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Estrogen induces estrogen receptor α expression and hepatocyte proliferation in late pregnancy
Biochemical and Biophysical Research Communications 511 (2019) 592e596
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Estrogen induces estrogen receptor a expression and hepatocyte proliferation in late pregnancy Yoji Tsugawa a, Masaki Hiramoto a, b, Takeshi Imai a, * a b
Department of Aging Intervention, National Center for Geriatrics and Gerontology, Obu, Aichi, 474-8511, Japan Department of Biochemistry, Tokyo Medical University, Shinjuku, Tokyo, 160-8402, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2019 Accepted 21 February 2019 Available online 28 February 2019
Partial hepatectomy (PH) induces estradiol production, and then hepatocyte proliferation. Estradiol may play a role in triggering hepatocyte proliferation after PH. In this study, estradiol was injected to the Estrogen Receptor alpha (ERa) or ERb KO mice. No increased hepatocyte proliferation was observed in ERa KO mice, indicates that ERa is involved in estradiol-induced hepatocyte proliferation. The ERa and ERb KO mice are sterile, hence it is impossible to study ERa and ERb function during pregnancy when the estrogen levels are highest. Using conditional mutagenesis technique, we made ERa hepatocyte KO mice, which are fertile. We used these mice for analyzing the hepatocyte ERa function during pregnancy. However, in the control mice, the maternal hepatocyte was proliferated higher in late pregnancy, but no pregnancy-induced hepatocyte proliferation was observed in KO mice. Hence, we conclude that the maternal hepatocyte ERa is involved in estradiol-induced hepatocyte proliferation in late pregnancy. © 2019 Elsevier Inc. All rights reserved.
Keywords: Pregnancy Estrogen receptor: estrogen Hepatocyte proliferation
1. Introduction
2. Materials and methods
Partial hepatectomy (PH) induces hepatocyte proliferation and liver regeneration [1,2]. We focused on the molecules that triggering hepatocyte proliferation. Several cytokine signals are involved in triggering liver regeneration. Liver regeneration is impaired/delayed in the Interleukin 6 (IL-6), and tumor necrosis factor a (TNFa) KO mice [1,2]. Our previous study showed that estradiol is also involved in triggering hepatocyte proliferation [3,4]. Moreover, hepatocyte proliferation was delayed and reduced because of ovariectomy and orchiectomy lacking estradiol [4]. Hepatocyte proliferation is accompanied by changing gene expression [1,2]. as two of the three estrogen receptors are transcription factors: Estrogen Receptor alpha (ERa) and ERb [3e5]. The most potent ligand estradiol, binds to ERa/ERb and exerts changing cognate gene transcription [5]. Our previous study showed that ERa expression is induced after PH [3,4]. Therefore, we hypothesize that estradiol-ERa signaling is indispensable for hepatocyte proliferation.
2.1. Materials
* Corresponding author. E-mail addresses: [email protected] (Y. Tsugawa), [email protected]. jp (M. Hiramoto), [email protected] (T. Imai). https://doi.org/10.1016/j.bbrc.2019.02.119 0006-291X/© 2019 Elsevier Inc. All rights reserved.
b-Estradiol (E2, 052e04041) was purchased from Wako Chemicals (Osaka, Japan). The estradiol enzyme immunoassay (EIA) kit (No. 582251) was purchased from Funakoshi (Tokyo, Japan). 2.2. Mice C57BL/6J and CD1 WT mice were obtained from Charles River Japan. The transgenic mice of ERa KO, ERb KO and hepatocyte ERa KO mice were described previously [3e6]. 2.3. Maternal hepatocyte proliferation in mice The hepatocyte proliferation rates among female mice were analyzed by bromodeoxyuridine (BrdU; M0744, Dako, Tokyo, Japan) and Ki67 (Funakoshi, Japan) immunohistochemistry (IHC, SK-4105, Vector). The mice were intraperitoneally injected with 50 mg/kg of BrdU 2 h before dissection, and the livers were then removed, rinsed, and embedded in Tissue-Tek OCT cryo embedding compound (Sakura Finetek Japan, Tokyo, Japan). Subsequently, 10mm cryosections were fixed with 4% paraformaldehyde, incubated
Y. Tsugawa et al. / Biochemical and Biophysical Research Communications 511 (2019) 592e596
Abbreviation BrdU DPC E2 ER FCS HPRT ISH RT-PCR SE WB WT
Bromodeoxyuridine day post coitum estrogen Estrogen Receptor fetal calf serum hypoxanthine phosphoribosyltransferase in situ hybridization reverse transcriptase polymerase chain reaction standard error Western blot wild type
with an anti-BrdU antibody (No. 11170376001, Roche Diagnostics Japan, Tokyo, Japan) diluted 50-fold in 0.1% bovine serum albumin/ phosphate-buffered saline, and then labelled with CY3-conjugated donkey anti-rabbit IgG antibody. The sections were then mounted on slides in Vectorshield medium (Vector Laboratories), and the
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number of BrdU/Ki67-positive hepatocyte nuclei in each sample (approximately 2000 hepatocytes) were counted in at least five low-magnification microscopic fields [7,8].
2.4. In situ hybridization The slides were incubated in the prehybridization buffer (50% formamide, 0.3 M NaCl, 10 mM Tris-HCl pH 6.8, 10 mM NaPO4 pH 6.8, 5 mM EDTA, 1 Denhart's solution, 10 mM DTT, 500-mg/mL yeast RNA, 100-mg/mL salmon sperm DNA, and 500-nmol/mL nonlabelled-thio-UTP; Du Pont) at 50 C for 2 h. After RNase A treatment, the slides were washed with the washing buffer (50% formamide, 0.3 M NaCl, 10 mM Tris-HCl pH 6.8, 10 mM NaPO4 pH 6.8, 5 mM EDTA, 1 Denhart's solution, and 10 mM DTT) at room temperature for 1 h, with 2 SSC at room temperature for 15 min, with 0.1 SSC at 50 C for 15 min, and with 0.1 SSC at room temperature for 30 min. After dehydrating the sections, they were coated with Kodak NTB-2 emulsion, dried, and stored at 4 C. The sections were then exposed for 12e15 days. The exposed slides were then developed in Kodak D19 developer at room temperature for 2 min, and the sections were stained in toluidine blue,
Fig. 1. ERa is involved in the estrogen-induced hepatocyte proliferation. a, Partial hepatectomy (PH) induces estradiol production, and then hepatocyte proliferation. Plasma estradiol (filled rectangle) is induced at 6 h after PH, and then hepatocyte proliferation (2 h-bromodeoxyuridine [BrdU] incorporation, open circle) starts at 26 h after PH. b, Estradiol administration induces liver to body weight ratio (filled circle) and hepatocyte proliferation (Ki67, open rectangle) in an estradiol dose dependent manner. Estradiol was administrated to C57BL/6J mice, and at 48 h later, liver and body weight and Ki67-immunohistochemistry was analyzed. c and d, ERa is involved in the estradiol-induced hepatocyte proliferation. One hundred mg estradiol was injected to mice, and then BrdU was injected after 46 h. Two hours later, BrdU (b) and Ki67 (c) immunohistochemistry was analyzed. Values are expressed as the mean ± SEM (n ¼ 5e7). NS non-significant, and *p < 0.05.
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dehydrated in ethanol, and mounted in Eukitt mounting medium under coverslips [3,4,9].
two-way analysis of variance test (*p < 0.05, **p < 0.005, and ***p < 0.0001). Non-statistical differences (p > 0.05) are denoted as NS (not significant).
2.5. Reverse transcriptase polymerase chain reaction (RT-PCR) Total liver RNA was extracted by the guanidium-thiocyanatephenol-chloroform method. cDNA was synthesized from 1-mg RNA using Moloney murine leukemia virus reverse transcriptase at 50 C for 20 min. The transcribed cDNAs from five mice were mixed, and were amplified via 30 cycles of PCR using the following primers: ERa, forward 50 -CGG CTG CCA CTT ACC TGG GAG CTC TCA GAT-30 and reverse 50 -GGG GAG CCT GGG AGC TCT CAG AT-3’; ERb, forward 50 -TCT CTG AGA GCA TCATGT CC-30 and reverse 50 -CAG CCT GGC CGT CAC TGT GA-3’; and hypoxanthine phosphoribosyltransferase, forward 50 -GTA ATG ATC AGT CAA CGG GGG AC-30 and reverse 50 -CCA GCA AGC TTG CAA CCT TAA CCA-30 ) [10,11]. 2.6. Animal study compliance All experiments were performed in accordance with the ethical guidelines for animal care established by the National Center for Geriatrics and Gerontology (NCGG), and the study was approved by the Animal Care Committee. 2.7. Statistical analysis Values are reported as the mean ± SEM (n ¼ 5e12). Statistical significance was assessed using the Student's t-test and one-way/
3. Result In our previous study, we observed that estrogen induces hepatocyte proliferation and ovariectomy delayed hepatocyte proliferation after partial hepatectomy (PH, [2,3]). We hypothesized that estrogen triggers hepatocyte proliferation after PH. Therefore, we analyzed both plasma estradiol and hepatocyte proliferation after PH (Fig. 1a). The estradiol concentration was elevated at 2 h after PH, and then hepatocytes proliferation started at 26 h, suggesting estradiol triggered hepatocyte proliferation after PH. Estradiol was administered to mice without PH, and hepatocyte proliferation was analyzed using proliferation specific marker Ki67 immunohistochemistry [12]. Both liver-body-weight-ratio (filled circle) and Ki67-positive hepatocyte rate (open rectangle) were increased in an estradiol-dependent manner (Fig. 1b). Estradiol binds to its receptor ERa or ERb as a ligand, and the estradiol exerts its genomic function (transcription) in the cell through ERa and ERb [3e5]. Estradiol was injected to the control (CT), ERa and ERb KO mice [5], and hepatocyte proliferation was analyzed with incorporated bromodeoxyuridine (BrdU) and Ki67 immunohistochemistry (Fig. 1c and d). The results showed that ERa is the estrogen target protein for estradiol-induced hepatocyte proliferation. In our previous study, hepatocyte proliferation was observed in the Estrus cycle [4], suggesting endogenous estradiol controls
Fig. 2. Maternal hepatocyte proliferation and estradiol concentration are increased in the late pregnancy. a, Postpartum CD-1 mice have bigger livers than non-pregnant mice. b, Maternal livers gained weight during pregnancy in CD-1 mice. Maternal body weight and the weight of three organs (the liver, kidney, and heart) of pregnant CD-1 mice were measured. Values are expressed as the mean ± SEM (n ¼ 7). The liver and body weights increased during pregnancy. c, Pregnancy induces maternal hepatocyte proliferation and estradiol concentration. Hepatocyte proliferation was analyzed by Ki67 (open rectangle) immunohistochemistry using liver sections obtained from pregnant CD-1 mice. Circulating estradiol concentration (gray bar) was analyzed with ELISA kit. Values are expressed as the mean ± SEM (n ¼ 7e12). *p < 0.05.
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Fig. 3. The ERa expression was induced in the late stage of pregnancy. a and b, ERa expression is induced in the late pregnancy. The RNA expression of ERa and ERb in the pregnant maternal livers was analyzed with RT-PCR. ERa and ERb expression positive control RNA was obtained from ovary, and HPRT was used for RT-PCR internal control c, ERa RNA expression on hepatocytes in zone III was induced in late pregnancy. The ERa in situ hybridization was performed in the maternal liver of non-pregnant (0 DPC) and pregnant (7.5 and 17.5 DPC) mice. The hybridized ERa transcripts are shown in white color. Induced expression was mainly observed in the hepatocytes located in the periportal area (zone III). Scale bar ¼ 1 mm.
Fig. 4. Hepatocyte ERa is involved in the estradiol-induced hepatocyte proliferation and in the late pregnancy. a, Normal estradiol production and less hepatocyte proliferation in hepatocyte ERa KO mice. Plasma estradiol concentration was analyzed from the ERa hepatocyte KO and control mice with ELISA kit. Hepatocyte proliferation was analyzed with proliferation marker Ki67 immunocytochemistry. b, Hepatocyte proliferation is induced in the Diestrus cycle, and hepatocyte ERa is involved in hepatocyte proliferation in the Diestrus cycle. Hepatocyte proliferation was analyzed with the ratio of mitotic figures % c and d, Hepatocyte ERa is involved in the estrogen-induced hepatocyte proliferation. Proliferation marker Ki67 (c) and BrdU (d) immunohistochemistry were performed with the livers from estradiol injected ERa hepatocyte KO and control mice. e, Plasma estradiol is elevated in the late pregnancy from the ERa hepatocyte KO and control mice. f and g, The maternal hepatocyte proliferate in the late preganacy, and ERa is involved hepatocyte proliferation in the late pregnancy. Hepatocyte proliferation was analyzed with Ki67 immunocytochemistry (f) and mitotic figure (%, g), respectively. Values are expressed as the mean ± SEM (n ¼ 5e7). *p < 0.05, and **p < 0.005.
hepatocyte proliferation. It is well known that the highest estrogen levels are seen during pregnancy. Therefore, we analyzed the livers in the pregnant CD-1 WT mice (Fig. 2), which are well-known as
prolific. The livers of pregnant mice were found to be bigger than the livers of non-pregnant mice (Fig. 2a). Body and liver weights increased approximately 60% and 70%, respectively, in pregnant
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mice (Fig. 2b). However, the weight of other organs, such as the kidney and heart, did not change during pregnancy (Fig. 2b). Further, the analysis of hepatocyte proliferation by proliferation marker Ki67 immunohistochemistry revealed maternal hepatocyte proliferation in late pregnancy, when plasma estradiol was increased (Fig. 2c). Maternal hepatocyte proliferation and plasma estradiol concentration are increased parallelly in the late pregnancy. Taken together, pregnancy induces estradiol, and estradiol triggers maternal hepatocyte proliferation. Our previous studies showed that PH and estradiol administration stimulate ERa expression in the periportal hepatocytes [3,4]. We analyzed estrogen receptors expression during pregnancy (Fig. 3), and ERa RNA expression is induced in the late pregnancy (Fig. 3a and b). Induced-ERa expressed hepatocytes located in the periportal area (Fig. 3c). Therefore, we analyzed ERa KO mice in the late pregnancy. Since the ERa KO mice (and ERb KO mice) are sterile [5], with conditional somatic mutagenesis, we established hepatocyte-ERa KO mice [5,6], which are fertile and have normal estradiol productivity. In these mice, the estradiol elevated in the Proestrus cycle (Fig. 4a, and [13]). Although WT-hepatocytes proliferate during the Diestrus cycle (Fig. 4a and b, and [3,4]), ERa ablated hepatocytes proliferate a low level (Fig. 4a and b). To see the effect of estradiol-induced hepatocyte proliferation, estradiol was injected to the ERa hepatocyte KO mice. The estradiol-induced hepatocyte proliferation was hardly observed in these mice (Fig. 4c and d). Finally, pregnant ERa hepatocyte KO mice were analyzed (Fig. 4eeg). Plasma estradiol was elevated to late pregnancy in both animals (Fig. 4e). The maternal WT-hepatocytes proliferation also increased in the late pregnancy. However, no induced proliferation was observed in ERa ablated hepatocyte during pregnancy (Fig. 4f and g). These data indicated that hepatocyte-ERa is involved in estradiol-induced hepatocyte proliferation in the late pregnancy in Diestrus cycle. 4. Discussion Estradiol is known as one of the stress signaling molecule [3,4,14]. Here, we showed that PH induced estradiol production before hepatocyte proliferation (Fig. 1). Conversely, the ovariectomized and orchitectomized mice produced less estradiol after PH, and had delayed liver regeneration [3,4]. We conclude that elevated estradiol triggers hepatocyte proliferation after PH. Circulating estradiol concentration is increased in the Estrus cycle (Fig. 4a, and b), after estradiol injection (Fig. 1bed, Fig. 4c and d), and in late pregnancy (Figs. 2c and 4c). The mRNA expression of ERa is also increased in the late pregnancy and in the hepatocyte located periportal area (Fig. 3). Generally, periportal hepatocytes are active and proliferative, and hepatocyte proliferation starts from the periportal area [3,4]. The elevated estradiol in the Estrus cycle induces ERa expression in the periportal hepatocytes, and proliferation. Milona et al. (2010) reported that pregnancy induces hepatocyte hypertrophy associated with cell-cycle progression without mitosis as they did not observe significant change mitotic figure (%) between non-pregnant and pregnant mice [15]. Here, we showed that more than three-fold differences of mitotic figure (%) were observed in the Estrus cycle (ca 0.08e0.3%; Fig. 4b), which indicate that non-pregnant mice should have all four stages of the Estrus cycle. If they use estrus cycle (just before ovulation) mice as a nonpregnant control, may have a different result of pregnancy-induced hepatocyte proliferation. Author contribution Y.T., and M.H. performed the experiments, and T.I. designed the
experiments, analyzed the data, and wrote the manuscript. Author information Reprints and permission information is available at (https:// www.journals.elsevier.com/biochemical-and-biophysicalresearch-communications). Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to TI ([email protected]). Conflicts of interest The authors declare no conflict of interest and competing financial interests. Acknowledgements We are grateful to our department members in the NCGG for their helpful discussions. This work was supported by a Grant-inAid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT 18659493), the Japan Science and Technology Agency (A-STEP-AS2312036G and FY2013-SICP) and NCGG (28-25) to T.I. References [1] C.J. Steer, Liver regeneration, FASEB J. 9 (1995) 1396e1400. [2] G.K. Michalpoules, M.C. Defrances, Liver regeneration Science, 276, 1997, pp. 60e66. [3] T. Uebi, M. Umeda, T. Imai, Estrogen induces Estrogen Receptor alpha expression and hepatocyte proliferation in the livers of the male mice, Genes Cells 20 (2015) 217e223. [4] M. Umeda, M. Hiramoto, T. Imai, Partial hepatectomy induces delayed hepatocyte proliferation and normal liver regeneration in ovariectomized mice, Clin. Exp. Gastroenterol. 8 (2015) 175e182. https://doi.org/10.2147/CEG. S80212. [5] S. Dupont, A. Krust, A. Gansmuller, A. Dierich, P. Chambon, M. Mark, Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes, Development 127 (2000) 4277e4291. [6] T. Imai, M. Jiang, P. Kastner, P. Chambon, D. Metzger, Selective ablation of retinoid X receptor a in hepatocytes impairs their lifespan and regenerative capacity, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 4581e4586, https://doi.org/ 10.1073/pnas.071056098. [7] T. Uebi, M. Umeda, N. Maekawa, S. Karasawa, H. Handa, T. Imai, Prohibitins, novel vitamin K2 target factors in osteoblast, J. Biosci. Med. 1 (2013) 1e4, https://doi.org/10.4236/jbm.2013.13001. [8] T. Imai, M. Jiang, P. Chambon, D. Metzger, Impaired adipogenesis and lipolysis in the mouse upon Cre-ERT2-mediated selective ablation of RXRa in adipocytes, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 224e228, https://doi.org/10.1073/ pnas.011528898. , D. Duboule, Two gene members of the murine HOX-5 complex show [9] P. Dolle regional and cell-type specific expression in developing limbs and gonads, EMBO J. 8 (1989) 1507e1515. [10] M. Umeda, T. Uebi, N. Maekawa, H. Handa, T. Imai, PGJIFs, new mitochondrial PGJ2 target proteins, regulate cell proliferation, J. Biosci. Med. 1 (2013) 11e15, https://doi.org/10.4236/jbm.2013.13003. [11] M. Umeda, T. Uebi, N. Maekawa, M. Isaki, Y. Miyama, Y. Masaike, H. Handa, T. Imai, Effective cofactor complex purification using nanobeads, J. Biosci. Med. 1 (2013) 5e10, https://doi.org/10.4236/jbm.2013.13003. [12] K.H. Kim, J.M. Sederstrom, Assaying cell cycle status using flow cytometry, Curr Protoc Mol Biol 28 (2015) 1e11, https://doi.org/10.1002/ 0471142727.mb2806s111. [13] B.R. Santmyire, V. Venkat, E. Beinder, C. Baylis, Impact of the estrus cycle and reduction in estrogen levels with aromatase inhibition, on renal function and nitric oxide activity in female rats, Steroids 75 (2010) 1011e1015. [14] P. Newhouse, K. Albert, Estrogen, stress, and depression: a neurocognitive model, JAMA Psychiatry 75 (2015) 727e729, https://doi.org/10.1001/ jamapsychiatry.2015.0487. [15] A. Milona, B.M. Owen, S. van Mil, D. Dormann, C. Mataki, M. Boudjelal, W. Cairns, K. Schoonjans, S. Milligan, M. Parker, R. White, C. Williamson, The normal mechanisms of pregnancy-induced liver growth are not maintained in mice lacking the bile acid sensor Fxr, Am. J. Physiol. Gastrointest. Liver Physiol. 298 (2010) G151eG158.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Estrogen induces estrogen receptor a expression and hepatocyte proliferation in late pregnancy Yoji Tsugawa a, Masaki Hiramoto a, b, Takeshi Imai a, * a b
Department of Aging Intervention, National Center for Geriatrics and Gerontology, Obu, Aichi, 474-8511, Japan Department of Biochemistry, Tokyo Medical University, Shinjuku, Tokyo, 160-8402, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2019 Accepted 21 February 2019 Available online 28 February 2019
Partial hepatectomy (PH) induces estradiol production, and then hepatocyte proliferation. Estradiol may play a role in triggering hepatocyte proliferation after PH. In this study, estradiol was injected to the Estrogen Receptor alpha (ERa) or ERb KO mice. No increased hepatocyte proliferation was observed in ERa KO mice, indicates that ERa is involved in estradiol-induced hepatocyte proliferation. The ERa and ERb KO mice are sterile, hence it is impossible to study ERa and ERb function during pregnancy when the estrogen levels are highest. Using conditional mutagenesis technique, we made ERa hepatocyte KO mice, which are fertile. We used these mice for analyzing the hepatocyte ERa function during pregnancy. However, in the control mice, the maternal hepatocyte was proliferated higher in late pregnancy, but no pregnancy-induced hepatocyte proliferation was observed in KO mice. Hence, we conclude that the maternal hepatocyte ERa is involved in estradiol-induced hepatocyte proliferation in late pregnancy. © 2019 Elsevier Inc. All rights reserved.
Keywords: Pregnancy Estrogen receptor: estrogen Hepatocyte proliferation
1. Introduction
2. Materials and methods
Partial hepatectomy (PH) induces hepatocyte proliferation and liver regeneration [1,2]. We focused on the molecules that triggering hepatocyte proliferation. Several cytokine signals are involved in triggering liver regeneration. Liver regeneration is impaired/delayed in the Interleukin 6 (IL-6), and tumor necrosis factor a (TNFa) KO mice [1,2]. Our previous study showed that estradiol is also involved in triggering hepatocyte proliferation [3,4]. Moreover, hepatocyte proliferation was delayed and reduced because of ovariectomy and orchiectomy lacking estradiol [4]. Hepatocyte proliferation is accompanied by changing gene expression [1,2]. as two of the three estrogen receptors are transcription factors: Estrogen Receptor alpha (ERa) and ERb [3e5]. The most potent ligand estradiol, binds to ERa/ERb and exerts changing cognate gene transcription [5]. Our previous study showed that ERa expression is induced after PH [3,4]. Therefore, we hypothesize that estradiol-ERa signaling is indispensable for hepatocyte proliferation.
2.1. Materials
* Corresponding author. E-mail addresses: [email protected] (Y. Tsugawa), [email protected]. jp (M. Hiramoto), [email protected] (T. Imai). https://doi.org/10.1016/j.bbrc.2019.02.119 0006-291X/© 2019 Elsevier Inc. All rights reserved.
b-Estradiol (E2, 052e04041) was purchased from Wako Chemicals (Osaka, Japan). The estradiol enzyme immunoassay (EIA) kit (No. 582251) was purchased from Funakoshi (Tokyo, Japan). 2.2. Mice C57BL/6J and CD1 WT mice were obtained from Charles River Japan. The transgenic mice of ERa KO, ERb KO and hepatocyte ERa KO mice were described previously [3e6]. 2.3. Maternal hepatocyte proliferation in mice The hepatocyte proliferation rates among female mice were analyzed by bromodeoxyuridine (BrdU; M0744, Dako, Tokyo, Japan) and Ki67 (Funakoshi, Japan) immunohistochemistry (IHC, SK-4105, Vector). The mice were intraperitoneally injected with 50 mg/kg of BrdU 2 h before dissection, and the livers were then removed, rinsed, and embedded in Tissue-Tek OCT cryo embedding compound (Sakura Finetek Japan, Tokyo, Japan). Subsequently, 10mm cryosections were fixed with 4% paraformaldehyde, incubated
Y. Tsugawa et al. / Biochemical and Biophysical Research Communications 511 (2019) 592e596
Abbreviation BrdU DPC E2 ER FCS HPRT ISH RT-PCR SE WB WT
Bromodeoxyuridine day post coitum estrogen Estrogen Receptor fetal calf serum hypoxanthine phosphoribosyltransferase in situ hybridization reverse transcriptase polymerase chain reaction standard error Western blot wild type
with an anti-BrdU antibody (No. 11170376001, Roche Diagnostics Japan, Tokyo, Japan) diluted 50-fold in 0.1% bovine serum albumin/ phosphate-buffered saline, and then labelled with CY3-conjugated donkey anti-rabbit IgG antibody. The sections were then mounted on slides in Vectorshield medium (Vector Laboratories), and the
593
number of BrdU/Ki67-positive hepatocyte nuclei in each sample (approximately 2000 hepatocytes) were counted in at least five low-magnification microscopic fields [7,8].
2.4. In situ hybridization The slides were incubated in the prehybridization buffer (50% formamide, 0.3 M NaCl, 10 mM Tris-HCl pH 6.8, 10 mM NaPO4 pH 6.8, 5 mM EDTA, 1 Denhart's solution, 10 mM DTT, 500-mg/mL yeast RNA, 100-mg/mL salmon sperm DNA, and 500-nmol/mL nonlabelled-thio-UTP; Du Pont) at 50 C for 2 h. After RNase A treatment, the slides were washed with the washing buffer (50% formamide, 0.3 M NaCl, 10 mM Tris-HCl pH 6.8, 10 mM NaPO4 pH 6.8, 5 mM EDTA, 1 Denhart's solution, and 10 mM DTT) at room temperature for 1 h, with 2 SSC at room temperature for 15 min, with 0.1 SSC at 50 C for 15 min, and with 0.1 SSC at room temperature for 30 min. After dehydrating the sections, they were coated with Kodak NTB-2 emulsion, dried, and stored at 4 C. The sections were then exposed for 12e15 days. The exposed slides were then developed in Kodak D19 developer at room temperature for 2 min, and the sections were stained in toluidine blue,
Fig. 1. ERa is involved in the estrogen-induced hepatocyte proliferation. a, Partial hepatectomy (PH) induces estradiol production, and then hepatocyte proliferation. Plasma estradiol (filled rectangle) is induced at 6 h after PH, and then hepatocyte proliferation (2 h-bromodeoxyuridine [BrdU] incorporation, open circle) starts at 26 h after PH. b, Estradiol administration induces liver to body weight ratio (filled circle) and hepatocyte proliferation (Ki67, open rectangle) in an estradiol dose dependent manner. Estradiol was administrated to C57BL/6J mice, and at 48 h later, liver and body weight and Ki67-immunohistochemistry was analyzed. c and d, ERa is involved in the estradiol-induced hepatocyte proliferation. One hundred mg estradiol was injected to mice, and then BrdU was injected after 46 h. Two hours later, BrdU (b) and Ki67 (c) immunohistochemistry was analyzed. Values are expressed as the mean ± SEM (n ¼ 5e7). NS non-significant, and *p < 0.05.
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dehydrated in ethanol, and mounted in Eukitt mounting medium under coverslips [3,4,9].
two-way analysis of variance test (*p < 0.05, **p < 0.005, and ***p < 0.0001). Non-statistical differences (p > 0.05) are denoted as NS (not significant).
2.5. Reverse transcriptase polymerase chain reaction (RT-PCR) Total liver RNA was extracted by the guanidium-thiocyanatephenol-chloroform method. cDNA was synthesized from 1-mg RNA using Moloney murine leukemia virus reverse transcriptase at 50 C for 20 min. The transcribed cDNAs from five mice were mixed, and were amplified via 30 cycles of PCR using the following primers: ERa, forward 50 -CGG CTG CCA CTT ACC TGG GAG CTC TCA GAT-30 and reverse 50 -GGG GAG CCT GGG AGC TCT CAG AT-3’; ERb, forward 50 -TCT CTG AGA GCA TCATGT CC-30 and reverse 50 -CAG CCT GGC CGT CAC TGT GA-3’; and hypoxanthine phosphoribosyltransferase, forward 50 -GTA ATG ATC AGT CAA CGG GGG AC-30 and reverse 50 -CCA GCA AGC TTG CAA CCT TAA CCA-30 ) [10,11]. 2.6. Animal study compliance All experiments were performed in accordance with the ethical guidelines for animal care established by the National Center for Geriatrics and Gerontology (NCGG), and the study was approved by the Animal Care Committee. 2.7. Statistical analysis Values are reported as the mean ± SEM (n ¼ 5e12). Statistical significance was assessed using the Student's t-test and one-way/
3. Result In our previous study, we observed that estrogen induces hepatocyte proliferation and ovariectomy delayed hepatocyte proliferation after partial hepatectomy (PH, [2,3]). We hypothesized that estrogen triggers hepatocyte proliferation after PH. Therefore, we analyzed both plasma estradiol and hepatocyte proliferation after PH (Fig. 1a). The estradiol concentration was elevated at 2 h after PH, and then hepatocytes proliferation started at 26 h, suggesting estradiol triggered hepatocyte proliferation after PH. Estradiol was administered to mice without PH, and hepatocyte proliferation was analyzed using proliferation specific marker Ki67 immunohistochemistry [12]. Both liver-body-weight-ratio (filled circle) and Ki67-positive hepatocyte rate (open rectangle) were increased in an estradiol-dependent manner (Fig. 1b). Estradiol binds to its receptor ERa or ERb as a ligand, and the estradiol exerts its genomic function (transcription) in the cell through ERa and ERb [3e5]. Estradiol was injected to the control (CT), ERa and ERb KO mice [5], and hepatocyte proliferation was analyzed with incorporated bromodeoxyuridine (BrdU) and Ki67 immunohistochemistry (Fig. 1c and d). The results showed that ERa is the estrogen target protein for estradiol-induced hepatocyte proliferation. In our previous study, hepatocyte proliferation was observed in the Estrus cycle [4], suggesting endogenous estradiol controls
Fig. 2. Maternal hepatocyte proliferation and estradiol concentration are increased in the late pregnancy. a, Postpartum CD-1 mice have bigger livers than non-pregnant mice. b, Maternal livers gained weight during pregnancy in CD-1 mice. Maternal body weight and the weight of three organs (the liver, kidney, and heart) of pregnant CD-1 mice were measured. Values are expressed as the mean ± SEM (n ¼ 7). The liver and body weights increased during pregnancy. c, Pregnancy induces maternal hepatocyte proliferation and estradiol concentration. Hepatocyte proliferation was analyzed by Ki67 (open rectangle) immunohistochemistry using liver sections obtained from pregnant CD-1 mice. Circulating estradiol concentration (gray bar) was analyzed with ELISA kit. Values are expressed as the mean ± SEM (n ¼ 7e12). *p < 0.05.
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Fig. 3. The ERa expression was induced in the late stage of pregnancy. a and b, ERa expression is induced in the late pregnancy. The RNA expression of ERa and ERb in the pregnant maternal livers was analyzed with RT-PCR. ERa and ERb expression positive control RNA was obtained from ovary, and HPRT was used for RT-PCR internal control c, ERa RNA expression on hepatocytes in zone III was induced in late pregnancy. The ERa in situ hybridization was performed in the maternal liver of non-pregnant (0 DPC) and pregnant (7.5 and 17.5 DPC) mice. The hybridized ERa transcripts are shown in white color. Induced expression was mainly observed in the hepatocytes located in the periportal area (zone III). Scale bar ¼ 1 mm.
Fig. 4. Hepatocyte ERa is involved in the estradiol-induced hepatocyte proliferation and in the late pregnancy. a, Normal estradiol production and less hepatocyte proliferation in hepatocyte ERa KO mice. Plasma estradiol concentration was analyzed from the ERa hepatocyte KO and control mice with ELISA kit. Hepatocyte proliferation was analyzed with proliferation marker Ki67 immunocytochemistry. b, Hepatocyte proliferation is induced in the Diestrus cycle, and hepatocyte ERa is involved in hepatocyte proliferation in the Diestrus cycle. Hepatocyte proliferation was analyzed with the ratio of mitotic figures % c and d, Hepatocyte ERa is involved in the estrogen-induced hepatocyte proliferation. Proliferation marker Ki67 (c) and BrdU (d) immunohistochemistry were performed with the livers from estradiol injected ERa hepatocyte KO and control mice. e, Plasma estradiol is elevated in the late pregnancy from the ERa hepatocyte KO and control mice. f and g, The maternal hepatocyte proliferate in the late preganacy, and ERa is involved hepatocyte proliferation in the late pregnancy. Hepatocyte proliferation was analyzed with Ki67 immunocytochemistry (f) and mitotic figure (%, g), respectively. Values are expressed as the mean ± SEM (n ¼ 5e7). *p < 0.05, and **p < 0.005.
hepatocyte proliferation. It is well known that the highest estrogen levels are seen during pregnancy. Therefore, we analyzed the livers in the pregnant CD-1 WT mice (Fig. 2), which are well-known as
prolific. The livers of pregnant mice were found to be bigger than the livers of non-pregnant mice (Fig. 2a). Body and liver weights increased approximately 60% and 70%, respectively, in pregnant
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mice (Fig. 2b). However, the weight of other organs, such as the kidney and heart, did not change during pregnancy (Fig. 2b). Further, the analysis of hepatocyte proliferation by proliferation marker Ki67 immunohistochemistry revealed maternal hepatocyte proliferation in late pregnancy, when plasma estradiol was increased (Fig. 2c). Maternal hepatocyte proliferation and plasma estradiol concentration are increased parallelly in the late pregnancy. Taken together, pregnancy induces estradiol, and estradiol triggers maternal hepatocyte proliferation. Our previous studies showed that PH and estradiol administration stimulate ERa expression in the periportal hepatocytes [3,4]. We analyzed estrogen receptors expression during pregnancy (Fig. 3), and ERa RNA expression is induced in the late pregnancy (Fig. 3a and b). Induced-ERa expressed hepatocytes located in the periportal area (Fig. 3c). Therefore, we analyzed ERa KO mice in the late pregnancy. Since the ERa KO mice (and ERb KO mice) are sterile [5], with conditional somatic mutagenesis, we established hepatocyte-ERa KO mice [5,6], which are fertile and have normal estradiol productivity. In these mice, the estradiol elevated in the Proestrus cycle (Fig. 4a, and [13]). Although WT-hepatocytes proliferate during the Diestrus cycle (Fig. 4a and b, and [3,4]), ERa ablated hepatocytes proliferate a low level (Fig. 4a and b). To see the effect of estradiol-induced hepatocyte proliferation, estradiol was injected to the ERa hepatocyte KO mice. The estradiol-induced hepatocyte proliferation was hardly observed in these mice (Fig. 4c and d). Finally, pregnant ERa hepatocyte KO mice were analyzed (Fig. 4eeg). Plasma estradiol was elevated to late pregnancy in both animals (Fig. 4e). The maternal WT-hepatocytes proliferation also increased in the late pregnancy. However, no induced proliferation was observed in ERa ablated hepatocyte during pregnancy (Fig. 4f and g). These data indicated that hepatocyte-ERa is involved in estradiol-induced hepatocyte proliferation in the late pregnancy in Diestrus cycle. 4. Discussion Estradiol is known as one of the stress signaling molecule [3,4,14]. Here, we showed that PH induced estradiol production before hepatocyte proliferation (Fig. 1). Conversely, the ovariectomized and orchitectomized mice produced less estradiol after PH, and had delayed liver regeneration [3,4]. We conclude that elevated estradiol triggers hepatocyte proliferation after PH. Circulating estradiol concentration is increased in the Estrus cycle (Fig. 4a, and b), after estradiol injection (Fig. 1bed, Fig. 4c and d), and in late pregnancy (Figs. 2c and 4c). The mRNA expression of ERa is also increased in the late pregnancy and in the hepatocyte located periportal area (Fig. 3). Generally, periportal hepatocytes are active and proliferative, and hepatocyte proliferation starts from the periportal area [3,4]. The elevated estradiol in the Estrus cycle induces ERa expression in the periportal hepatocytes, and proliferation. Milona et al. (2010) reported that pregnancy induces hepatocyte hypertrophy associated with cell-cycle progression without mitosis as they did not observe significant change mitotic figure (%) between non-pregnant and pregnant mice [15]. Here, we showed that more than three-fold differences of mitotic figure (%) were observed in the Estrus cycle (ca 0.08e0.3%; Fig. 4b), which indicate that non-pregnant mice should have all four stages of the Estrus cycle. If they use estrus cycle (just before ovulation) mice as a nonpregnant control, may have a different result of pregnancy-induced hepatocyte proliferation. Author contribution Y.T., and M.H. performed the experiments, and T.I. designed the
experiments, analyzed the data, and wrote the manuscript. Author information Reprints and permission information is available at (https:// www.journals.elsevier.com/biochemical-and-biophysicalresearch-communications). Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to TI ([email protected]). Conflicts of interest The authors declare no conflict of interest and competing financial interests. Acknowledgements We are grateful to our department members in the NCGG for their helpful discussions. This work was supported by a Grant-inAid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT 18659493), the Japan Science and Technology Agency (A-STEP-AS2312036G and FY2013-SICP) and NCGG (28-25) to T.I. References [1] C.J. Steer, Liver regeneration, FASEB J. 9 (1995) 1396e1400. [2] G.K. Michalpoules, M.C. Defrances, Liver regeneration Science, 276, 1997, pp. 60e66. [3] T. Uebi, M. Umeda, T. Imai, Estrogen induces Estrogen Receptor alpha expression and hepatocyte proliferation in the livers of the male mice, Genes Cells 20 (2015) 217e223. [4] M. Umeda, M. Hiramoto, T. Imai, Partial hepatectomy induces delayed hepatocyte proliferation and normal liver regeneration in ovariectomized mice, Clin. Exp. Gastroenterol. 8 (2015) 175e182. https://doi.org/10.2147/CEG. S80212. [5] S. Dupont, A. Krust, A. Gansmuller, A. Dierich, P. Chambon, M. Mark, Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes, Development 127 (2000) 4277e4291. [6] T. Imai, M. Jiang, P. Kastner, P. Chambon, D. Metzger, Selective ablation of retinoid X receptor a in hepatocytes impairs their lifespan and regenerative capacity, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 4581e4586, https://doi.org/ 10.1073/pnas.071056098. [7] T. Uebi, M. Umeda, N. Maekawa, S. Karasawa, H. Handa, T. Imai, Prohibitins, novel vitamin K2 target factors in osteoblast, J. Biosci. Med. 1 (2013) 1e4, https://doi.org/10.4236/jbm.2013.13001. [8] T. Imai, M. Jiang, P. Chambon, D. Metzger, Impaired adipogenesis and lipolysis in the mouse upon Cre-ERT2-mediated selective ablation of RXRa in adipocytes, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 224e228, https://doi.org/10.1073/ pnas.011528898. , D. Duboule, Two gene members of the murine HOX-5 complex show [9] P. Dolle regional and cell-type specific expression in developing limbs and gonads, EMBO J. 8 (1989) 1507e1515. [10] M. Umeda, T. Uebi, N. Maekawa, H. Handa, T. Imai, PGJIFs, new mitochondrial PGJ2 target proteins, regulate cell proliferation, J. Biosci. Med. 1 (2013) 11e15, https://doi.org/10.4236/jbm.2013.13003. [11] M. Umeda, T. Uebi, N. Maekawa, M. Isaki, Y. Miyama, Y. Masaike, H. Handa, T. Imai, Effective cofactor complex purification using nanobeads, J. Biosci. Med. 1 (2013) 5e10, https://doi.org/10.4236/jbm.2013.13003. [12] K.H. Kim, J.M. Sederstrom, Assaying cell cycle status using flow cytometry, Curr Protoc Mol Biol 28 (2015) 1e11, https://doi.org/10.1002/ 0471142727.mb2806s111. [13] B.R. Santmyire, V. Venkat, E. Beinder, C. Baylis, Impact of the estrus cycle and reduction in estrogen levels with aromatase inhibition, on renal function and nitric oxide activity in female rats, Steroids 75 (2010) 1011e1015. [14] P. Newhouse, K. Albert, Estrogen, stress, and depression: a neurocognitive model, JAMA Psychiatry 75 (2015) 727e729, https://doi.org/10.1001/ jamapsychiatry.2015.0487. [15] A. Milona, B.M. Owen, S. van Mil, D. Dormann, C. Mataki, M. Boudjelal, W. Cairns, K. Schoonjans, S. Milligan, M. Parker, R. White, C. Williamson, The normal mechanisms of pregnancy-induced liver growth are not maintained in mice lacking the bile acid sensor Fxr, Am. J. Physiol. Gastrointest. Liver Physiol. 298 (2010) G151eG158.