effect of ES1 on L2-GHR promoter activity, we transiently cotransfected BNL CL.2 (mouse liver) and Hep G2 (human hepatocellular liver carcinoma) cells with ES1 expressing plasmid and L2-GHR promoter-luciferase constructs and measured luciferase activity in the cell lysates 48 h after transfection. These results indicate that ES1 inhibits the activity of the full-length L2-GHR promoter (L2-GHR[⫺2.0 kb]) in both BNL CL.2 and Hep G2 cells (Fig. 1, B and C). To verify specificity of this effect, we tested the effect of ectopically expressed ES1 on the L1-GHR promoter and a deletion construct of L2-GHR (L2-GHR[⫺43 bp]) devoid of FARE1. These results (Fig. 1, B and C) indicate that ES1 did not significantly alter the activity of these two reporter constructs, thus supporting the specificity of the observed effect of ES1 on L2-GHR promoter activity. To further verify the specificity of ES1 effect on L2-GHR promoter activity, we also tested the effect of ES1 on the promoter of an unrelated gene, c-fos. These results (Fig. 1B) demonstrate that ES1 does not significantly alter the activity of the c-fos promoter, thereby indicating the specificity of ES1’s effect on the L2GHR promoter. The mammalian carboxyesterases constitute a multigene superfamily with more than 50 members (18). We reasoned that if the effect of ES1 on L2-GHR promoter activity was specific, then it should be restricted to ES1 or to a select group of carboxyesterases. Hence, to further establish the specificity the effect of ES1 on L2-GHR promoter activity, we tested the effect of an unrelated carboxyesterase, murine arylacetamide deacetylase (esterase) (mAadac), for which the cDNA was readily available. Ectopic expression of mAadac in BNL CL.2 cells established that, unlike ES1, mAadac did not significantly alter L2-GHR promoter activity (Fig. 1D). These results further support the specificity of ES1’s effects on L2-GHR promoter activity. FARE in the L2-GHR promoter are necessary for ES1 effects on L2-GHR promoter activity Having established the specificity of ES1’s effects on the L2-GHR promoter, we next sought to localize the cis-element(s) on the L2-GHR that transduce the effect of ES1 on the L2-GHR promoter. A previous report had identified two FARE, L2-D1 and L2-A2, in the L2-GHR promoter (17). The probe (FARE1) used in the cDNA phage screening protocol described above overlapped with L2-D1 (Fig. 2A). Hence, we first tested whether ES1’s effect on the L2-GHR promoter was transduced via FARE1 by transiently cotransfecting BNL CL.2 cells with the ES1 expressing plasmid and a L2-GHR promoterluciferase construct devoid of L2-D1 (pGL3B-L2[
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GHR promoter (Fig. 2B). In contrast, deletion of both L2-D1 and L2-A2 (pGL3B-L2[2kb⌬D1/A2]) resulted in abrogation of ES1’s effect of the L2GHR promoter. We conclude from these results that both L2-D1 and L2-A2 cis-elements transduce ES1’s effects on the L2-GHR promoter, and there is functional redundancy between these two sites with either one of these sites being able to compensate for the loss of the other site. Effect of catalytically inactive ES1 on L2-GHR promoter activity There are two principal mechanisms that could explain the observed inhibitory effect of ES1 on L2-GHR promoter activity. First, it is known that ES1, via its carboxyl esterase activity, catalyzes FIG. 1. Ectopically expressed ES1 inhibits L2-GHR promoter activity. A, Schematic representation of the location and nucleotide sequence of the L2–D1 FARE. The relative the hydrolysis of endogenous short- and locations (vis-a`-vis the transcription start site) of the L2–D1 (stippled box) FARE of the L2 long-chain acylglycerols and long-chain promoter is indicated. The nucleotide sequence of the probe (FARE1) used to screen the cDNA acyl-coenzyme A (CoA) esters (18). phage expression library is underlined. B and C, BNL CL.2 (B) and Hep G2 (C) cells were Thus, these products of ES1’s catalytic cotransfected with either ES1 expression vector or empty vector (pcDNA), and luciferase reporter plasmid for L2-GHR promoter [either the full-length 2.0 kb or deletion construct (⫺43 activity could directly inhibit L2-GHR bp)], L1-GHR promoter, or c-fos promoter, and an internal control, pRL-TK, expressing the promoter activity. Alternatively, ES1 Renilla luciferase, which was used to normalize for transfection efficiency. The normalized could repress GHR promoter activity by luciferase activity of the cells transfected with ES1 expression vector (open bars) is depicted relative to activity of cells transfected with the empty expression vector (pcDNA 3.1; solid bars) either binding directly to the cognate designated as 1. Error bars indicate mean ⫾ SEM (n ⫽ 5–7); *, P ⬍ 0.05 compared with cells DNA element or by serving as a cofactor transfected with empty vector. D, BNL CL.2 cells were cotransfected with empty expression for a cognate DNA-binding protein(s) vector (pcDNA 3.1), ES1 expression plasmid (0.05– 0.2 g), or mAadac expression plasmid, and luciferase reporter plasmid for full-length 2.0-kb L2-GHR promoter, and an internal that interacts with the FARE. To invescontrol, pRL-TK, expressing the Renilla luciferase, which was used to normalize for tigate these two possible mechanisms, transfection efficiency. The normalized luciferase activity of the cells transfected with ES1 (gray ES1 [S221A/H445A] protein construct bar) or mAadac (open bar) expression vector is depicted relative to activity of cells transfected was engineered with mutations in the with the empty expression vector (pcDNA 3.1; solid bars) designated as 1. Error bars indicate mean ⫾ SEM (n ⫽ 4 –5); *, P ⬍ 0.05 compared with cells transfected with empty expression two canonical catalytic sites in ES1 vector. Inset, Western blot analysis verifying expression of mAadac. BNL CL.2 cells transfected predicted by an in silico analysis of ES1 with expression plasmid for c myc-tagged ES1 or mAadac were harvested, whole-cell lysates protein structure (Fig. 3A). An in vitro size fractionated by electrophoresis and transferred onto nitrocellulose membrane by Western blotting, and the membrane probed sequentially with anti-c myc and antiactin antibodies. enzymatic assay (20) confirmed that Specific signals were visualized using the ECL system. The identities of the proteins and the the ES1 [S221A/H445A] mutant propositions of the molecular weight markers are indicated. tein was devoid of catalytic activity (data not shown). We next investi2kb⌬D1]) and measuring luciferase activity in the cell gated the effect of the mutant ES1 on the L2-GHR prolysates 48 h after transfection. These results indicated that moter activity by transiently cotransfecting the ES1 muL2-D1 (and thus FARE1) was not essential for ES1’s actant construct and the L2-GHR promoter-reporter tion on the L2-GHR promoter (Fig. 2B). construct into CHO cells and measuring the luciferase In addition to L2-D1, the L2-GHR promoter has a second FARE, L2-A2 (Fig. 2A) (17). To test the possibility activity. These results reveal that the loss of the catalytic that ES1’s actions on the L2-GHR promoter is transduced activity of ES1 did not alleviate the inhibitory effect on via L2-A2, BNL CL.2 cells were transiently cotransfected L2-GHR promoter activity (Fig. 3B), suggesting that the with ES1 expressing plasmid and L2-GHR promoter- inhibitory effect of ES1 on L2-GHR promoter activity is luciferase construct devoid of L2-A2 (pGL3B-L2[2kb⌬A2]). neither mediated by its carboxyl esterase activity nor Similar to the L2-D1 site, these results also indicate that the through catalyzed products, such as acylglycerols and L2-A2 site was not essential for ES1’s actions on the L2- acyl-CoA esters.
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FIG. 2. Functional analysis of cis-elements that transduce ES1’s effects on the L2-GHR promoter. A, Schematic representation of the location and nucleotide sequence of the L2–D1 and L2–A2 FARE. The relative locations (vis-a`-vis the transcription start site) of the L2–D1 (stippled box) and L2–A2 (solid box) FARE of the L2 promoter are indicated. The nucleotide sequence of the probe (FARE1) used to screen the cDNA phage expression library is underlined. B, Combined deletion of L2–D1 and L2–A2 abolishes ES1’s effect on the L2 promoter. Luciferase expression plasmids containing either the full-length promoter (L2[2kb]), or internal deletion of L2–D1 (L2[2kb⌬D1]), L2–A2 (L2[2kb⌬A2), or L2–D1 and L2–A2 (L2[2kb⌬D1/A2]) were transiently transfected in BNL CL.2 (mouse liver) cells either with ES1 expression plasmid or empty vector. Cotransfection of Renilla luciferase was used to normalize transfection efficiency. The normalized luciferase activity of the cells transfected with ES1 expression plasmid (open bars) is depicted relative to activity of cells transfected with the empty expression vector (pcDNA 3.1; solid bars) designated as 1. Results represent mean ⫾ SEM; n ⫽ 4 –5; *, P ⬍ 0.05 compared with cells transfected with empty expression vector.
Effect of ES1 and catalytically inactive ES1 on L2-GHR mRNA and protein expression To further confirm whether wild-type and mutant forms of ES1 exert effects on endogenous L2-GHR expression, we transiently transfected c-myc-tagged ES1 and ES1 [S221A/H445A] constructs into 3T3-F442A preadipocytes. Total RNA and whole-cell protein lysates isolated from these cells were analyzed for L2-GHR mRNA levels and GHR protein expression by real-time quantitative RT-PCR (RT-qPCR) and Western blotting, respectively. These results demonstrate that both the wild-type ES1 and the catalytically inactive ES1 [S221A/ H445A] inhibited expression of the endogenous GHR mRNA (Fig. 3C) and protein (Fig. 3, D and E) in 3T3F442A preadipocytes. ES1 localizes both to the cytoplasm and the nucleus In silico analysis of protein domains of ES1 predicts ES1 to be a secreted protein, devoid of a canonical nuclear localization signal (NLS) sequence. However, in view of our results indicating direct effect of ES1 on GHR promoter activity, we posited that ES1 could enter the nuclear compartment via processes, such as importin-independent transport, that are not dependent on the presence of a canonical NLS sequence (21). We investigated this possibility by transiently transfecting CHO cells with c-
myc-tagged ES1 and examining for nuclear localization of ES1 using anti-cmyc antibody. Confocal scanning light microscopy revealed the presence of cmyc tag in the nucleus of transfected CHO cells (Fig. 4A), suggesting that a fraction of the cellular complement of ES1 is localized in the nucleus. The presence of ES1 in the nucleus was further confirmed by subcellular fractionation of CHO cells transiently transfected with c-myc-tagged ES1. These experiments revealed the presence of 72- and 64-kDa ES1 isoforms in whole-cell and cytoplamic extracts. In contrast, only the 64-kDa isoform could be detected in the nuclear fraction (Fig. 4B). The purity of the cytoplasmic and nuclear fractions was verified by probing for the presence of canonical cytoplasmic (tubulin) and nuclear poly(ADP-ribose) polymerase (PARP)proteins (Fig. 4B).
Localization of nuclear export signal (NES) in ES1 The chromosome region maintenance 1 (CRM1) export receptor-specific inhibitor leptomycin B (22) was used to probe for CRM1-dependent export of ES1 from the nucleus. These results revealed that leptomycin B treatment of CHO cells transfected with a green fluorescent protein (GFP)-epitope-tagged ES1 resulted in time-dependent accumulation of ES1 in the nucleus (Fig. 5A). Examination of the amino acid sequence of ES1 revealed three putative hydrophobic-rich nuclear sequences (Fig. 5B) that matched putative export signal (NES) sequence motif of LX2–3 LX2– 4 LXL (23– 25). The biological significance of these sequences was assessed by examining the effect of mutating these sequences on nuclear-cytoplasmic partitioning of ES1 (Fig. 5C). In contrast to mutations of NES1 and NES2 that did not alter the nuclear-cytoplasmic partitioning of ES1, mutation of NES3 rendered ES1 defective in nuclear export, resulting in nuclear accumulation of ES1 (Fig. 5D). These results support CRM1-dependent export of ES1 from the nucleus and the presence of a functional NES in the ES1 sequence. Effect of truncated ES1 on L2-GHR promoter activity The results (Fig. 4B) presented above established the preferential presence in the nucleus of the 64-kDa isoform (vs. the 72-kDa isoform) of ectopically expressed ES1.
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FIG. 3. Ectopically expressed catalytically inactive ES1 inhibits L2-GHR promoter activity and endogenous GHR expression. A, Location of mutations in the catalytic domains of ES1. Using strategies outlined in Materials and Methods, mutations were introduced in the indicated amino acids in the ES1 catalytic sites to engineer catalytically inactive ES1 [S221A/H455A]. B, The effect of catalytically inactive ES1 [S221A/H455A] mutant on the L2-GHR promoter activity. BNL CL.2 cells were cotransfected with empty expression vector (pcDNA 3.1), ES1 expression plasmid, or catalytically inactive ES1 [S221A/H455A] expression plasmid, and luciferase reporter plasmid for full-length 2.0-kb L2-GHR promoter, and an internal control, pRL-TK, expressing the Renilla luciferase, which was used to normalize for transfection efficiency. The normalized luciferase activity of the cells transfected with ES1 (gray bar) or ES1 [S221A/H455A] (open bar) expression vector is depicted relative to activity of cells transfected with the empty expression vector (solid bar) designated as 1. Error bars indicate mean ⫾ SEM (n ⫽ 5– 6); *, P ⬍ 0.05 compared with cells transfected with empty expression vector. C, Ectopically expressed ES1 inhibits endogenous L2-GHR mRNA expression. 3T3-F442A preadipocytes were transiently transfected with either ES1 expression plasmid (gray bar), catalytically inactive ES1 [S221A/H455A] expression plasmid (open bar), or empty pcDNA expression plasmid (solid bar), and 48 h after transfection, the cells were harvested for isolation of total RNA. The steady-state abundance of the L2-GHR mRNA was measured by real-time RT-PCR using methods previously described; mRNA expression was normalized to GAPDH and depicted relative to abundance in cells transfected with the empty expression vector (solid bar) designated as 1. D, Ectopically expressed ES1 inhibits endogenous GHR expression. 3T3-F442A preadipocytes were transiently transfected with either ES1, catalytically inactive expression plasmid ES1 [S221A/H455A], or empty pcDNA expression vector. Fortyeight hours after transfection, the cells were harvested, and whole-cell lysates were size fractionated by electrophoresis, transferred onto nitrocellulose membrane by Western blotting, and the membrane probed sequentially with anti-c myc, anti-GHR, and antiactin antibodies. Specific signals were visualized using the ECL system. The identity and molecular weights of the proteins are indicated. E, Densitometric measurements (mean ⫾ SEM; n ⫽ 4) of the Western blot analysis of cells transfected with either ES1 (gray bar) or catalytically inactive expression plasmid ES1 [S221A/H455A] (open bar) are depicted. *, P ⬍ 0.05 compared with empty expression vector control (solid bar) depicted as 1. WT, Wild type.
The predicted molecular mass of ES1 is approximately 72 kDa, suggesting that the 64-kDa isoform results from posttranslational modification(s) of ES1 and that this modification plays a role in determining the intracellular localization of ES1. To ascertain the nature of the potential posttranslational modification(s) yielding the 64-kDa isoform, we first tested the possibility of N-linked glycosylation. However, experiments testing susceptibility to N-glycosidase failed to support this possibility (data not shown). We next chose to investigate alternate possibili-
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ties, such as use of an alternate translational start site or cleavage of the ES1 protein at an appropriate cleavage site, that result in an isoform with the same size as the 64-kDa isoform detected in the nucleus. We therefore engineered two truncated c-myc-tagged chimeric species of ES1, a 60- to 65-kDa (ES1 [103–554]) fragment and the other, (ES1 [1–102]) fragment representing the remaining 8 –10 kDa (Fig. 6A). We studied the effect of these truncated forms of ES1 on L2-GHR promoter activity by cotransfecting the constructs expressing the truncated ES1 proteins with the L2-GHR luciferase reporter construct and then assaying for luciferase activity. These results demonstrate that only ES1 [103–554], and not ES1 [1–102], retained the inhibitory effect of the full-length ES1 on L2-GHR promoter activity (Fig. 6B). In agreement with these functional results, transiently transfecting CHO cells with c-myc-tagged truncated ES1 constructs revealed that, in contrast to absence of ES1 [1–102] in the nucleus, ES1[103–554] was present in the nucleus (Fig. 6C). Thus, these results support the model wherein ES1[103–554] enters the nucleus and represses L2GHR promoter activity.
The 64-kDa isoform of ES1 binds to the FARE on the L2-GHR promoter Having demonstrated that the 64kDa truncated form of ES1 represses L2-GHR promoter activity, we next investigated binding of ES1 to FARE, L2-D1, and L2-A2. For these experiments, we transiently transfected BNL CL.2 cells with c-myc-tagged ES1, ES1[1–102], and ES1[103–554] constructs and used chromatin immunoprecipitation (ChIP) assay to investigate the direct binding of ES1 to L2-D1 and L2-A2. These results reveal that the full-length ES1 and ES1[103–554] were associated with the chromatin at the L2-D1 and L2-A2 sites (Fig. 7, A and B). The specificity of these associations was verified by demonstrating lack of binding to an adjacent region of the L2-GHR promoter (Fig. 7C). In contrast, we were unable to demonstrate associ-
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(Fig. 8C) was decreased in the HFD cohort. Our results support a model wherein HFD inhibits GHR gene expression, in part, by increasing ES1 expression.
Discussion The cardinal finding of the current study is the identification of a novel biological role for a member of the mammalian carboxyesterase family of proteins, ES1, as a transcriptional repressor of GHR promoter activity. Our results indicate that this action of ES1, transduced via two noncontiguous FARE in the promoter of the dominant L2 transcript of the murine GHR gene, is independent of ES1’s catalytic activity. The noncanonical importinFIG. 4. Intracellular localization of ES1. A, Intracellular localization analyzed by confocal microscopy. CHO cells were transiently transfected with expression vector independent transport of ES1 into the nucleus for c-myc-tagged ES1 and incubated for 24 h before fixation with 4% is regulated via a nucleus export signal in ES1. paraformaldehyde and permeabilization with 0.1% Triton X-100; the nuclei were Our studies also demonstrate an association visualized by DAPI staining. Images were captured using Olympus FluoView 500 laser scanning confocal microscope using FluoView imaging software. B, Intracellular between HFD-induced decrease in liver GHR localization analyzed via cell fractionation. CHO cells transiently transfected with expression and concomitant increase in liver expression vector for c-myc-tagged ES1 were subjected to fractionation into ES1 expression, supporting a biological role cytoplasmic and nuclear fractions as detailed in Materials and Methods. Aliquots of for ES1’s effects on GHR expression. whole-cell lysate (WCL), cytoplasmic (CF), and nuclear fractions (NF) were size fractionated via electrophoresis, transferred onto nitrocellulose membrane by Although the effects of FFA on the GH/ Western blotting, and the membrane probed sequentially with anti-c myc, antiGHR axis are well known (17, 30 –32), our PARP, and antitubulin antibodies. Specific signals were visualized using the ECL understanding of the molecular basis for these system. The identity and molecular weights of the proteins are indicated. Results are representative of three experiments. effects is incomplete. Our previous studies had identified two noncontiguous FARE in the L2 ation of ES1[1–102] with either L2-D1 (Fig. 7A) or L2-A2 GHR promoter (17). ChIP studies demonstrate that ES1 (data not shown). Thus, these results support the model associates with the chromatin in the vicinity of these wherein ES1[103–554] enters the nucleus and associates FARE. However, we were unable to demonstrate ES1 with FARE, L2-D1 and L2-A2, to represses L2-GHR pro- binding to these FARE either via Southwestern blotting or moter activity. EMSA (data not shown). One explanation for this lack of binding is that the conditions of the Southwestern blotHigh-fat diet (HFD) increases ES1 expression with ting/EMSA are not conducive to the association between concomitant decrease in L2-GHR expression ES1 and these FARE. Another explanation is that ES1 It has been reported that ES1 activity in plasma in- does not directly bind to FARE but rather functions as a creases with HFD (26 –29). To discern the biological sig- corepressor via association with a factor(s) that binds to nificance(s) of the observed effect of ES1 on L2-GHR, we these cis-elements. In silico analysis of protein domains of investigated the effect of HFD on ES1 and L2-GHR ex- ES1 did not yield clues as to the identity/type of proteins pression in mice. For these studies male C57BL/6 mice that could interact with ES1. Our previous studies had were fed HFD (45 kcal% fat) for 16 wk; controls were fed established physical and functional interactions between normal chow diet (ND). As expected, the HFD mice the DNA-binding protein ZBP-89 and FARE, L2-D1, and gained additional weight as compared with the ND-fed L2-A2 (17). Hence, we tested the possibility that ES1 cohort [24 ⫾ 0.7 vs. 42 ⫾ 3.3 g (mean ⫾ SEM; n ⫽ 6)]. At associates with ZBP-89. However, we were unable to the end of wk 16, the mice were euthanized and the livers demonstrate such an association via immunoprecipitawere harvested for RNA and protein extraction. RT- tion experiments of ectopically expressed proteins (data qPCR analysis revealed a significant increase in the not shown). These results still leave open the possibility steady-state abundance of ES1 mRNA in the HFD cohort that ES1 associates with these FARE as part of a larger compared with the ND cohort (Fig. 8A). In contrast, the complex and physically associates with, an as yet unidenexpression of L2-GHR mRNA (Fig. 8B) and GHR protein tified, member of this complex.
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FIG. 5. Localization of NES in ES1. A, Leptomycin B (LMB) inhibits nuclear export of ES1. CHO cells transiently transfected with expression vector for GFP-tagged ES1 were incubated without (⫺) and with (⫹) Leptomcycin B (20 nM) for 16 h, counter stained with DAPI, and immunofluorescence visualized by confocal microscopy. B, Location of putative NES sequences (boxed) in ES1. C, Mutations introduced in putative NES sequences in ES1. D, CHO cells transiently transfected with expression vector for GFP-tagged ES1 constructs without or with mutations in the three putative NES sequences in ES1 and immunofluorescence visualized by confocal microscopy. AA, Amino acid.
Mammalian carboxyesterases are ␣,-hydrolase fold proteins that constitute a multigene superfamily with ubiquitous expression (18). Five mammalian carboxyesterase gene families have been identified (19). Carboxyesterase genes are ancient in their genetic origins and were established before the appearance of mammals during evolution (19). Carboxylesterases catalyze the hydrolysis of endogenous short- and long-chain acyl glycerols, longchain acylcarnitine, and long-chain acyl-CoA esters. The canonical role of these enzymes is in the hydrolytic biotransformation of a diverse cadre of drugs or prodrugs containing ester- and amide-bonds, to the respective free acids and alcohols (18). Our results from functional (i.e. repression of GHR gene expression), structural (i.e. intracellular localization via cell fractionation and confocal microscopy), and mechanistic (i.e. CRM1-dependent nuclear export) studies indicate the presence of ES1 in the nucleus. This finding is contrary to the dogma that proteins of the carboxylesterases family are cytoplasmic proteins, with some members of the family being secreted into the plasma, whereas others remain associated with cell membranes, such as the endoplasmic reticulum (18). It is noteworthy that recent studies have reported on other enzyme proteins, such as acyl citrase lysase, that localize
to both the cytoplasm and the nucleus (33). The precise mechanism(s) that enable the transport of ES1 into the nucleus is unclear. The primary sequence of ES1 does not predict a canonical NLS required for importin/ karyopherin-dependent transport to the nucleus (34). However, recent studies have established that a number of proteins use nonconventional, and in some instances, importin-independent pathways to accumulate in the nucleus (21). Whether such mechanisms are involved in the nuclear-cytoplasmic partitioning of ES1 remains to be determined. One caveat to our findings is that, because of the nonavailability of a specific anti-ES1 antibody, our results are based on ectopically expressed proteins. Our results support a novel role for ES1 as a transcriptional repressor with the transcriptionally active domain of ES1 being located toward the C-terminal portion of the protein. Our studies in CHO cells reveal two distinct isoforms of ES1 distinguishable by molecular weights of approximately 64 and 72 kDa, putatively derived from proteolytic cleavage of the full-length ES1. However, the identity of the cleaved fragment, the protease(s) involved, and the stimulus for such a cleavage remain to be identified. There are many examples, exemplified by sterol regulatory element-binding proteins (35), wherein a cyto-
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activity in circulation is increased by HFD (26 –29), and our studies demonstrate that ES1’s expression is increased in the liver of mice on a HFD. GHR expression is inhibited by FFA (17). Our results demonstrate increased ES1 expression in liver that correlates with decrease in hepatic GHR mRNA in mice on a HFD. These results raise the possibility that one of the mechanisms responsible for FFA’s effect on GHR could be ES1’s effect on GHR promoter activity. There are two direct clinical implications of the nexus between ES1 and GHR expression revealed in the current study. The GH/ GHR axis plays a critical role in statural growth and regulation of metabolism. Abnormalities in the GH/ GHR axis are implicated in the short stature associated with small for gestation and very low birth weight babies (38 – 40), both conditions that are also FIG. 6. Localization of transcriptional activity to C-terminal portion of ES1. A, Cartoon associated with dysregulation of lipid depicting the organization of full-length ES1 [FL], ES1 [1–102], and ES1 [103–554] constructs. homeostasis (41, 42). The regulation B, BNL CL.2 cells were cotransfected with either expression vector for indicated ES1 protein or of ES1 in these conditions is not empty expression vector (pcDNA 3.1), the full-length 2.0-kb GHR promoter luciferase reporter plasmid, and an internal control, pRL-TK, expressing the Renilla luciferase, which was used to known, but our results support the hynormalize for transfection efficiency. The normalized luciferase activity of the cells transfected pothesis that changes in ES1 expreswith ES1 expression vector (gray bars) is depicted relative to activity of cells transfected with sion could play a role in the GHR regthe empty expression vector (solid bar) designated as 1. Error bars indicate mean ⫾ SEM (n ⫽ 5); *, P ⬍ 0.05 compared with cells transfected with empty vector. C, Intracellular localization ulation in these states. A second of truncated ES1 proteins. CHO cells were transiently transfected with expression vector for clinical situation where the results of c-myc-tagged ES1 [1–102] or ES1 [103–554] and incubated for 24 h before fixation with 4% the current study could have mechanisparaformaldehyde and permeabilization with 0.1% Triton X-100; the nuclei were visualized by tic implications is in the syndrome of DAPI staining. Images were captured using Olympus FluoView 500 laser scanning confocal microscope using FluoView imaging software. FL, Full length; N-SP, N terminal signal peptide; nonalcoholic steatohepatitis (NASH). aa, amino acid. Recent studies have highlighted the importance of GH action on hepatic plasmic protein is cleaved to release a transcriptionally lipid metabolism (43, 44). Thus humans with GH defiactive fragment. Whether the posttranslational processciency exhibit fatty infiltration of the liver (45), and patients ing of ES1 parallels the sterol regulatory element-binding with NASH have low plasma GH levels (46). Furthermore, protein paradigm remains to be elucidated. An alternate cessation of GH therapy in children with GH deficiency is possibility is that the smaller isoform results from the associated with the increased incidence of NASH (47). In use of an in-frame downstream AUG codon located at position 86 or 100 with the AUG codon at position 100 this context, it is particularly noteworthy that a recent study being a stronger candidate, because it is in an optimal reported that hepatic ES 1 activity is increased in patients context (purine in position ⫺3) for translational initi- with NASH (48). Hence, we speculate that increased ES 1 ation (36). It is noteworthy that the size of either of activity is one of the mechanisms that results in reduced these two putative N-truncated isoforms would be GHR expression and consequent decrease in GH action in compatible with the 64-kDa size of the ES1 isoform the liver in NASH. In summary, the current report establishes a novel role detected in the nucleus in our experiments. The precise physiological role(s) of the novel transcrip- for ES1 as a transcriptional repressor of GHR promoter tional activity of ES1 described in the current report is activity. Our studies demonstrate that despite the absence unclear. ES1 is one of the few carboxyesterases that are of a canonical NLS, a fraction of the cellular component secreted into circulation and present in plasma (37). ES1’s of ES1 is localized to the nucleus. We demonstrate that the
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FIG. 7. ES1 binds to FARE in the L2-GHR promoter. ChIP assays with chromatin fractions from BNL CL.2 cells ectopically expressing c-myc-tagged ES1 were performed using anti-c myc (4 g), or anti-RNA polymerase (Pol) II (4 g) antibody; normal mouse IgG served as control. Input DNA and DNA from each of the immunoprecipitated fractions were PCR amplified for FARE, L2–D1 (A) and L2–A2 (B) or unrelated sequence approximately 2000-bp 5⬘ of FARE (C) as described in Materials and Methods; 1% of the input is shown. PCR products (20 l aliquots from each reaction) were resolved on a 2% agarose gel and viewed by staining with ethidium bromide. TSS, Transcription start site.
DMEM, penicillin/streptomycin/amphotericin B mixture, and Trizol reagent were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum, heat-inactivated fetal bovine serum, and calf serum were purchased from Atlanta Biologicals (Lawrenceville, GA). Esterase enzymatic assay kit was bought from Sigma (St. Louis, MO). Anti-c-myc, antiactin, antitubulin, and anti-PARP antibodies were procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
FARE1 (upper strand) 5⬘-AGG GAA GTG GGG GGG AAG GGT ATG and FARE1 (lower strand) 5⬘-CAT ACC CTT CCC CCC CAC TTC CCT. The double-stranded DNA probes with mutations (lower case) in the putative FARE were: mutated FARE1 (upper strand) 5⬘-Att ctt GTG GGG ctt tcG GGT ATG and mutated FARE1 (lower strand) 5⬘-CAT ACC Cga aag CCC CAC aag aat. Real-time quantitative RT-PCR: L2-GHR forward (F), 5⬘-GTC CAC GCG GCC TGA G-3⬘; L2-GHR reverse (R), 5⬘-TCG CCT CGG GAG ACA GAA C-3⬘; L2-GHR probe, 5⬘-CAG CCC CCA AGC GGA CAC GA-3⬘; ES1 Exon 2/3 F, 5⬘-TTA CAG TCC TGC CGA TTT GAC A-3⬘; ES1 Exon 2/3 R, 5⬘- CCT ATC ACC AGT CCA CCT CCAT3⬘; and ES1 probe, AAA GCA GCC AAT TGC CTG TGA TGG TG. ChIP: L2-D1 F, 5⬘-GTA CCC CGG AGC TCT TGA CT-3⬘; L2-D1 R, 5⬘-GCC TTG GGA AGT GTG ATG AG-3⬘; L2-A2 F, 5⬘-CCA CCC CTC CCC TCT CTT-3⬘; L2-A2 R, 5⬘-CAG CTC GTG GGT TGT CAG-3⬘; L2 (unrelated) F, 5⬘-gca ttc ctt ttc tag ggt cca-3⬘; and L2 (unrelated) R, 5⬘-AG GGC AGC TGA ATA CAG AA-3⬘.
Cell culture
Expression plasmids and reporter gene constructs
Hep G2 cells, BNL CL.2, CHO, and 3T3-F442A cells were cultured at 37 C with either 5% (Hep G2 cells, BNL CL.2, and CHO) or 10% (3T3-F442A) CO2 in DMEM supplemented with 10% calf serum and penicillin/streptomycin/amphotericin B mixture. Primers: cDNA phage library screening:
ES1 expression plasmid was engineered by cloning the ES1 cDNA into the pcDNA3.1/myc-His (⫹) vector (Invitrogen). The catalytically inactive ES1 [S221A/S455A] was engineered by introducing point mutations into the ES1 sequence using QuikChange site directed mutagenesis kit (Stratagene, La Jolla, CA) and verified by sequencing to ensure directionality and nucleotide fidelity. The expression plasmids for truncated ES1 proteins (ES1[1–102],
effect of ES1 on the GHR promoter activity, transduced via two noncontiguous FAREs in the GHR promoter, is independent of ES1’s catalytic activity, and this effect of ES1 is localized to the C-terminal portion of ES1. Studies with a murine model of HFD feeding support a physiological role for ES1-dependent suppression of GHR activity in high-fat feeding. To the best of our knowledge, this is the first report to establish a nonenzymatic role for a member of the mammalian carboxyesterase family of proteins and thus expands the potential biological roles of this protein superfamily.
Materials and Methods Materials
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Assessing the activity of ES1 ES1 and ES [S221A/445A] were expressed in CHO cells and affinity purified via the His tag. The property of esterase to catalyze the conversion of p-nitrophenyl acetate to nitrophenol (20) was used to assay the enzymatic activity of the purified protein using reagents from Sigma-Aldrich (St. Louis, MO).
Transient transfection and luciferase assay
FIG. 8. HFD-induced decrease in hepatic GHR expression correlates with increase in hepatic ES1 expression. Eight-week-old male C57/BL6 mice were fed either ND (regular chow; solid bars) or HFD (45 kcal% fat) (open bars) for 16 wk, before harvesting of liver tissue for mRNA and protein extraction. A and B, Steady-state abundance of ES1 mRNA (A) and L2 transcript of GHR (B) was measured via RT-qPCR. mRNA abundance was normalized to GAPDH levels. The results (mean ⫾ SEM; n ⫽ 5) are depicted relative to mRNA abundance in ND cohort designated as 1. P ⬍ 0.05 compared with respective ND cohort. C, Western blot analysis for GHR protein in whole-cell lysates from liver of either C57/BL6 mice fed ND (lanes 2– 4) or HFD (lanes 5– 8). Equal amounts of protein were size fractionated by SDS-PAGE and Western blotted with anti-GHR AL47 antibody. Whole-cell lysate from liver of GHR⫺/⫺ (Laron) mice (lane 1) was analyzed as a negative control to demonstrate specificity of GHR antibody. The position of the specific GHR (⬃115 kDa) and nonspecific (NS) (⬃82 kDa) bands are indicated.
ES1[103–554]) and for ES1 proteins with mutations in the putative NES (mNES1; mNES1,2; and mNES1,2,3) were engineered using PCR-based strategies. The expression plasmid for mAadac (UniGene Mm.24547) was engineered by cloning cDNA clone MGC: 28468 (IMAGE:4162194; American Type Culture Collection, Manassas, VA) into the pcDNA3.1/myc-His (⫹) vector (Invitrogen). Luciferase reporter gene constructs engineered to contain various portions of the GHR 5⬘-flanking region, pGL3B-L2[⫺2.0], pGL3B-L2[⫺43], pGL3B-L2[2kb⌬D1], pGL3B-L2[2kb⌬A2], and pGL3B-L2[2kb⌬D1/A2] have been described previously (17). The reporter plasmid fos-Luc containing the mouse c-fos enhancer (379 to ⫹1) upstream of the luciferase gene was provided by J. Schwartz.
cDNA phage screening A mouse liver cDNA phage expression library (CLONTECH, Mountain View, CA) was screened using a previously reported protocol (49). Briefly, the phage library was plated on 0.7% agarose 100-mm plates followed by overlay of isopropyl -D1-thiogalactoside-saturated nitrocellulose filters. After incubation for 4 h at 37 C, the filters were lifted and probed with 32P labeled DNA probe. Positive clones were identified by autoradiography, and sequential rounds of plaque purification were carried out to obtain a single phage clone.
Hep G2 and BNL CL.2 cells (1 ⫻ 105 cells/well in 12-well plates) were cotransfected with ES1 constructs (0.05– 0.2 g/ well), L2-GHR luciferase reporter constructs (1 g/well), and internal control expressing the Renilla luciferase, pRL-TK (0.25– 0.1 g/well; Promega, Madison, WI). Hep G2 and BNL CL.2 cells were transfected using FuGENE (Roche Applied Sciences, Indianapolis, IN); 3T3-F442A preadipocytes were transfected using the Amaxa Nucleofector V (Lonza, Basel, Switzerland) protocol. Forty-eight hours after transfection, cells were washed twice with PBS and cells harvested with 100 l passive lysis buffer (Promega). After a brief freeze-thaw cycle, the insoluble debris was removed by centrifugation at 4 C for 2 min at 14,000 ⫻ g, and 20 l aliquots of the supernatant were processed for sequential quantitation of firefly and Renilla luciferase activity (Dual Luciferase Assay System; Promega) using a Monolight TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA). The activity of the cotransfected Renilla reporter plasmid was used to normalize for transfection efficiency. Transfections experiments were routinely performed in triplicates.
Real-time quantitative reverse-transcription PCR Total RNA was extracted using TRI-reagent (Invitrogen). RT-qPCR using the ABI Prism 7000 sequence detection system (PE Applied Biosystems, Foster City, CA) was performed in triplicate using either the QuantiTect SYBR Green RT-PCR kit (QIAGEN, Valencia, CA) or via the TaqMan protocol as described previously (12). Primers were designed using Primer Express 2.0 software. mRNA expression of each gene was normalized using the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene and data analyzed via the comparative threshold cycle method (50).
Subcellular fractionation Nuclear and cytoplasmic subcellular fractionation was carried out using the NE-PER kit (Pierce, Rockland, IL). Briefly, 48 h after transiently transfecting CHO cells (4 ⫻ 106) with the ES1 c-myc construct, cells were harvested and centrifuged at 500 ⫻ g for 2 min. The resulting cell pellet was resuspended in 400 l of cytoplasmic extraction reagent I and 22 l of cytoplasmic extraction reagent II and centrifuged at 14,000 ⫻ g for 5 min and the supernatant collected as the cytoplasmic fraction. The insoluble pellet was dissolved in 100 l of nuclear extraction reagent and represented the nuclear fraction. Fractionated cytoplasmic and nuclear aliquots were stored at ⫺80 C until Western blot analysis, wherein tubulin and PARP were used as markers for cytoplasmic and nuclear fractions, respectively.
Immunobloting Aliqouts of total cell lysate (routinely 15 g) were heated for 5 min at 100 C in 62.5 mM Tris HCl, 10% (vol/vol) glycerol, 5% (vol/vol) 2-mercaptoethanol, 1.05% sodium dodecyl sulfate
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(SDS), and 0.004% bromophenol blue. The protein samples were then electrophoresed through a 4% stacking, 8% resolving, discontinuous SDS-polyacrylamide gel in 25 mM Tris HCl, 192 mM glycine, and 0.1% SDS buffer. BenchMark prestained protein ladder (Invitrogen) was also concurrently electrophoresed. After electrophoresis, the proteins were transferred to nitrocellulose membrane by electroblotting (Bio-Rad Laboratories, Hercules, CA) in transfer buffer [10 mM N-cyclohexyl-3aminopropanesulfonic acid, 3 mM dithiothreitol, and 15% methanol (pH 10.5)] for 1.5 h. The nitrocellulose membrane were then soaked overnight at 4 C in 5% nonfat dry milk, 1⫻ TBS, and 0.1% Tween 20 and subsequently probed with the indicated antibody [anti-c myc (1:500), antiactin (1:1000), or anti-GHR AL-47 antibody (1:1000 dilution)] using the enhanced chemiluminescence (ECL) Western blotting substrate (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions.
Confocal microscopy CHO cells were transfected with ES1 c-myc plasmid construct using FuGENE (Roche), and cells were incubated for 24 h before they were fixed with 4% paraformaldehyde. The subcellular distribution of the various ES1 c-myc proteins was determined using anti-c myc antibody (Santa Cruz Biotechnology, Inc.). Cells were mounted with ProLong Gold antifade reagent with 4⬘,6-diamidino-2-phenylindole (DAPI) (Invitrogen) and visualized by confocal microscopy (Olympus FluoView 500 Laser Scanning Confocal Microscope; Olympus, Tokyo, Japan). Images were captured and viewed using FluoView software.
ChIP assay ChIP experiments were conducted on BNL CL.2 cells, either naive or transiently transfected with ES1 expression vectors. Cells were subjected to ChIP using the EZ-ChIP kit (Upstate Biotechnology, Lake Placid, NY) with minor modifications. Briefly, cultured BNL CL.2 cells were cross-linked by exposure to 1% formaldehyde in PBS for 10 min, followed by addition of 1 ml 10⫻ glycine to quench unreacted formaldehyde. The cells were then scraped in ice cold PBS containing a protease inhibitor cocktail, centrifuged, and the cell pellet resuspended in SDS lysis buffer. The cell lysate was subjected to shearing [7 ⫻ 15 sec, at 4.5 output of a sonicator (Heat Systems-Ultrasonics, Farmingdale, NY)] to generate approximately 500-bp DNA fragments. The samples were diluted 1:10 with ChIP dilution buffer [16.7 mM Tris-HCl (pH 8.0), 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, and protease inhibitor cocktail] and precleaned with 10 g of salmon sperm DNA and 60-l-packed protein G-agarose beads/ml ChIP dilution buffer. For immunoprecipitation, samples containing 100 g of nuclear protein were incubated overnight at 4 C with the antibodies individually: anti-c myc (Santa Cruz Biotechnology, Inc.), antiacetyl H4 (Upstate Biotechnology), or RNA Pol 2 (Upstate Biotechnology). Normal rabbit and mouse IgG served as negative controls. After incubation of individual immunoprecipitate for 1 h with 10 g of salmon sperm DNA and 60 l of protein G agarose beads, the beads were washed, eluted, reverse cross-linked, and free DNA purified with a PCR purification kit.
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Animals Animal care and experimental conditions were approved by the Institutional Animal Care and Use Committee at the University of Michigan Medical School. Eight- to 10-wk-old male C57BL/6J mice, housed in a temperature-controlled room with 12-h light, 12-h dark cycle, were divided into two cohorts (n ⫽ 6 in each cohort): regular chow fed and HFD [D12451 (24 g%; 45 kcal% fat); Research Diets, New Brunswick, NJ]. Food and water were provided ad libitum. After 16 wk of either regular chow or HFD feeding, mice were euthanized and tissues harvested for processing as indicated.
Data analysis Data are presented as mean ⫾ SEM unless otherwise indicated. The Mann-Whitney and Kruskal-Wallis nonparametric tests were performed to analyze statistical significance of the difference between the distribution of two and multiple independent samples, respectively. P values equal to or less than 0.05 were considered significant.
Acknowledgments We thank the generous provision of reagents by Dr. Stuart Frank (GHR antibody) and Dr. Jessica Schwartz (c-fos promoterluciferase construct). Address all correspondence and requests for reprints to: Ram K. Menon, M.D., University of Michigan Medical School, D1205 MPB/SPC 5718, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-5718. E-mail:
[email protected]. This work was supported National Institutes of Health Grants T32 DK071212 (to N.S.), DK49845 (to R.K.M.), and P60DK-20572 (to the Michigan Diabetes Research and Training Center) and by the Diabetes Research and Education Advocates of Michigan. Disclosure Summary: The authors have nothing to disclose.
References 1. Møller N, Jørgensen JO 2009 Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects. Endocr Rev 30:152–177 2. Baumann G 1995 Editorial: growth hormone binding protein— errant receptor or active player. Endocrinology 136:377–378 3. Richmond E, Rogol A 2010 Current indications for growth hormone therapy for children and adolescents. In: Hindmarsh P, ed. Current indications for growth hormone therapy. 2nd ed. Basel: Karger; 92–108 4. Binder G, Weidenkeller M, Blumenstock G, Langkamp M, Weber K, Franz AR 2010 Rational approach to the diagnosis of severe growth hormone deficiency in the newborn. J Clin Endocrinol Metab 95:2219 –2226 5. Almqvist EG, Groop LC, Manhem PJ 1999 Growth hormone response to the insulin tolerance test and clonidine tests in type 1 diabetes. Scand J Clin Lab Invest 59:375–582 6. Schrijvers BF, De Vriese AS, Flyvbjerg A 2004 From hyperglycemia to diabetic kidney disease: the role of metabolic, hemodynamic, intracellular factors and growth factors/cytokines. Endocr Rev 25: 971–1010 7. Flyvbjerg A 2004 The role of growth hormone in the pathogenesis
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 September 2014. at 11:31 For personal use only. No other uses without permission. . All rights reserved.
1362
8.
9. 10.
11.
12.
13.
14.
15.
16.
17.
18. 19.
20.
21. 22.
23.
24.
25.
26.
Sun et al.
Inhibition of GH Receptor Gene Expression by ES1
of diabetic kidney disease. Pediatr Endocrinol Rev 1 (Suppl 3):525– 529 Defalque D, Brandt N, Ketelslegers JM, Thissen JP 1999 GH insensitivity induced by endotoxin injection is associated with decreased liver GH receptors. Am J Physiol 276:E565–E572 Edens A, Talamantes F 1998 Alternative processing of growth hormone receptor transcripts. Endocr Rev 19:559 –582 Menon RK, Stephan DA, Singh M, Morris Jr SM, Zou L 1995 Cloning of the promoter-regulatory region of the murine growth hormone receptor gene. Identification of a developmentally regulated enhancer element. J Biol Chem 270:8851– 8859 Menon RK, Cheng H, Singh M 1997 Identification and characterization of single strand DNA-binding protein that represses growth hormone receptor gene expression. Mol Endocrinol 11:1291–1304 Menon RK, Shaufl A, Yu JH, Stephan DA, Friday RP 2001 Identification and characterization of a novel transcript of the murine growth hormone receptor gene exhibiting development- and tissuespecific expression. Mol Cell Endocrinol 172:135–146 Moffat JG, Edens A, Talamantes F 1999 Structure and expression of the mouse growth hormone receptor/growth hormone binding protein gene. J Mol Endocrinol 23:33– 44 Schwartzbauer G, Yu JH, Cheng H, Menon RK 1998 Transcription factor MSY-1 regulates expression of the murine growth hormone receptor gene. J Biol Chem 273:24760 –24769 Yu JH, Schwartzbauer G, Kazlman A, Menon RK 1999 Role of the Sp family of transcription factors in the ontogeny of growth hormone receptor gene expression. J Biol Chem 274:34327–34336 Southard JN, Barrett BA, Bikbulatova L, Ilkbahar Y, Wu K, Talamantes F 1995 Growth hormone (GH) receptor and GH-binding protein messenger ribonucleic acids with alternative 5⬘-untranslated regions are differentially expressed in mouse liver and placenta. Endocrinology 136:2913–2921 Thimmarayappa J, Sun J, Schultz LE, Dejkhamron P, Lu C, Giallongo A, Merchant JL, Menon RK 2006 Inhibition of growth hormone receptor gene expression by saturated fatty acids: role of Kruppel-like zinc finger factor, ZBP-89. Mol Endocrinol 20:2747– 2760 Satoh T, Hosokawa M 2006 Structure, function and regulation of carboxylesterases. Chem-Biol Interact 162:195–211 Holmes RS, Wright MW, Laulederkind SJ, Cox LA, Hosokawa M, Imai T, Ishibashi S, Lehner R, Miyazaki M, Perkins EJ, Potter PM, Redinbo MR, Robert J, Satoh T, Yamashita T, Yan B, Yokoi T, Zechner R, Maltais LJ 2010 Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins. Mamm Genome 21:427– 441 Erlanson C 1970 p-nitrophenylacetate as a substrate for a carboxylester hydrolase in pancreatic juice and intestinal content. Scand J Gastroenterol 5:333–336 Wagstaff KM, Jans DA 2009 Importins and beyond: non-conventional nuclear transport mechanisms. Traffic 10:1188 –1198 Kudo N, Wolff B, Sekimoto T, Schreiner EP, Yoneda Y, Yanagida M, Horinouchi S, Yoshida M 1998 Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp Cell Res 242:540 –547 McBride KM, McDonald C, Reich NC 2000 Nuclear export signal located within the DNA-binding domain of the STAT1transcription factor. EMBO J 19:6196 – 6206 Brunet A, Kanai F, Stehn J, Xu J, Sarbassova D, Frangioni JV, Dalal SN, DeCaprio JA, Greenberg ME, Yaffe MB 2002 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. J Cell Biol 156:817– 828 Bogerd HP, Fridell RA, Benson RE, Hua J, Cullen BR 1996 Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay. Mol Cell Biol 16:4207– 4214 Van Lith HA, Meijer GW, Van der Wouw MJ, Den Bieman M, Van Tintelen G, Van Zutphen LF, Beynen AC 1992 Influence of amount
Mol Endocrinol, August 2011, 25(8):1351–1363
27.
28.
29.
30.
31.
32.
33.
34. 35.
36. 37.
38.
39.
40.
41.
42.
43.
44.
of dietary fat and protein on esterase-1 (ES-1) activities of plasma and small intestine in rats. Br J Nutr 67:379 –390 Van Lith HA, Meijer GW, Van Zutphen LF, Beynen AC 1990 Specific effect of the amount of dietary fat on esterase-1 (ES-1) activity of rat plasma. Z Ernahrungswiss 29:129 –134 Van Lith HA, Meijer GW, Van Zutphen LF, Beynen AC 1989 Plasma esterase-1 (ES-1) activity is increased in rats fed high-fat diets. Lipids 24:86 – 88 Van Lith HA, Haller M, van Tintelen G, Van Zutphen LF, Beynen AC 1992 Plasma esterase-1 (ES-1) activity in rats Is influenced by the amount and type of dietary fat, and butyryl cholinesterase activity by the type of dietary fat. J Nutr 122:2109 –2120 Cordido F, Peino R, Pen˜alva A, Alvarez CV, Casanueva FF, Dieguez C 1996 Impaired growth hormone secretion in obese subjects is partially reversed by acipimox-mediated plasma free fatty acid depression. J Clin Endocrinol Metab 81:914 –918 Kok P, Buijs MM, Kok SW, Van Ierssel IH, Fro¨lich M, Roelfsema F, Voshol PJ, Meinders AE, Pijl H 2004 Acipimox enhances spontaneous growth hormone secretion in obese women. Am J Physiol Regul Integr Comp Physiol 286:R693–R698 Dieguez C, Carro E, Seoane L, Garcia M, Camina J, Senaris R, Popovic V, Casanueva F 2000 Regulation of somatotroph cell function by the adipose tissue. Int J Obes Relat Metab Disord 24(Suppl 2):S100 –S103 Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui TV, Cross JR, Thompson CB 2009 ATP-citrate lyase links cellular metabolism to histone acetylation. Science 324:1076 –1080 Poon IK, Jans DA 2005 Regulation of nuclear transport: central role in development and transformation? Traffic 6:173–186 Takeuchi Y, Yahagi N, Izumida Y, Nishi M, Kubota M, Teraoka Y, Yamamoto T, Matsuzaka T, Nakagawa Y, Sekiya M, Iizuka Y, Ohashi K, Osuga J, Gotoda T, Ishibashi S, Itaka K, Kataoka K, Nagai R, Yamada N, Kadowaki T, Shimano H 2010 Polyunsaturated fatty acids selectively suppress sterol regulatory element-binding protein-1 through proteolytic processing and autoloop regulatory circuit. J Biol Chem 285:11681–11691 Kozak M 2002 Pushing the limits of the scanning mechanism for initiation of translation. Gene 299:1–34 Kadner SS, Katz J, Finlay TH 1992 Esterase-1: developmental expression in the mouse and distribution of related proteins in other species. Arch Biochem Biophys 296:435– 441 Leger J, Oury JF, Noel M, Baron S, Benali K, Blot P, Czernichow P 1996 Growth factors and intrauterine growth retardation. I. Serum growth hormone, insulin-like growth factor (IGF)-I, IGF-II, and IGF binding protein 3 levels in normally grown and growth-retarded human fetuses during the second half of gestation. Pediatr Res 40:94 –100 Ali O, Cohen P 2003 Insulin-like growth factors and their binding proteins in children born small for gestational age: implication for growth hormone therapy. Horm Res 60:115–123 Cutfield WS, Regan FA, Jackson WE, Jefferies CA, Robinson EM, Harris M, Hofman PL 2004 The endocrine consequences for very low birth weight premature infants. Growth Horm IGF Res 14: S130 –S135 Boiko J, Jaquet D, Chevenne D, Rigal O, Czernichow P, LevyMarchal C 2005 In situ lipolytic regulation in subjects born small for gestational age. Int J Obes 29:565–570 Bazaes RA, Salazar TE, Pittaluga E, Pen˜a V, Alegría A, In˜iguez G, Ong KK, Dunger DB, Mericq MV 2003 Glucose and lipid metabolism in small for gestational age infants at 48 hours of age. Pediatrics 111:804 – 809 Barclay JL, Nelson CN, Ishikawa M, Murray LA, Kerr LM, McPhee TR, Powell EE, Waters MJ 2011 GH-dependent STAT5 signaling plays an important role in hepatic lipid metabolism. Endocrinology 152:181–192 Fan Y, Menon RK, Cohen P, Hwang D, Clemens T, DiGirolamo DJ, Kopchick JJ, Le Roith D, Trucco M, Sperling MA 2009 Liverspecific deletion of the growth hormone receptor reveals essential
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Mol Endocrinol, August 2011, 25(8):1351–1363
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role of growth hormone signaling in hepatic lipid metabolism. J Biol Chem 284:19937–19944 45. Ichikawa T, Hamasaki K, Ishikawa H, Ejima E, Eguchi K, Nakao K 2003 Non-alcoholic steatohepatitis and hepatic steatosis in patients with adult onset growth hormone deficiency. Gut 52:914 46. Langendonk JG, Meinders AE, Burggraaf J, Fro¨lich M, Roelen CA, Schoemaker RC, Cohen AF, Pijl H 1999 Influence of obesity and body fat distribution on growth hormone kinetics in humans. Am J Physiol 277:E824 –E829 47. Fukuda I, Hizuka N, Yasumoto K, Morita J, Kurimoto M, Takano K 2008 Metabolic co-morbidities revealed in patients with childhood-onset adult GH deficiency after cessation of GH replacement therapy for short stature. Endocr J 55:977–984
1363
48. Takami Y, Uto H, Tamai T, Sato Y, Ishida Y, Morinaga H, Sakakibara Y, Moriuchi A, Oketani M, Ido A, Nakajima T, Okanoue T, Tsubouchi H 2010 Identification of a novel biomarker for oxidative stress induced by hydrogen peroxide in primary human hepatocytes using the 2-nitrobenzenesulfenyl chloride isotope labeling method. Hepatol Res 40:438 – 445 49. Singh H, LeBowitz JH, Baldwin JAS, Sharp PA 1988 Molecular cloning of an enhancer binding protein:Isolation by screening of an expression library with a recognition site DNA. Cell 52:415– 423 50. Schmittgen TD, Livak KJ 2008 Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108
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