Identification of mitochondrial hormone receptors in avian muscle cells

Identification of mitochondrial hormone receptors in avian muscle cells Abstract The major objective of this study was to assess the expression of mitochondrial hormone receptors for progesterone (PR), estrogen (ER), glucocorticoid (GR), thyroid (TR), and insulin (IR) in avian muscle cells (quail muscle 7, QM7) and in breast muscle of quail and broilers. Visualization of receptor location in QM7 cells was accomplished by immunofluorescence. QM7 cells were stained with Mito Tracker Deep Red CMX, fixed in methanol, immune stained with anti-PR, -GR, -TR, -IR, and -ER primary antibodies overnight at 4°C, and visualized with Alexa Fluor 488-conjugated secondary antibody. After staining the nucleus with 4',6-diamidino-2-phenylindole, images were obtained by immunofluorescence microscopy. Merged images revealed the presence of all 5 hormone receptors on mitochondria in QM7 cells. Western blot analysis identified; (a) the β-isoform of the PR, (b) the α-isoform of GR, (c) the α−receptor of TR, (d) the β−subunit of IR, and (e) the α-isoform of the ER on mitochondria isolated from broiler breast muscle. Similar results were obtained in quail breast muscle mitochondria with the exception that the α-isoform of the GR was not detected. To our knowledge, this is the first report of hormone receptors (PR, TR, GR, IR, and ER) on mitochondria in avian cells. We hypothesize that these receptors could play important roles in regulating mitochondrial function in avian muscle cells. INTRODUCTION Steroid hormones, thyroid hormone, and insulin are well known to influence fundamental cellular and organismal processes such as metabolism, growth, and development. There is accumulating evidence in mammals that these hormones may act on mitochondria by 2 general mechanisms. The first is an indirect mechanism in which the hormone is conveyed to the nucleus, where it binds to hormone response elements followed by cell signaling that, among other cell processes, enhance mitochondrial protein transcription, mitochondrial biogenesis, and subsequently, mitochondrial function (see reviews by Wrutniak-Cabello et al., 2001; Chen et al., 2005; Psarra et al., 2006). In the second mechanism, the hormone binds directly to mitochondrial receptors to initiate effects on mitochondrial gene expression and function (e.g., Tsiriyotis et al., 1997; Psarra and Sekeris, 2008; Du et al., 2009; Dai et al., 2013; Feng et al., 2014). From global gene and global protein expression studies conducted in muscle tissue, we have evidence that several hormones or hormone receptors or both including progesterone, estrogen, thyroid hormone, corticosterone, and components of insulin signaling are associated with the phenotypic expression of feed efficiency in a Pedigree Male broiler line (Bottje et al., 2012; 2016; Kong et al., 2011, 2016, 2017). Progesterone receptor and 2 other transcription factors involved in progesterone signaling were identified as having a major impact on phenotypic expression of feed efficiency (Bottje et al., 2017). This latter finding is somewhat curious as these studies were conducted in immature male broiler breeders in which endogenous progesterone levels would not typically be considered as having major effects on cell function. Studies investigating progesterone receptors in mammalian mitochondria have shown that the hormone plays a role in controlling oxidative phosphorylation during periods of high metabolic activity or in metabolically active tissues (Dai et al., 2013; Price and Dai, 2015). The binding of hormones to mitochondrial receptors stimulates the activation of transcription factors and genes encoding components of the oxidative phosphorylation system, which increases mitochondrial biogenesis and the capacity for cellular respiration (Psarra et al., 2006). Progesterone and estrogen reduced mitochondrial reactive oxygen species production and oxidative damage in brain cells (Irwin et al., 2008). Pretreatment of animals with progesterone reduced the effects of traumatic brain injury by preserving mitochondrial function in nerve tissue (Robertson and Sarawati, 2015). Progesterone was also effective in maintaining mitochondrial function by initiating transcription when the hormone-receptor complex binds to hormone-response elements located on both nuclear and mitochondrial DNA (Psarra et al., 2006). Although mitochondrial hormone receptors have been identified in mammalian tissues, to our knowledge, there are no reports of avian mitochondrial hormone receptors. The presence of hormone receptors in avian mitochondria would give additional insight into how mitochondrial function is controlled and if this might be related to genetic traits such as feed efficiency. Therefore, the purpose of this study is to determine whether the mitochondrial hormone receptors for progesterone, estrogen, glucocorticoid, thyroid, and insulin are present in both an avian muscle cell line and intact avian muscle tissues. MATERIALS AND METHODS Animals and Breast Muscle Tissue Collection Breast muscle tissue used in this study was obtained from male Cobb-500 broilers and male Japanese quail. The animals used in this study were raised under standard conditions, with a standard diet and provided access to feed and water ad libitum. Birds were humanely euthanized, breast muscle tissue (Pectoralis major) was quickly excised and flash frozen in liquid nitrogen, and stored at −80°C. All procedures for animal care complied with the University of Arkansas Institutional Animal Care and Use Committee (IACUC Protocols #13,039 and #14,012). Cell Culture Quail muscle 7 (QM7) cells were grown in cell culture medium M199 (ThermoFisher, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Life Technologies), 10% tryptose phosphate broth (Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin (Life Technologies) at 37°C under a humidified atmosphere of 95% air and 5% CO2 as previously described (Lassiter et al., 2015). Separation of Nuclear, Cytoplasmic and Mitochondrial Components Nuclear and cytoplasmic proteins were extracted from QM7 cells and breast muscle using the Membrane, Nuclear, and Cytoplasmic Protein Extraction Kit (BSP002) (Bio Basic Inc., Ontario, Canada), according to the manufacturer's recommendations. Mitochondria were isolated from QM7 cells and broiler/quail breast muscle using Thermo Scientific Mitochondria Isolation Kits 89874 and 89801 (Pierce Biotechnology, Rockford, IL), respectively, according to the manufacturer's recommendations. Antibodies Immunofluorescence assessment of mitochondrial hormone receptors in QM7 cells utilized affinity purified rabbit polyclonal antibodies for antiprogesterone receptor (PR), antiestrogen receptor (ER), antiglucocorticoid receptor (GR), antithyroid hormone receptor (TR), and antiinsulin receptor (IR) used in this study were obtained from Santa Cruz Biotechnology (Dallas, TX). The anti-PR was raised against a peptide mapping at the C-terminus of PR of human origin, and the anti-GR was raised against a peptide mapping at the C-terminus of GRα of human origin. The anti-TR was raised against amino acids 1 to 408 that represents the full length TRα1 of chicken origin, yet this antibody is able to detect both α and β isoforms. The anti-IR was raised against amino acids 128 to 205 that represent the α-isoform of human origin. With the exception of the TR polyclonal antibody, initial studies revealed extensive nonspecific binding of polyclonal antibodies for the remaining hormone receptors in breast muscle tissue. Thus, monoclonal antibodies were used to detect the PR, IR, and GR hormone receptors in broiler and quail breast muscle by Western blot analysis. Protein G purified mouse monoclonal anti-estrogen receptor (ER) (Thermo Fisher Scientific, Rockford, IL) was raised against the C-terminus (aa 302 to 595) of human ER expressed in E. coli. Mouse monoclonal anti-IR and anti-GR (Santa Cruz Biotechnology, Dallas, TX) were raised against the C-terminus of IR of human origin and amino acids 121 to 420 of GR of human origin, respectively. The protein G purified mouse monoclonal anti-PR (Abcam Inc., Cambridge, MA) was raised against PR originating from chick oviduct cytosol. To verify the separation of mitochondria from cytosolic and nuclear compartments, western analysis was conducted on each cell fraction using polyclonal antibodies (Cell Signaling Technology Inc., Danvers MA) for: a) the 32 kDa voltage-dependent anion channel (VDAC) protein found exclusively in mitochondria (Columbini et al., 1996), b) the 75 kDa glucose regulated protein 75 (GRP-75), a protein found in the mitochondria and cytosol, but not in the nucleus (Mizzen et al., 1991; Mazkereth et al., 2016), c) glyceraldehyde -3-phosphate dehydrogenase (GAPDH) located primarily in the cytosol (Lodish et al., 2000), and d) the 110 kDa phosphoprotein nucleolin that is expressed in the nucleolus (Tutega and Tutega, 1999). The VDAC protein was detected using protein A/peptide affinity chromatography-purified polyclonal antibody for human VDAC (Cell Signaling Technology Inc., Danvers, MA). GRP-75 was detected with a human GRP-75 monoclonal antibody (Thermo Fisher Scientific, Waltham MA). GAPDH was detected using a polyclonal anti-GAPDH raised against amino acids 1 to 335 that represent full length GAPDH of human origin (Santa Cruz Biotechnology, Dallas, TX). Nucleolin was detected using a polyclonal anti-nucleolin raised against amino acids 271 to 520 of nucleolin of human origin (Santa Cruz Biotechnology, Dallas, TX). Immunofluorescence QM7 cells were grown to 50 to 60% confluence in chamber slides (Lab-Tek, Hatfield, PA) and stained with Mitotracker deep red CMX dye (dihydro-X-rosamine, ThermoFisher, Molecular Probes, Life Technologies, Grand Island, NY) according to manufacturer specifications. The MitoTracker dye passively diffuses across the cell membrane and accumulates in active mitochondria. The cells were then fixed in methanol for 10 min at −20°C with protein block serum-free blocking buffer (Dako, Carpinteria, CA). The cells were then incubated with rabbit anti-PR, anti-GR, anti-TR, anti-IR (1:200; Santa Cruz Biotechnology, Dallas, TX) or mouse anti-ER (Thermo Fisher Scientific, Rockford, IL) overnight at 4°C and visualized with Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Life Technologies). The slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and cover-slipped in Vectashield (Vector Laboratories, Burlingame, CA). Fluorescent images were obtained using a Zeiss Imager M2 microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY) with an attached CCD camera (Hamamatsu, Orca ER, Bridgewater, NJ), and Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD). Protein Expression QM7 cells and breast muscle tissue were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Pierce Biotechnology) containing Halt Protease and Phosphatase Inhibitor Cocktail (ethylenediaminetetraacetic acid (EDTA)-free, Pierce Biotechnology). Protein concentrations were determined using a Synergy HT multimode microplate reader (BioTek, Winooski, VT) and a Bradford assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin (BSA) as a standard. Proteins (ranging from 30 to 125 μg loaded on the gel) were separated on 10% Mini-PROTEAN TGX gels (Bio-Rad). The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked for 1 h at room temperature, and incubated with primary antibodies (dilutions ranging from 1:100 to 1:1,000) at 4°C overnight. Due to nonspecific binding observed with polyclonal antibodies, monoclonal antibodies (mouse anti-ER, anti-PR, anti-IR, and anti-GR) were used for western analysis. A prestained molecular weight marker (Precision Plus Protein Dual Color) was used as a standard (Bio-Rad). The PVDF membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000) (Cell Signaling Technology) for 1 h at room temperature. The signal was visualized by enhanced chemiluminescence (ECL plus) (GE Healthcare Bio-Sciences, Pittsburgh, PA) and captured by FluorChem M MultiFluor System (ProteinSimple, San Jose, CA). Image acquisition was performed using AlphaView software (ProteinSimple). Immunoprecipitation Immunoprecipitation was performed prior to the detection of the ER in order to reduce incidences of nonspecific binding by the primary antibody observed in pilot studies. The primary antibody was first conjugated to Dynabeads M-280 Tosylactivated magnetic beads using a DynaMag-2 magnet (Thermo Fisher Scientific, Rockford, IL) following the manufacturer's instructions. The antibody-coupled beads were mixed with the protein sample and incubated at 35°C for 30–40 min to capture the specific target protein. The bead–antibody–protein complex was resuspended in Laemmli sample buffer before proceeding directly to Western blotting. RESULTS Immunofluorescence Imaging Immunofluorescence imaging of QM7 cells is presented in Figure 1. In the third panel for each hormone, functional mitochondria were evident in the QM7 myoblast cells as indicated by their ability to import MitoTracker Red into the organelles. The yellow-orange color in the merged images in the fourth panel indicates that hormone receptors were colocalized on mitochondria of QM7 cells. Figure 1. View largeDownload slide Immunofluorescent staining of QM7 cells for visualization of mitochondrial receptors for; (A) Progesterone, (B) Glucocorticoid, (C) Thyroid, (D) Insulin and Estrogen. The nucleus was visualized with DAPI (blue), cytoplasm with Alexafluor (green), and mitochondria with Mitotracker deep red CMX (red). The merged images with orange to yellow coloring represent the presence of mitochondrially located hormone receptors. Figure 1. View largeDownload slide Immunofluorescent staining of QM7 cells for visualization of mitochondrial receptors for; (A) Progesterone, (B) Glucocorticoid, (C) Thyroid, (D) Insulin and Estrogen. The nucleus was visualized with DAPI (blue), cytoplasm with Alexafluor (green), and mitochondria with Mitotracker deep red CMX (red). The merged images with orange to yellow coloring represent the presence of mitochondrially located hormone receptors. Western Blot Analysis To verify that the mitochondrial fraction did not contain nuclear proteins, western analysis was conducted on each of the cell fractions for GRP-75, VDAC, and GAPDH (Figure 2). Clear separation of the mitochondrial from the nuclear fraction is shown in Figure 2A by the absence of binding to GRP-75 and VDAC1. GAPDH was primarily detected in the cytoplasmic fraction with a small amount present in the nuclear fraction and a faint detection in the mitochondrial fraction (Figure 2B). There was no band present in the mitochondrial fraction at the appropriate molecular weight for nucleolin (110 kDa), but there was considerable nonspecific binding in bands below 100 kDa (data not shown). Figure 2. View largeDownload slide Demonstration of cell fractionation procedures. Gels are shown for (A) Nucleolin (C23) (110 kDa) that was detected in nuclear fraction but not the mitochondrial fraction. (B) Voltage dependent activation channel (VDAC) that was detected in the mitochondrial fraction only, and (C) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—present primarily in the cytoplasmic fraction but not in the mitochondrial fraction. Figure 2. View largeDownload slide Demonstration of cell fractionation procedures. Gels are shown for (A) Nucleolin (C23) (110 kDa) that was detected in nuclear fraction but not the mitochondrial fraction. (B) Voltage dependent activation channel (VDAC) that was detected in the mitochondrial fraction only, and (C) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—present primarily in the cytoplasmic fraction but not in the mitochondrial fraction. The presence of a specific isoform, subunit, or receptor for each mitochondrial hormone receptor at the target molecular weight was determined for all 5 hormones examined in broilers (Figure 3) and for PR, TR, IR, and ER in quail (Figure 4). It is worth mentioning that the gel provided in the product description for the GR antibody provided by the company (Santa Cruz Biotechnology) indicated the presence of 2 bands between approximately 90 to 110 kDa and the gel was cutoff below 80 kDa. Thus, the second band shown in Figure 3b could either be the result of nonspecific binding or possibly the presence of a receptor protein fragment. No literature citation was provided for this gel, so we assume it was conducted by the company. A second band at ∼30 to 32 kDa in the ER blot (Figure 3e) was also present in the background information on the ER antibody obtained from Thermo Fisher. In quail muscle, several bands in addition to the β isoform band of PR was apparent (Figure 4a). A very gel image was presented in the background information for the Thermo Fisher PR antibody. Figure 3. View largeDownload slide Hormone receptor expression in mitochondria isolated from breast muscle obtained from broiler for; (A) progesterone, (B) glucocorticoid, (C) thyroid hormone, (D) insulin, and (E) estrogen. The amount of protein loaded onto the gel for each lane was 75 μg. Figure 3. View largeDownload slide Hormone receptor expression in mitochondria isolated from breast muscle obtained from broiler for; (A) progesterone, (B) glucocorticoid, (C) thyroid hormone, (D) insulin, and (E) estrogen. The amount of protein loaded onto the gel for each lane was 75 μg. Figure 4. View largeDownload slide Hormone receptor expression in mitochondria isolated from breast muscle obtained from Japanese Quail for; (A) progesterone, (B) glucocorticoid, (C) thyroid hormone, (D) insulin, and (E) estrogen. The amount of protein loaded onto the gel for each lane was 75 μg. Figure 4. View largeDownload slide Hormone receptor expression in mitochondria isolated from breast muscle obtained from Japanese Quail for; (A) progesterone, (B) glucocorticoid, (C) thyroid hormone, (D) insulin, and (E) estrogen. The amount of protein loaded onto the gel for each lane was 75 μg. DISCUSSION The economic impact of feed efficiency has been illustrated in production animal agriculture (e.g., Emmerson 1997; Robinson and Oddy, 2004; Patience et al., 2015). Commercial broilers have been highly selected for growth and feed efficiency when compared to previous, less intensely selected lines of birds (Zuidhof et al., 2014). Progesterone was reported to play a role in the phenotypic expression of feed efficiency in pedigree male broilers (Bottje et al., 2017). Progesterone was also predicted to be activated in the high feed efficiency pedigree male broiler phenotype based on downstream target protein expression (Kong et al., 2016). As mammalian mitochondria receptors for many hormones including progesterone (Tsiriyotis et al., 1997; Psarra, et al. 2008; Du et al., 2009; Dai et al., 2013; Feng et al., 2014), we initiated studies to determine whether mitochondrial hormone receptors for progesterone, estrogen, glucocorticoid, thyroid hormone, and insulin were present in avian muscle cells in vitro and in vivo. To our knowledge, this is the first time that mitochondrial hormone receptors have been reported in any avian tissue or species. The binding of progesterone to its receptor is typically associated with the physiological role in the reproductive function of females; i.e., development of the mammary glands and uterus, ovarian function, and sexual behavior (Connelly and Lydon, 2000). The progesterone–receptor complex functions as a transcription factor through its binding to hormone response elements in the nucleus, and the activation of Src signaling pathways in the cytoplasm that promote cell proliferation, cell differentiation, and mitochondrial function (Edwards et al., 2002). Using rat brain mitochondria, Irwin et al. (2008) reported that progesterone-treated mitochondria exhibited increased cytochrome c oxidase activity, reduced oxidative damage, and enhanced antioxidant protection. Dai et al. (2013) demonstrated that treating cells with progestin increased cellular respiration and mitochondrial membrane potential. Therefore, the action of progesterone signaling appears to be beneficial to mitochondrial function, and supports findings in recent proteogenomic studies (Kong et al., 2016; Bottje et al., 2017). The glucocorticoid receptor is a part of the superfamily of nuclear receptors (Psarra et al., 2006). The presence of glucocorticoid receptors in the mitochondria of mammalian cells has been clearly shown using immunofluorescence and immunogold electron microscopy (Scheller et al., 2000). There have been a number of reports illustrating how the presence of glucocorticoid receptors affects mitochondrial activity and the regulation of energy production. When HepG2 cells were stably transfected to overexpress mitochondrial-targeted glucocorticoid receptors, these cells showed an increase in RNA synthesis, expression of the mitochondrial protein cytochrome oxidase subunit I, and mitochondrial ATP production (Psarra and Sekeris, 2011). Treating rat skeletal muscle and C2C12 muscle cells with dexamethasone, a synthetic glucocorticoid, was shown to stimulate mitochondrial biogenesis (Weber et al., 2002). In a recent study, Morgan et al. (2016) suggested that a specific isoform of the glucocorticoid receptor (GRγ) is specialized in regulating mitochondrial function, causing an increase in oxygen consumption, ATP production, and mitochondrial mass. Steroid receptors are translocated to the mitochondria following hormonal stimulation (Psarra et al., 2006). Translocation of the glucocorticoid receptor to the mitochondria is thought to occur by its interaction with chaperone proteins such as HSP70 (Du et al., 2009). Therefore, mitochondrial glucocorticoid receptors in avian muscle cells may play a role in regulating mitochondrial function and energy production. Thyroid hormone plays a significant role in energy metabolism. This occurs when the hormone response element is activated by binding of the ligand–receptor complex, affecting transcription of both nuclear and mitochondrial genes that encode for subunits of the mitochondrial oxidative phosphorylation system (Scheller et al., 2003; Psarra and Sekeris, 2008). Due to this ability to interact with DNA-binding sites that modulate gene and protein expression, the thyroid hormone receptor functions as a ligand-activated transcription factor (Psarra et al., 2006). Several isoforms of the thyroid hormone receptor are present in mammalian cells. The variation in number and position of amino acids in these isoforms can determine whether ligand binding promotes or inhibits transcription (Bassett et al., 2003). Two truncated thyroid receptor α isoforms (p28 and p43) that are specifically targeted to the mitochondria increase mitochondrial gene expression, oxidative phosphorylation, and thermogenesis (Bassett et al., 2003). The p43 isoform is of particular interest as it is located in the mitochondrial matrix, and therefore in direct contact with the hormone response elements of mitochondrial DNA (Wrutniak et al., 1995). Interestingly, studies in which QM7 cells were transfected to overexpress p43 showed that mitochondrial activity was stimulated by this receptor isoform (Casas et al., 1999; Rochard et al., 2000). Based on the molecular weight of the thyroid receptor detected in our study that corresponds to the β isoform, it is not likely to be one of these mitochondria-specific isoforms. However, it is possible that this β isoform is associated with nongenomic actions linked to secondary messenger signaling pathways (i.e., protein kinase and Ca2+ pathways) to elicit a hormone response (Losel and Wehling, 2003). In the present study, immunofluorescence microscopy demonstrated that the insulin receptor colocalized with mitochondria in QM7 cells (Figure 1D), and that the β-subunit precursor was detected in the mitochondrial fraction of broiler (Figure 3D) and quail breast muscle (Figure 4C). However, a search of the literature does not indicate that the insulin receptor is located on the mitochondrial membrane. A relationship between mitochondrial function and insulin resistance has been reported (Montgomery and Turner, 2015). In neurons, signaling between the insulin receptor and mitochondria occurs through the generation of reactive oxygen species in the mitochondria, primarily via succinate oxidation (Pomytkin, 2012). The insulin receptor belongs to the family of receptor tyrosine kinases, where ligand binding stimulates a phosphorylation cascade that helps regulate cell metabolism, proliferation, and differentiation. Recent studies have shown that certain members of the receptor tyrosine kinase family translocate to the mitochondria where the phosphorylation of mitochondrial proteins regulates metabolism and function (Ding et al., 2012; Salvi 2013). In the review by Lemmon and Schlessinger (2010), the extracellular structural domain of the human insulin receptor is very similar to that of the epidermal growth factor receptor which another receptor tyrosine kinase member that is known to translocate to the mitochondria (Salvi et al., 2005). The extracellular domains of both the epidermal growth factor receptor and insulin receptor subfamilies contain large amounts of leucine and cysteine regions (Lemmon and Schlessinger, 2010). In summary, the results of this study indicate that the avian muscle cells and breast muscle tissue express mitochondrial hormone receptors for progesterone, glucocorticoid, thyroid hormone, and insulin. 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This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Identification of mitochondrial hormone receptors in avian muscle cells

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey126
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Abstract

Abstract The major objective of this study was to assess the expression of mitochondrial hormone receptors for progesterone (PR), estrogen (ER), glucocorticoid (GR), thyroid (TR), and insulin (IR) in avian muscle cells (quail muscle 7, QM7) and in breast muscle of quail and broilers. Visualization of receptor location in QM7 cells was accomplished by immunofluorescence. QM7 cells were stained with Mito Tracker Deep Red CMX, fixed in methanol, immune stained with anti-PR, -GR, -TR, -IR, and -ER primary antibodies overnight at 4°C, and visualized with Alexa Fluor 488-conjugated secondary antibody. After staining the nucleus with 4',6-diamidino-2-phenylindole, images were obtained by immunofluorescence microscopy. Merged images revealed the presence of all 5 hormone receptors on mitochondria in QM7 cells. Western blot analysis identified; (a) the β-isoform of the PR, (b) the α-isoform of GR, (c) the α−receptor of TR, (d) the β−subunit of IR, and (e) the α-isoform of the ER on mitochondria isolated from broiler breast muscle. Similar results were obtained in quail breast muscle mitochondria with the exception that the α-isoform of the GR was not detected. To our knowledge, this is the first report of hormone receptors (PR, TR, GR, IR, and ER) on mitochondria in avian cells. We hypothesize that these receptors could play important roles in regulating mitochondrial function in avian muscle cells. INTRODUCTION Steroid hormones, thyroid hormone, and insulin are well known to influence fundamental cellular and organismal processes such as metabolism, growth, and development. There is accumulating evidence in mammals that these hormones may act on mitochondria by 2 general mechanisms. The first is an indirect mechanism in which the hormone is conveyed to the nucleus, where it binds to hormone response elements followed by cell signaling that, among other cell processes, enhance mitochondrial protein transcription, mitochondrial biogenesis, and subsequently, mitochondrial function (see reviews by Wrutniak-Cabello et al., 2001; Chen et al., 2005; Psarra et al., 2006). In the second mechanism, the hormone binds directly to mitochondrial receptors to initiate effects on mitochondrial gene expression and function (e.g., Tsiriyotis et al., 1997; Psarra and Sekeris, 2008; Du et al., 2009; Dai et al., 2013; Feng et al., 2014). From global gene and global protein expression studies conducted in muscle tissue, we have evidence that several hormones or hormone receptors or both including progesterone, estrogen, thyroid hormone, corticosterone, and components of insulin signaling are associated with the phenotypic expression of feed efficiency in a Pedigree Male broiler line (Bottje et al., 2012; 2016; Kong et al., 2011, 2016, 2017). Progesterone receptor and 2 other transcription factors involved in progesterone signaling were identified as having a major impact on phenotypic expression of feed efficiency (Bottje et al., 2017). This latter finding is somewhat curious as these studies were conducted in immature male broiler breeders in which endogenous progesterone levels would not typically be considered as having major effects on cell function. Studies investigating progesterone receptors in mammalian mitochondria have shown that the hormone plays a role in controlling oxidative phosphorylation during periods of high metabolic activity or in metabolically active tissues (Dai et al., 2013; Price and Dai, 2015). The binding of hormones to mitochondrial receptors stimulates the activation of transcription factors and genes encoding components of the oxidative phosphorylation system, which increases mitochondrial biogenesis and the capacity for cellular respiration (Psarra et al., 2006). Progesterone and estrogen reduced mitochondrial reactive oxygen species production and oxidative damage in brain cells (Irwin et al., 2008). Pretreatment of animals with progesterone reduced the effects of traumatic brain injury by preserving mitochondrial function in nerve tissue (Robertson and Sarawati, 2015). Progesterone was also effective in maintaining mitochondrial function by initiating transcription when the hormone-receptor complex binds to hormone-response elements located on both nuclear and mitochondrial DNA (Psarra et al., 2006). Although mitochondrial hormone receptors have been identified in mammalian tissues, to our knowledge, there are no reports of avian mitochondrial hormone receptors. The presence of hormone receptors in avian mitochondria would give additional insight into how mitochondrial function is controlled and if this might be related to genetic traits such as feed efficiency. Therefore, the purpose of this study is to determine whether the mitochondrial hormone receptors for progesterone, estrogen, glucocorticoid, thyroid, and insulin are present in both an avian muscle cell line and intact avian muscle tissues. MATERIALS AND METHODS Animals and Breast Muscle Tissue Collection Breast muscle tissue used in this study was obtained from male Cobb-500 broilers and male Japanese quail. The animals used in this study were raised under standard conditions, with a standard diet and provided access to feed and water ad libitum. Birds were humanely euthanized, breast muscle tissue (Pectoralis major) was quickly excised and flash frozen in liquid nitrogen, and stored at −80°C. All procedures for animal care complied with the University of Arkansas Institutional Animal Care and Use Committee (IACUC Protocols #13,039 and #14,012). Cell Culture Quail muscle 7 (QM7) cells were grown in cell culture medium M199 (ThermoFisher, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Life Technologies), 10% tryptose phosphate broth (Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin (Life Technologies) at 37°C under a humidified atmosphere of 95% air and 5% CO2 as previously described (Lassiter et al., 2015). Separation of Nuclear, Cytoplasmic and Mitochondrial Components Nuclear and cytoplasmic proteins were extracted from QM7 cells and breast muscle using the Membrane, Nuclear, and Cytoplasmic Protein Extraction Kit (BSP002) (Bio Basic Inc., Ontario, Canada), according to the manufacturer's recommendations. Mitochondria were isolated from QM7 cells and broiler/quail breast muscle using Thermo Scientific Mitochondria Isolation Kits 89874 and 89801 (Pierce Biotechnology, Rockford, IL), respectively, according to the manufacturer's recommendations. Antibodies Immunofluorescence assessment of mitochondrial hormone receptors in QM7 cells utilized affinity purified rabbit polyclonal antibodies for antiprogesterone receptor (PR), antiestrogen receptor (ER), antiglucocorticoid receptor (GR), antithyroid hormone receptor (TR), and antiinsulin receptor (IR) used in this study were obtained from Santa Cruz Biotechnology (Dallas, TX). The anti-PR was raised against a peptide mapping at the C-terminus of PR of human origin, and the anti-GR was raised against a peptide mapping at the C-terminus of GRα of human origin. The anti-TR was raised against amino acids 1 to 408 that represents the full length TRα1 of chicken origin, yet this antibody is able to detect both α and β isoforms. The anti-IR was raised against amino acids 128 to 205 that represent the α-isoform of human origin. With the exception of the TR polyclonal antibody, initial studies revealed extensive nonspecific binding of polyclonal antibodies for the remaining hormone receptors in breast muscle tissue. Thus, monoclonal antibodies were used to detect the PR, IR, and GR hormone receptors in broiler and quail breast muscle by Western blot analysis. Protein G purified mouse monoclonal anti-estrogen receptor (ER) (Thermo Fisher Scientific, Rockford, IL) was raised against the C-terminus (aa 302 to 595) of human ER expressed in E. coli. Mouse monoclonal anti-IR and anti-GR (Santa Cruz Biotechnology, Dallas, TX) were raised against the C-terminus of IR of human origin and amino acids 121 to 420 of GR of human origin, respectively. The protein G purified mouse monoclonal anti-PR (Abcam Inc., Cambridge, MA) was raised against PR originating from chick oviduct cytosol. To verify the separation of mitochondria from cytosolic and nuclear compartments, western analysis was conducted on each cell fraction using polyclonal antibodies (Cell Signaling Technology Inc., Danvers MA) for: a) the 32 kDa voltage-dependent anion channel (VDAC) protein found exclusively in mitochondria (Columbini et al., 1996), b) the 75 kDa glucose regulated protein 75 (GRP-75), a protein found in the mitochondria and cytosol, but not in the nucleus (Mizzen et al., 1991; Mazkereth et al., 2016), c) glyceraldehyde -3-phosphate dehydrogenase (GAPDH) located primarily in the cytosol (Lodish et al., 2000), and d) the 110 kDa phosphoprotein nucleolin that is expressed in the nucleolus (Tutega and Tutega, 1999). The VDAC protein was detected using protein A/peptide affinity chromatography-purified polyclonal antibody for human VDAC (Cell Signaling Technology Inc., Danvers, MA). GRP-75 was detected with a human GRP-75 monoclonal antibody (Thermo Fisher Scientific, Waltham MA). GAPDH was detected using a polyclonal anti-GAPDH raised against amino acids 1 to 335 that represent full length GAPDH of human origin (Santa Cruz Biotechnology, Dallas, TX). Nucleolin was detected using a polyclonal anti-nucleolin raised against amino acids 271 to 520 of nucleolin of human origin (Santa Cruz Biotechnology, Dallas, TX). Immunofluorescence QM7 cells were grown to 50 to 60% confluence in chamber slides (Lab-Tek, Hatfield, PA) and stained with Mitotracker deep red CMX dye (dihydro-X-rosamine, ThermoFisher, Molecular Probes, Life Technologies, Grand Island, NY) according to manufacturer specifications. The MitoTracker dye passively diffuses across the cell membrane and accumulates in active mitochondria. The cells were then fixed in methanol for 10 min at −20°C with protein block serum-free blocking buffer (Dako, Carpinteria, CA). The cells were then incubated with rabbit anti-PR, anti-GR, anti-TR, anti-IR (1:200; Santa Cruz Biotechnology, Dallas, TX) or mouse anti-ER (Thermo Fisher Scientific, Rockford, IL) overnight at 4°C and visualized with Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Life Technologies). The slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and cover-slipped in Vectashield (Vector Laboratories, Burlingame, CA). Fluorescent images were obtained using a Zeiss Imager M2 microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY) with an attached CCD camera (Hamamatsu, Orca ER, Bridgewater, NJ), and Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD). Protein Expression QM7 cells and breast muscle tissue were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Pierce Biotechnology) containing Halt Protease and Phosphatase Inhibitor Cocktail (ethylenediaminetetraacetic acid (EDTA)-free, Pierce Biotechnology). Protein concentrations were determined using a Synergy HT multimode microplate reader (BioTek, Winooski, VT) and a Bradford assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin (BSA) as a standard. Proteins (ranging from 30 to 125 μg loaded on the gel) were separated on 10% Mini-PROTEAN TGX gels (Bio-Rad). The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked for 1 h at room temperature, and incubated with primary antibodies (dilutions ranging from 1:100 to 1:1,000) at 4°C overnight. Due to nonspecific binding observed with polyclonal antibodies, monoclonal antibodies (mouse anti-ER, anti-PR, anti-IR, and anti-GR) were used for western analysis. A prestained molecular weight marker (Precision Plus Protein Dual Color) was used as a standard (Bio-Rad). The PVDF membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000) (Cell Signaling Technology) for 1 h at room temperature. The signal was visualized by enhanced chemiluminescence (ECL plus) (GE Healthcare Bio-Sciences, Pittsburgh, PA) and captured by FluorChem M MultiFluor System (ProteinSimple, San Jose, CA). Image acquisition was performed using AlphaView software (ProteinSimple). Immunoprecipitation Immunoprecipitation was performed prior to the detection of the ER in order to reduce incidences of nonspecific binding by the primary antibody observed in pilot studies. The primary antibody was first conjugated to Dynabeads M-280 Tosylactivated magnetic beads using a DynaMag-2 magnet (Thermo Fisher Scientific, Rockford, IL) following the manufacturer's instructions. The antibody-coupled beads were mixed with the protein sample and incubated at 35°C for 30–40 min to capture the specific target protein. The bead–antibody–protein complex was resuspended in Laemmli sample buffer before proceeding directly to Western blotting. RESULTS Immunofluorescence Imaging Immunofluorescence imaging of QM7 cells is presented in Figure 1. In the third panel for each hormone, functional mitochondria were evident in the QM7 myoblast cells as indicated by their ability to import MitoTracker Red into the organelles. The yellow-orange color in the merged images in the fourth panel indicates that hormone receptors were colocalized on mitochondria of QM7 cells. Figure 1. View largeDownload slide Immunofluorescent staining of QM7 cells for visualization of mitochondrial receptors for; (A) Progesterone, (B) Glucocorticoid, (C) Thyroid, (D) Insulin and Estrogen. The nucleus was visualized with DAPI (blue), cytoplasm with Alexafluor (green), and mitochondria with Mitotracker deep red CMX (red). The merged images with orange to yellow coloring represent the presence of mitochondrially located hormone receptors. Figure 1. View largeDownload slide Immunofluorescent staining of QM7 cells for visualization of mitochondrial receptors for; (A) Progesterone, (B) Glucocorticoid, (C) Thyroid, (D) Insulin and Estrogen. The nucleus was visualized with DAPI (blue), cytoplasm with Alexafluor (green), and mitochondria with Mitotracker deep red CMX (red). The merged images with orange to yellow coloring represent the presence of mitochondrially located hormone receptors. Western Blot Analysis To verify that the mitochondrial fraction did not contain nuclear proteins, western analysis was conducted on each of the cell fractions for GRP-75, VDAC, and GAPDH (Figure 2). Clear separation of the mitochondrial from the nuclear fraction is shown in Figure 2A by the absence of binding to GRP-75 and VDAC1. GAPDH was primarily detected in the cytoplasmic fraction with a small amount present in the nuclear fraction and a faint detection in the mitochondrial fraction (Figure 2B). There was no band present in the mitochondrial fraction at the appropriate molecular weight for nucleolin (110 kDa), but there was considerable nonspecific binding in bands below 100 kDa (data not shown). Figure 2. View largeDownload slide Demonstration of cell fractionation procedures. Gels are shown for (A) Nucleolin (C23) (110 kDa) that was detected in nuclear fraction but not the mitochondrial fraction. (B) Voltage dependent activation channel (VDAC) that was detected in the mitochondrial fraction only, and (C) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—present primarily in the cytoplasmic fraction but not in the mitochondrial fraction. Figure 2. View largeDownload slide Demonstration of cell fractionation procedures. Gels are shown for (A) Nucleolin (C23) (110 kDa) that was detected in nuclear fraction but not the mitochondrial fraction. (B) Voltage dependent activation channel (VDAC) that was detected in the mitochondrial fraction only, and (C) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—present primarily in the cytoplasmic fraction but not in the mitochondrial fraction. The presence of a specific isoform, subunit, or receptor for each mitochondrial hormone receptor at the target molecular weight was determined for all 5 hormones examined in broilers (Figure 3) and for PR, TR, IR, and ER in quail (Figure 4). It is worth mentioning that the gel provided in the product description for the GR antibody provided by the company (Santa Cruz Biotechnology) indicated the presence of 2 bands between approximately 90 to 110 kDa and the gel was cutoff below 80 kDa. Thus, the second band shown in Figure 3b could either be the result of nonspecific binding or possibly the presence of a receptor protein fragment. No literature citation was provided for this gel, so we assume it was conducted by the company. A second band at ∼30 to 32 kDa in the ER blot (Figure 3e) was also present in the background information on the ER antibody obtained from Thermo Fisher. In quail muscle, several bands in addition to the β isoform band of PR was apparent (Figure 4a). A very gel image was presented in the background information for the Thermo Fisher PR antibody. Figure 3. View largeDownload slide Hormone receptor expression in mitochondria isolated from breast muscle obtained from broiler for; (A) progesterone, (B) glucocorticoid, (C) thyroid hormone, (D) insulin, and (E) estrogen. The amount of protein loaded onto the gel for each lane was 75 μg. Figure 3. View largeDownload slide Hormone receptor expression in mitochondria isolated from breast muscle obtained from broiler for; (A) progesterone, (B) glucocorticoid, (C) thyroid hormone, (D) insulin, and (E) estrogen. The amount of protein loaded onto the gel for each lane was 75 μg. Figure 4. View largeDownload slide Hormone receptor expression in mitochondria isolated from breast muscle obtained from Japanese Quail for; (A) progesterone, (B) glucocorticoid, (C) thyroid hormone, (D) insulin, and (E) estrogen. The amount of protein loaded onto the gel for each lane was 75 μg. Figure 4. View largeDownload slide Hormone receptor expression in mitochondria isolated from breast muscle obtained from Japanese Quail for; (A) progesterone, (B) glucocorticoid, (C) thyroid hormone, (D) insulin, and (E) estrogen. The amount of protein loaded onto the gel for each lane was 75 μg. DISCUSSION The economic impact of feed efficiency has been illustrated in production animal agriculture (e.g., Emmerson 1997; Robinson and Oddy, 2004; Patience et al., 2015). Commercial broilers have been highly selected for growth and feed efficiency when compared to previous, less intensely selected lines of birds (Zuidhof et al., 2014). Progesterone was reported to play a role in the phenotypic expression of feed efficiency in pedigree male broilers (Bottje et al., 2017). Progesterone was also predicted to be activated in the high feed efficiency pedigree male broiler phenotype based on downstream target protein expression (Kong et al., 2016). As mammalian mitochondria receptors for many hormones including progesterone (Tsiriyotis et al., 1997; Psarra, et al. 2008; Du et al., 2009; Dai et al., 2013; Feng et al., 2014), we initiated studies to determine whether mitochondrial hormone receptors for progesterone, estrogen, glucocorticoid, thyroid hormone, and insulin were present in avian muscle cells in vitro and in vivo. To our knowledge, this is the first time that mitochondrial hormone receptors have been reported in any avian tissue or species. The binding of progesterone to its receptor is typically associated with the physiological role in the reproductive function of females; i.e., development of the mammary glands and uterus, ovarian function, and sexual behavior (Connelly and Lydon, 2000). The progesterone–receptor complex functions as a transcription factor through its binding to hormone response elements in the nucleus, and the activation of Src signaling pathways in the cytoplasm that promote cell proliferation, cell differentiation, and mitochondrial function (Edwards et al., 2002). Using rat brain mitochondria, Irwin et al. (2008) reported that progesterone-treated mitochondria exhibited increased cytochrome c oxidase activity, reduced oxidative damage, and enhanced antioxidant protection. Dai et al. (2013) demonstrated that treating cells with progestin increased cellular respiration and mitochondrial membrane potential. Therefore, the action of progesterone signaling appears to be beneficial to mitochondrial function, and supports findings in recent proteogenomic studies (Kong et al., 2016; Bottje et al., 2017). The glucocorticoid receptor is a part of the superfamily of nuclear receptors (Psarra et al., 2006). The presence of glucocorticoid receptors in the mitochondria of mammalian cells has been clearly shown using immunofluorescence and immunogold electron microscopy (Scheller et al., 2000). There have been a number of reports illustrating how the presence of glucocorticoid receptors affects mitochondrial activity and the regulation of energy production. When HepG2 cells were stably transfected to overexpress mitochondrial-targeted glucocorticoid receptors, these cells showed an increase in RNA synthesis, expression of the mitochondrial protein cytochrome oxidase subunit I, and mitochondrial ATP production (Psarra and Sekeris, 2011). Treating rat skeletal muscle and C2C12 muscle cells with dexamethasone, a synthetic glucocorticoid, was shown to stimulate mitochondrial biogenesis (Weber et al., 2002). In a recent study, Morgan et al. (2016) suggested that a specific isoform of the glucocorticoid receptor (GRγ) is specialized in regulating mitochondrial function, causing an increase in oxygen consumption, ATP production, and mitochondrial mass. Steroid receptors are translocated to the mitochondria following hormonal stimulation (Psarra et al., 2006). Translocation of the glucocorticoid receptor to the mitochondria is thought to occur by its interaction with chaperone proteins such as HSP70 (Du et al., 2009). Therefore, mitochondrial glucocorticoid receptors in avian muscle cells may play a role in regulating mitochondrial function and energy production. Thyroid hormone plays a significant role in energy metabolism. This occurs when the hormone response element is activated by binding of the ligand–receptor complex, affecting transcription of both nuclear and mitochondrial genes that encode for subunits of the mitochondrial oxidative phosphorylation system (Scheller et al., 2003; Psarra and Sekeris, 2008). Due to this ability to interact with DNA-binding sites that modulate gene and protein expression, the thyroid hormone receptor functions as a ligand-activated transcription factor (Psarra et al., 2006). Several isoforms of the thyroid hormone receptor are present in mammalian cells. The variation in number and position of amino acids in these isoforms can determine whether ligand binding promotes or inhibits transcription (Bassett et al., 2003). Two truncated thyroid receptor α isoforms (p28 and p43) that are specifically targeted to the mitochondria increase mitochondrial gene expression, oxidative phosphorylation, and thermogenesis (Bassett et al., 2003). The p43 isoform is of particular interest as it is located in the mitochondrial matrix, and therefore in direct contact with the hormone response elements of mitochondrial DNA (Wrutniak et al., 1995). Interestingly, studies in which QM7 cells were transfected to overexpress p43 showed that mitochondrial activity was stimulated by this receptor isoform (Casas et al., 1999; Rochard et al., 2000). Based on the molecular weight of the thyroid receptor detected in our study that corresponds to the β isoform, it is not likely to be one of these mitochondria-specific isoforms. However, it is possible that this β isoform is associated with nongenomic actions linked to secondary messenger signaling pathways (i.e., protein kinase and Ca2+ pathways) to elicit a hormone response (Losel and Wehling, 2003). In the present study, immunofluorescence microscopy demonstrated that the insulin receptor colocalized with mitochondria in QM7 cells (Figure 1D), and that the β-subunit precursor was detected in the mitochondrial fraction of broiler (Figure 3D) and quail breast muscle (Figure 4C). However, a search of the literature does not indicate that the insulin receptor is located on the mitochondrial membrane. A relationship between mitochondrial function and insulin resistance has been reported (Montgomery and Turner, 2015). In neurons, signaling between the insulin receptor and mitochondria occurs through the generation of reactive oxygen species in the mitochondria, primarily via succinate oxidation (Pomytkin, 2012). The insulin receptor belongs to the family of receptor tyrosine kinases, where ligand binding stimulates a phosphorylation cascade that helps regulate cell metabolism, proliferation, and differentiation. Recent studies have shown that certain members of the receptor tyrosine kinase family translocate to the mitochondria where the phosphorylation of mitochondrial proteins regulates metabolism and function (Ding et al., 2012; Salvi 2013). In the review by Lemmon and Schlessinger (2010), the extracellular structural domain of the human insulin receptor is very similar to that of the epidermal growth factor receptor which another receptor tyrosine kinase member that is known to translocate to the mitochondria (Salvi et al., 2005). The extracellular domains of both the epidermal growth factor receptor and insulin receptor subfamilies contain large amounts of leucine and cysteine regions (Lemmon and Schlessinger, 2010). In summary, the results of this study indicate that the avian muscle cells and breast muscle tissue express mitochondrial hormone receptors for progesterone, glucocorticoid, thyroid hormone, and insulin. We hypothesize that the steroid hormones investigated in the current study could have direct effects on mitochondrial function in avian muscle through binding to specific mitochondrial hormone receptors and may play fundamentally important roles in avian physiology and genetic phenotypes. Acknowledgements This research was supported through funding by USDA-NIFA (USDA-NIFA #2013-01953), Arkansas Biosciences Institute (Little Rock, AR), and by the Director of the Arkansas Agriculture Experiment, Division of Agriculture, University of Arkansas. REFERENCES Bassett J. H., Harvey C. B., Williams G. R.. 2003. Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol. Cell Endocrinol.  213: 1– 11. Google Scholar CrossRef Search ADS PubMed  Bottje W., Kong B-W., Reverter A., Waarendenberg A. J., Hudson N. J.. 2017. Progesterone signalling in broiler skeletal muscle is associated with divergent feed efficiency. BMC Syst. Biol.  11: 29. 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Poultry ScienceOxford University Press

Published: May 10, 2018

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