Regulation of JAK2 protein expression by chronic, pulsatile GH administration in vivo: A possible mechanism for ligand enhancement of signal transduction

General and Comparative Endocrinology 144 (2005) 128–139

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Regulation of JAK2 protein expression by chronic, pulsatile GH administration in vivo: A possible mechanism for ligand

enhancement of signal transduction

Yuan Zhou a, Xiaohong Wang a, Jill Hadley a, Seth J. Corey b, Regina Vasilatos-Younken a,¤

a Department of Poultry Science, The Pennsylvania State University, University Park, PA 16802, USAb Department of Pediatrics, U.T.-M.D. Anderson Cancer Center, Houston, TX 77030, USA

Received 9 June 2004; revised 28 April 2005; accepted 2 May 2005Available online 1 July 2005

Abstract

Growth hormone (GH) is a key factor controlling postnatal growth and development. Despite growth-promoting eVects in mam-mals, GH is not associated with muscle growth in the chicken. Janus kinase 2 (JAK2) has been identiWed as the Wrst intracellular stepin GH receptor (GHR) signaling in many species, however, there is limited knowledge regarding the GH signaling pathway in thechicken. In this study, GH-responsive, JAK2 immunoreactive proteins were Wrst assessed in an avian hepatoma cell line (LMH).Tyrosine phosphorylation of a 120–122 kDa JAK2 immunoreactive protein was GH dose-dependent. In addition to in vitro studies,the timecourse of JAK2 activation in liver and skeletal muscle (Pectoralis superWcialis) in response to a single intravenous (i.v.) injec-tion of chicken GH (cGH), and the eVect of chronic exposure to GH in a physiologically relevant pattern on JAK2 protein expres-sion and tyrosine phosphorylation in vivo were assessed. At a dose of GH that was previously demonstrated to elicit a maximalmetabolic response (6.25 �g/kg BW), maximum tyrosine phosphorylation of JAK2 appeared at 10 min post-GH administration inthe pectoralis muscle, but was not detectable in liver. To assess whether chronic enhancement of GH would alter expression of JAK2,we utilized a dynamic model of pulsatile GH infusion that mimicked the early pattern of circulating GH expressed in younger, rap-idly growing birds (high amplitude peaks with an inter-peak interval of 90 min). A 120–122 kDa protein in liver and muscle, and adominant 130–136 kDa protein in the muscle, that was phosphorylated in response to GH, were speciWcally recognized by the JAK2antibody. Chronic, pulsatile infusion of cGH into 8-week-old chickens was associated with increased abundance and tyrosine phos-phorylation of JAK2 protein in both liver and muscle (P < 0.05), which were GH dose-dependent, and mirrored previously reportedbiological responses for the same birds [Vasilatos-Younken, R., Zhou, Y., Wang, X., McMurtry, J.P., Rosebrough, R.W., Decuypere,E., Buys, N., Darras, V.M., Van Der Geyten, S., Tomas, F., 2000. Altered chicken thyroid hormone metabolism with chronic GHenhancement in vivo: Consequences for skeletal muscle growth. Journal of Endocrinology 166, 609–620.]. In summary (1) JAK2immunoreactive proteins that associate with the GHR and are tyrosine phosphorylated in response to GH were identiWed in anavian hepatoma cell line and expressed in both GH responsive (liver) and “non-responsive” (skeletal muscle) tissues; (2) tyrosinephosphorylation of JAK2 occurred within minutes of exposure to a single i.v. injection of GH in vivo in muscle but not liver of 8-week-old birds; and 3) there were GH dose-dependent increases in abundance of JAK2 protein and tyrosine phosphorylation in bothtissues when chronically exposed to GH in a physiologically relevant pattern, that mirrored dose-dependent biological responses,including alterations in the pathway of thyroid hormone metabolism, previously reported. Enhanced JAK2 suggests one possiblemechanism whereby chronic, physiologically appropriate exposure to the ligand enhances GH biological action via increasedabundance of a key upstream component of the signal transduction pathway. 2005 Elsevier Inc. All rights reserved.

0016-6480/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2005.05.001

* Corresponding author. Fax: +1 814 863 4627.E-mail address: [email protected] (R. Vasilatos-Younken).

mailto: [email protected]

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Y. Zhou et al. / General and Comparative Endocrinology 144 (2005) 128–139 129

Keywords: JAK2; GH; Muscle; Liver; Tyrosine phosphorylation; Protein expression

1. Introduction there are speciWc tissue diVerences in the nature of this

Growth hormone (GH) has long been recognized asone of the principle factors that control postnatalgrowth. Intracellular signaling mechanisms for GHdepend upon ligand binding. The earliest event in GHsignaling appears to be the binding of a single moleculeof GH to a pair of GH receptors (GHR) (KossiakoV,1995). Binding of GH to the GHR leads to the rapidactivation of JAK2, a cytoplasmic tyrosine kinase thatassociates with the cytoplasmic domain of the GHR(Argetsinger et al., 1993; Campbell et al., 1993; Sotiro-poulos et al., 1994). Receptor activation leads to tyro-sine phosphorylation of a variety of protein kinasesubstrates including insulin receptor substrate-1 (IRS-1),Src homology-containing protein (SHC), phosphati-dylinositol 3� phosphate kinase (PI-3), and mitogen-activated protein (MAP) kinases, as well as severalmembers of the STAT (signal transducers and activa-tors of transcription) family of transcriptional regula-tors (STATs 1, 3, 5a, and 5b) (see Carter-Su et al., 1996;Frank, 2002; Herrington and Carter-Su, 2001; Sand-berg et al., 2004, for reviews). Phosphorylated STATproteins, possibly in complex, translocate to thenucleus, bind to DNA, and activate the transcription ofspeciWc genes (Han et al., 1996; Xu et al., 1996; also, seeHerrington and Carter-Su, 2001, for reviews). In addi-tion, GH induces the expression of SOCS (suppressorsof cytokine signaling)-1, -2, -3, and CIS (cytokine-inducible SH2 (domain-containing) protein), a familyof kinase-inducible genes that inhibit the activity ofJAKs (see Herrington and Carter-Su for review, 2001).Recently, two additional molecules were identiWed thatbind to JAK2: SH2-B (Src homology-containing pro-tein 2B), a cytoplasmic adaptor protein, and SIRP (sig-nal-related protein), that is likely a negative regulatorof GH action (see Carter-Su et al., 2000, for review). Itis thought that all GH signaling downstream of theGHR depends upon initial activation of JAK2, includ-ing activation of the MAP kinase pathway (Zhu andLobie, 2000).

There is in vitro evidence that other JAKs (besidesJAK2) may be important for GHR signaling. Johnstonet al. (1994) showed that GH induces tyrosine phosphor-ylation of JAK3, and Smit et al. (1996) showed GH-induced tyrosine phosphorylation of JAK1. In vivo, GHstimulates tyrosine phosphorylation of JAK2 andSTAT5, but not IRS-1, SHC or other STAT proteins inliver and skeletal muscle of normal rats (Chow et al.,1996), and Davey et al. (2001) demonstrated thatSTAT5b is required for GH-induced liver IGF-I geneexpression in the rat. However, very little is known aboutthe interaction in vivo between the GHR and JAK2, or if

interaction.Domestic poultry, along with the guinea pig, are

unique among vertebrates with respect to GH action, inthat all evidence in the literature suggests that GH doesnot have anabolic eVects in these species (see Baumann,1997; Clayton and Worden, 1960; Mitchell et al., 1954;Vasilatos-Younken and Scanes, 1991). In the case ofpoultry, this is particularly true with respect to lack ofenhancement of skeletal muscle deposition by exogenousGH (see Vasilatos-Younken, 1999, for review). Althoughit has been suggested that intense genetic selection of thecommercial broiler chicken for rapid growth hasresulted in achievement of maximum genetic potential sothat further improvements are not possible, lack of agrowth response to exogenous GH in slow-growing, ran-dombred lines of chickens would argue against this (Pee-bles et al., 1988). In addition, it is generally accepted thatthe anabolic eVects of GH are mediated by the action ofinsulin-like growth factor (IGF)-1, however, chicken (c)GH administered to pituitary-intact birds fails toincrease circulating IGF-I concentrations or skeletalmuscle growth (see Vasilatos-Younken, 1999; Vasilatos-Younken et al., 1999, for reviews).

The Wrst step in GH signal transduction is binding ofthe hormone to receptors on the cell membrane. Detec-tion of functional GHR in the young, rapidly growingbroiler chicken by conventional radioreceptor assayshas, in general, been unrewarding, with speciWc bindinglow to undetectable in young birds during the period ofmost rapid growth (Leung et al., 1984, 1987). Given theimportance of JAK2 activation to subsequent down-stream signaling events, assessment of JAK2 tyrosinephosphorylation in response to GH in the chicken mayprovide a better indicator of functional GHR bindingand upstream signaling events.

The pulsatile nature of GH secretion has long beenrecognized in the chicken (Johnson et al., 1987; Vasila-tos-Younken and Leach, 1986; Vasilatos-Younken andZarkower, 1987), including its importance to GH biolog-ical action (Vasilatos-Younken et al., 1988). Accumulat-ing evidence in rodent and in vitro models suggests thatGH signaling events are episodic under physiologicalconditions, and that pulsatile GH activates STAT5which, along with other factors, mediates changes ingene transcription (see Schwartz, 2001, for review).Alternatively, continuous exposure to GH has beenshown to down-regulate GHR-JAK2 signaling to STAT5b (Ram and Waxman, 2000). Thus, a pulsatile patternof exposure to GH may be critical for eVective GH sig-naling and, ultimately, biological action.

The purpose of this study was to conWrm that anavian homologue of JAK2 is expressed in relevant GH

130 Y. Zhou et al. / General and Comparative Endocrinology 144 (2005) 128–139

target tissues and cell lines; to assess tissue-speciWc diVer-ences in expression of JAK2 and phosphotyrosine acti-vation of JAK2 in response to GH; and to determinewhether chronic enhancement of circulating GH altersthe expression of JAK2 protein in vivo using a pulsatilepattern of GH enhancement to ensure desensitization ofsignaling does not occur.

2. Materials and methods

2.1. Reagents

Rabbit anti-recombinant chicken growth hormoneantiserum (�-rcGH Ab, 5198) was prepared in our labo-ratory. Recombinant chicken GH (rcGH) (Cat. No.CGB002) was purchased from Lucky Biotech Corp/LGChemical (Science Town, Taejon, Korea, and Engle-wood CliVs, NJ). The immunological and biologicalpotency of this rcGH preparation has been describedpreviously (Vasilatos-Younken et al., 2000). JAK2Immunizing Peptide (Cat. No. 12-148, Lot. 15329),JAK2 polyclonal antibody (Ab) (Cat. No. 06-255, Lot.15690) and anti-mouse-JAK3 polyclonal Ab (Cat. No.06-342, Lot. 16193) were purchased from Upstate Bio-technology, (Lake Placid, NY). Monoclonal anti-phos-photyrosine (�-PT) Ab (Cat. No. K01111M, Lots.2H2205 and 2A02204, clone PY20) was purchased fromBiodesign International (Saco, ME). Rabbit polyclonalantiserum against the extracellular domain of the bovineGHR/GH binding protein (�-GHBP/GHR, F296;Staten et al., 1993), which cross-reacts with the chickenGHR, was kindly provided by Dr. Nicholas R. Staten,Monsanto Company (Monsanto, St. Louis, MO). Anti-rabbit IgG-HRP (Cat. No. NA931), anti-mouse-IgG-HRP (Cat. No. NA934), streptavidin-HRP (Cat. No.RPN1231), and ECL protein molecular weight markers(Cat. No. RPN2107) were obtained from AmershamBiosciences (Piscataway, NJ). Bovine serum albumin(Bovuminar Lot. U73506) was from Intergen/Serologi-cals (Norcross, GA). Aprotonin (Cat. No. 981532) andleupeptin (Cat. No. 1017128) were from Boehringer–Mannheim (Mannheim, Germany). Pepstatin A (Cat.No. P-4265), protein A–agarose (Cat. No. P-1406, Lot.56H6811), buVer salts, and all other chemicals were pur-chased from Sigma Chemical (St. Louis, MO).

2.2. Animals and timecourse study

All animal experiments were approved by the PennState University Institutional Animal Care and UseCommittee (IACUC) under approval # 89R1389G197.Chickens used in the Wrst (timecourse) study were RossArbor Acre females, and in the second (infusion) study,Petersen£Arbor Acre females, hatched and reared at thePennsylvania State University Poultry Education and

Research Center (PERC). Birds were fed a commercialbroiler starter (30% crude protein; 0.78% methionine,1.57% lysine; and 3200 kcal ME/kg calculated analysis)ad libitum throughout the experiments, and maintainedunder a 16-h light and 8-h dark cycle with constant tem-perature (23 °C) throughout rearing and experimentalperiods.

Recombinant chicken GH was dissolved in a sterile,buVered saline solution containing 0.025 M NaHCO3and 0.025 M Na2CO3, pH 9.4 (Peel et al., 1981; Roseb-rough et al., 1991), which, with the addition of 0.1%chicken serum albumin, served as the vehicle for injec-tion. For single-injection timecourse studies, 8-week-oldbirds were intravenously injected (brachial vein) withvehicle (control solution) or rcGH at a dosage of 6.25 �gcGH/kg body weight. This dosage results in plasma con-centrations approximating maximal GH secretory pulseamplitudes documented in 4-week-old, rapidly growingbroiler chickens (Vasilatos-Younken and Zarkower,1987). Birds were rapidly killed by i.v. barbiturate over-dose (69 mg/ml Na-pentobarbital; 1 ml/kg BW) immedi-ately prior to or at 0, 5, 10, 20, 30, or 60 min followinginjection of cGH. Separate, individual birds were usedfor each time point, and the full timecourse was repli-cated twice. Liver and the Pectoralis superWcialis musclewere immediately removed and frozen in liquid nitrogenand stored at ¡80 °C until analysis.

2.3. Chronic pulsatile infusion of GH in vivo

Full details of the in vivo study are described in Vasi-latos-Younken et al., 2000. BrieXy, birds with similarbody weight were used, and any birds showing externalevidence of disease were eliminated from this study.Birds were fed a commercial starter diet and maintainedas described above. Before surgical preparation, chick-ens were fasted overnight, and the following morninganaesthetized with sodium pentobarbital (31 mg/kg BW,i.v.). The right jugular vein was exposed and catheterizedfor chronic intravenous delivery of cGH as previouslydescribed (Cravener and Vasilatos-Younken, 1989).Birds recovered for at least 72 h, and exhibited pre-sur-gery behavior and levels of voluntary feed intake priorto initiation of infusions. Recombinant chicken growthhormone was dissolved in vehicle as described above.Intravenous infusions [0, 10, 50, 100 or 200 �g GH/kgBW/day (0–200 GH); 9–10 birds per dosage groupinfused in two experiment replicates over time] began at8 weeks of age and continued for 7 consecutive days.Treatments were delivered in a pulsatile manner (50 �L/pulse, 2 min/pulse delivery, 90 min/interpulse interval,24 h/day for a total volume of 0.8 ml/day) by micropro-cessor-controlled infusion pumps (AutoSyringe, Model.AS20A), connected to the birds via a Xuid swivel/springtether/harness system that allowed 360° unrestrictedmovement inside the cage. On the Wnal day of the 7-day


infusion period, blood samples were removed from eachbird by brachial venipuncture immediately (within1 min) after delivery of the Wnal GH pulse. Blood wascollected into chilled, heparinized tubes, centrifuged at2000g for 20 min at 4 °C, and plasma was harvested andstored at ¡80 °C until analyzed. Each bird was killed bycervical dislocation. The liver and P. superWcialis musclewere removed and immediately weighed and plungedinto liquid nitrogen at 10–12 (liver) or 15 (skeletal mus-cle) min following the Wnal pulse of GH, and stored at¡80 °C for further analyses.

Because voluntary feed intake of birds receiving thehigher dosages of cGH (100 and 200 GH) was reduced(see Vasilatos-Younken et al., 2000; Wang et al., 2001),replicate groups of surgically prepared birds were pre-pared and infused with vehicle for 7 days. A controlgroup was provided with feed ad libitum. Additionalgroups were pair-fed to the reduced level of voluntaryintake of the 100 and 200 GH treatment groups, and allmeasurements, including JAK2 protein abundance andphosphotyrosine activity in liver and breast muscleassessed to discern if observed responses were secondaryto alterations in feed intake, rather than primary GHeVects per se.

2.4. Plasma cGH radioimmunoassay

To conWrm cGH delivery, plasma GH concentrationswere detected by a homologous chicken GH radioimmu-noassay (Vasilatos-Younken, 1986). BrieXy, the assayentailed a sample volume of 100 �L and a Wnal incuba-tion volume of 400 �L. Assay buVer consisted of phos-phate buVered saline (PBS; 0.01 M phosphate/0.14 MNaCl) with 0.02 M EDTA, 0.1% BSA, and 0.01% thimer-osal, pH 7.4. Following incubation of unknowns with200 �l primary antibody (�-cGH Ab, 1:16,200, in assaybuVer without BSA) for 24 h at 4 °C,125I-cGH tracer(13,400 cpm in 100 �L assay buVer/tube) was added andincubated for an additional 24 h at 4 °C. Precipitatingantiserum [200 �L of a 1:10 dilution of goat anti-rabbit�-globulin (GARGG); Lot. 910263, Calbiochem, LaJo-lla, CA], and 100 �L of a 1:20 dilution of normal rabbitserum (NRS; Lot. 022 A, ICN Biomedicals, Irvine, CA)were then added and incubated further for 2 h at roomtemperature (RT), then at 4 °C for 0.5 h. Finally, 2 ml ofice-cold PBS (without EDTA, thimerosal or BSA) wereadded, the assay tubes were centrifuged at 2000g at 4 °Cfor 30 min, and the supernatants were discarded. Radio-activity in the precipitate was counted for 2 min in agamma counter (Gamma Master, Pharmacia LKBNuclear, Gaithersburg, MA).

2.5. Protein determinations

Before analysis, frozen tissues were homogenizedusing a Tekmar Tissumizer (Tekmar–Dohrmann,

Cincinnati, OH) in Lysis BuVer (1 g wet tissue per 3 mlLysis BuVer for muscle; 1:1 for liver) consisting of10 mM Tris-buVer containing 5 mM EDTA, 50 mMNaCl, 1% Triton-X 100, 30 mM Na-pyrophosphate,50 mM Na-Xuoride, 200 �M Na-orthovanadate (toinhibit phosphotyrosine phosphatases), and proteaseinhibitors (1 mM phenylmethylsulfonyl Xuoride(PMSF), 5�g/ml aprotonin, 1 �g/ml pepstatin-A, and2 �g/ml leupeptin), and extracted for 60 min at 4 °C withagitation. Insoluble material was pelleted by centrifuga-tion at 50,000g for 30 min at 4 °C. The supernatants werecollected and protein concentration was determinedusing a standard Lowry assay (Lowry et al., 1951), withBSA as standard.

2.6. Cell culture

In addition to liver and muscle tissues, JAK2 activa-tion in response to cGH was explored in a chicken hepa-toma cell line. The chicken Leghorn Male Hepatoma(LMH) cell line, available from ATCC (Manassas, VA;ATCC # CRL-2117), is readily transfectable (Binderet al., 1990) and retains hepatocyte characteristics in cul-ture (Kawaguchi et al., 1987). Cells were grown on 6 or10 cm cell culture dishes coated with 0.1% gelatin, inWaymouth’s MB 752/1 containing 10% fetal bovineserum (FBS) (Hyclone Laboratories, Logan, UT),1%chicken serum (CS; Sigma Chemical), and 100 IU/mlpenicillin and 100 �g/ml streptomycin (1% of a100£Pen-Strep solution; Gibco/Invitrogen Life Tech-nologies, Carlsbad, CA) and maintained in a 37 °C incu-bator under 5% CO2. Cells were split into fresh dishescontaining fresh media when they reached 60–80% con-Xuence.

2.7. Culture treatments

LMH cells (100% conXuent) were preincubated with aserum-free medium (SF) consisting of Williams EMedium supplemented with sodium bicarbonate(2.2 mg/ml), porcine insulin (0.1 �g/ml), porcine glucagon(1.0�g/ml), transferrin (10 �g/ml), bovine serum albumin(BSA) (Serologicals (formerly Intergen), Norcross, GA)(1.6 mg/ml), 1% Pen-Strep solution, 17-�-estradiol(10¡9 M), NaCl (30 mM), glucose (1.0 mg/ml), and traceelements (100£Trace Element Mix, Formula No. 96-0438DG, Gibco-Invitrogen Life Technologies, GrandIsland, NY) (1 ml/100 ml) for 4 h at 37 °C in an incubatorunder 5% CO2. Media were removed and cells were incu-bated for 10 min at 37 °C with either SF medium (con-trol) or SF containing either 5 �g/ml cGH or varyingamounts of cGH for the dose–response study. Immedi-ately following treatment, media were removed by vac-uum, and cell dishes were placed on slabs of dry ice toquick-freeze the cells. Cells were then scraped, washedonce in 4 ml Hepatocyte Wash BuVer (Invitrogen Life


Technologies), and resuspended in 1.0 ml lysis buVer(described above). Cell pellets were lysed by sonication(Sonic Dismembrator 60, Fisher ScientiWc, Pittsburgh,PA), and extracted for 60 min at 4 °C with agitation.Insoluble material was pelleted by centrifugation at15,000g for 30 min at 4 °C in a microfuge. Supernatantswere assayed for protein content by the method ofLowry as described above, or by quantitation via Gene-quantPro (Amersham Biosciences, Piscataway, NJ).

Samples (6 or 10 mg total protein) of LMH cellextracts were prepared for immunoprecipitation andimmunoblotting as described further below.

2.8. Immunoprecipitation (IP) and immunoblotting

Prior to assessment of tissues from chronicallyinfused birds, an extensive series of preliminary experi-ments was Wrst conducted using tissues from replicatebirds, to determine optimal conditions for Western blot-ting in terms of amount of tissue lysate, antibody dilu-tions, etc. Final conditions selected are described below.SpeciWcity of immunodetection was also evaluated andno immunoreactive bands were detected with the use of:(1) non-speciWc serum; (2) when �-JAK2 was pre-absorbed with JAK2 immunizing peptide (24 �g antigenper 10 �l �-JAK2; antigen corresponds to amino acidresidues 758–776 of murine JAK2) prior to immunoblot-ting; or (3) when tissue lysates were immunoprecipitatedand/or detected with �-JAK3.

Equal amounts of tissue or cellular protein (6 or10 mg) in lysis buVer were incubated with primary Abs(4�l of �-JAK2, 2�l of �-cGH, or 6 �l �-GHBP/GHRAbs) at 4 °C overnight. The Abs were then adsorbed ontoprotein A–agarose beads (100�l slurry containing 50�lpacked beads) for 2 h at 4 °C. The resulting immunocom-plexes were washed six times by centrifugation and resus-pended with 1 ml lysis buVer each time. After the Wnalwash and centrifugation, immunoprecipitates were resus-pended in 2£ treatment buVer (TB; 50�l) (0.125 M Tris–Cl, 4% SDS, 20% glycerol, 10% of 2-mercaptoethanol,and 1% phenol red, pH 6.8), heated at 100 °C for 5 min,and centrifuged. Forty microliters of each supernatantwas loaded onto 11 cm £14 cm £ 0.75 mm, 7.5% acrylam-ide gels (Hoefer ScientiWc Instruments, San Francisco,CA), and along with biotinylated standards (Cat. No.161-0319, Bio-Rad Laboratories, Hercules, CA), proteinsseparated by SDS–PAGE according to Laemmli (1970),and electroblotted (400 mA for approximately 1–1.5 h)onto supported nitrocellulose membranes. Preliminaryexperiments were conducted to optimize blotting condi-tions, including primary and secondary antibody dilu-tions, amount of membrane protein, and other variables.Based on these preliminary experiments, Wnal blots wererun under the following conditions, with washing andincubation steps performed as indicated on an orbitalshaker: membranes were blocked overnight at 4 °C in

TBS-T (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, and0.1% Tween 20) with 3% BSA plus 1% non-fat dried milk(NFDM) for �-GHBP/GHR or �-JAK2 detection; or for2 h at 4 °C in 10 mM TBS-T with 3% BSA (no NFDM)for �-PT detection. The blots were subsequentlyincubated with primary antibodies (1:1000 dilution of�-JAK2 or 1:1500 dilution of �-GHBP/GHR) in TBS-Twith 0.1% BSA for 2 h at RT; or 1:200 dilution of �-PTAb (clone PY20) in TBS-T with 3% BSA overnight at4 °C), washed four times for 5 min each, and then incu-bated with horseradish peroxidase (HRP) labeledsecondary antibodies (1:1000 dilution of anti-rabbit IgG-HRP for 50 min at RT or 1:2000 dilution of anti-mouseIgG-HRP for 2 h at 4 °C). After washing, labeled proteinswere visualized using enhanced chemiluminescence(ECL, Amersham Biosciences, Cat. No. RPN2108).BrieXy, blots from tissue preparations were incubatedwith ECL chemiluminescence substrate mixture (1:1,Reagent 1:Reagent 2) for precisely 1 min and exposed toX-ray Wlm (ECL HyperWlm, RPN3103H, Amersham Bio-sciences) for 10 s (7 s for phosphotyrosine detection).Blots from cell culture preparations were incubated withECL detection reagents as above, and exposed to KodakBiomax Imaging Film (Eastman Kodak, Rochester, NY)for 3–5 min (�-JAK2 or �-PT (clone PY20) detectionantibodies). All Wlms were manually developed (GBXDeveloper/Replenisher; Sigma–Aldrich) and Wxed (GBXFixer/Replenisher; Sigma–Aldrich) and washed in a run-ning water bath. When stripping was required to reprobe,membranes were incubated with stripping buVer(62.5 mM Tris–HCl, pH 6.8, 2% SDS, and 0.1 M of 2-mercaptoethanol) for 30 min at 50 °C, and washed exten-sively with TBS-T. Films were analyzed using an EagleEye II Still Video System (Stratagene, La Jolla, CA), andONE-Dscan one-dimensional electrophoresis analysissoftware (Scanalytics, Billerica, MA) or Eagle Sight soft-ware (Stratagene).

JAK2 phosphorylation in LMH cells stimulated with0, 10, 200, or 1000 ng/ml cGH for 10 min was also deter-mined. Ten milligrams of LMH cell lysate from eachtreatment was immunoprecipitated with �-JAK2 anti-body (Upstate USA, Waltham, MA), separated on 7.5%SDS–PAGE gels, transferred to supported nitrocellulosemembrane, and detected with �-PT antibody (clonePY20, Biodesign). The same blot was then stripped andre-probed with �-JAK2 antibody (Upstate USA).

2.9. Statistical analyses

Quantitative data are presented as means § standarderror of the mean (SEM). Dose–response data were ana-lyzed by regression analysis and linear, quadratic, andcubic functions tested for each response. Initially, eachresponse was tested for a treatment£experiment repli-cate interaction, which was not signiWcant for any mea-surement, therefore experiments were pooled and


replicate was dropped from the model. The statisticalmodel for pair-feeding data was based on a one-wayclassiWcation (Steel and Torrie, 1980), with terms in themodel for analysis of variance (ANOVA) being treat-ment (ad libitum fed, or restricted to the level of volun-tary intake of the 100 or 200 GH treatment groups) andbird (within treatment).

3. Results

3.1. Chicken GHR signaling in avian LMH hepatoma cells

The presence of a functional GH-R was conWrmed inLMH hepatoma cells via immunoprecipitation (IP) ofcell lysates with �-GHBP/GHR and detection by West-ern blotting (Fig. 1). Additional immunoanalysis of

Fig. 1. Western blot analysis of cell lysates from LMH cells incubatedwith or without exposure to cGH. LMH cells were grown to conXu-ence on 100 mm plates and exposed to cGH (5 �g/ml) in serum-freemedium (lanes 2, 4, 6, and 8). Duplicate control plates (0 cGH; lanes 1,3, 5, and 7) were incubated simultaneously. After a 10 min exposure,control and cGH-treated cells were immediately frozen and extractedin lysis buVer. Extracts were immunoprecipitated (IP) with anti-JAK2or anti-bGHBP/GHR antibodies (Ab), separated on 7.5% SDS–PAGE gels, transferred to nitrocellulose membranes, and detectedwith anti-phosphotyrosine or anti-bovine GH binding protein (whichcross-reacts with the chicken GHR) Abs.

LMH cells indicated that upon exposure to GH, a 120–122 kDa JAK2 immunoreactive protein associated withthe cGHR and was tyrosine phosphorylated, whereasJAK2 was not phosphorylated in control (vehicle-stimu-lated) cells (Fig. 1). Tyrosine phosphorylation of cGHRwas also stimulated by GH (Fig. 1). JAK2 phosphoryla-tion increased when cells were stimulated with increasingamounts of cGH for 10 min (Fig. 2A). There was not anassociated increase in JAK2 protein when cells weretreated acutely with GH (Fig. 2B).

3.2. In vivo GH receptor signaling and eVects of chronic infusion of ligand

The functional expression JAK2 in avian tissues wasexplored by Wrst assessing the timecourse of JAK2 phos-phorylation in liver and breast muscle of 8-week-oldbroilers following a single i.v. injection of cGH. At a dos-age that was previously shown to elicit biologicalresponses, maximum tyrosine phosphorylation of JAK2appeared at 10 min in muscle, and remained elevated upto 60% above basal levels through 60 min post-injection(Fig. 3). No response was observed in liver.

Because phosphorylation of JAK2 in response toacute GH exposure was not observed in liver in theabsence of previous exposure to signiWcant levels ofendogenous circulating GH, a chronic GH infusionstudy was designed in which GH was infused for 7 daysin a pulsatile pattern to simulate the GH plasma proWleof early post-hatch, rapidly growing birds.

Prior to assessment of tissues from chronicallyinfused birds, an extensive series of preliminary experi-ments was Wrst conducted using tissues from replicatebirds, to determine optimal conditions for Western blot-ting in terms of amount of tissue lysate, antibody dilu-tions, etc. Final conditions selected are described abovein Section 2. SpeciWcity of immunodetection was alsoevaluated and no immunoreactive bands were detected(not shown) with the use of: (1) non-speciWc serum; (2)

Fig. 2. JAK2 phosphorylation in LMH cells stimulated with 0, 10.0, 200.0, or 1000.0 ng/ml cGH for 10 min. (A) Immediately following a 10 min expo-sure, control and cGH-treated cells were frozen and extracted in lysis buVer. Lysates were clariWed by centrifugation, and 10 mg total protein fromeach extract was IP with anti-JAK2. Forty microliters of each sample was subjected to SDS–PAGE (7.5% acrylamide), transferred to supportednitrocellulose membranes and detected with anti-phosphotyrosine antibody (clone PY20, Biodesign International). (B) The same blot was stripped
and re-probed with anti-JAK2 (Upstate USA, Waltham, MA).


when �-JAK2 was pre-absorbed with JAK2 immunizingpeptide (24�g antigen per 10 �l �-JAK2; antigen corre-sponds to amino acid residues 758–776 of murine JAK2)prior to immunoblotting; or (3) when tissue lysates wereimmunoprecipitated and/or detected with �-JAK3.

To further demonstrate that JAK2 forms a complexwith the chicken GHR, tissue lysates from birds infusedwith cGH were immunoprecipitated (IP) with �-cGH or�-JAK2, and detected with �-JAK2. JAK2 was detectedin lysates IP with �-cGH, consistent with association ofJAK2 with the GHR–GH–GHR complex that formsupon binding of cGH to the cGHR (Fig. 4).

Our optimized Western blotting procedure was uti-lized for detection of JAK2 in liver and breast muscleof 8-week-old broilers infused with cGH at 0, 10, 50,100 or 200 �g/kg BW/day for 7 days in a pulsatile pat-tern (see Vasilatos-Younken et al., 2000 for circulatingGH concentrations resulting from infusions). JAK2immunoreactive proteins were detectable at Mr ofapproximately 120–122 kDa (120JAK2) in liver (Fig.5A, upper panel), and 120–122 kDa (120JAK2) and130–136 kDa bands (130JAK2) in skeletal muscle (Fig.6A, upper panel), with predominantly the 130JAK2being tyrosine phosphorylated in response to cGH inmuscle (Fig. 6A, lower panel). In both tissues, there

Fig. 3. Timecourse of JAK2 activation in chicken skeletal muscle (P.superWcialis). Eight-week-old, female broiler chickens were rapidlyinjected i.v. with either 0 or 6.25 �g cGH/kg BW, and tissue samplesremoved at 0, 5, 10, 20, 30, and 60 min post-injection, and rapidly fro-zen in liquid nitrogen. DiVerent birds were used for each time point,and the full timecourse was replicated twice. Cellular proteins wereextracted in lysis buVer and 6 mg extracted protein were immunopre-cipitated with 4 �l anti-JAK2 Ab. Immunoprecipitated proteins wereelectrophoresed on 7.5% SDS–PAGE gels, electrotransferred to nitro-cellulose membranes, and probed with anti-phosphotyrosine antibody,as described in Section 2 (A, top panel). The same blots were thenstripped and re-probed with anti-JAK2 antibody as an internal con-trol of loading (A, bottom panel). Blots were subjected to densitomet-ric analysis (B), and values represent means § SEM of two birds pertime point expressed as densitometry units. Densitometry of JAK2phosphotyrosine activity revealed maximal activation occurred at10 min post-cGH injection, and remained slightly elevated through60 min.

were signiWcant GH–dependent increases in JAK2 pro-tein abundance and tyrosine phosphorylation (Figs. 5Aand 6A, upper and lower panels, respectively). Densi-tometry conWrmed that a maximal response occurred

Fig. 4. Evidence of association of JAK2 with the GH–R2–GH com-plex. Tissue lysates from birds infused for 7 days with 50 �g/kg BW/day cGH were immunoprecipitated with either anti-JAK2 (�-JAK2)or anti-cGH (�-GH) antibodies (Ab), separated on 7.5% SDS–PAGE,electrotransferred to nitrocellulose membranes, and detected with�-JAK2 Ab. JAK2 immunoreactive bands in lysates precipitated with�-GH are presumed to be JAK2 that has associated with the GH–R2–GH complex upon binding.

Fig. 5. Hepatic JAK2 protein abundance and tyrosine phosphoryla-tion in birds chronically infused with cGH (A; upper and lower panel,respectively). Tissue lysates from female broilers infused with 0, 10, 50,100, or 200 �g/kg BW/day cGH in a pulsatile pattern for 7 days begin-ning at 8 weeks of age were immunoprecipitated with anti-JAK2 anti-body, separated on 7.5% SDS–PAGE, electrotransferred tonitrocellulose membranes, and detected with either anti-JAK2 (A;upper panel) or anti-phosphotyrosine (anti-PT; A: lower panel). Lanelabeled +JAK2 is a positive control lysate of Cos-M9 cells transfectedwith pBOSHAI containing murine wild-type JAK2, generously pro-vided by Dr. Don Wojchowski, Department of Veterinary Sciences,Penn State University. Densitometry (B) revealed a maximal responseat the 100 �g/kg BW/day cGH dosage for both JAK2 protein abun-dance and tyrosine phosphorylation. Densitometry values are repre-sented as the means § SEM. N D 9–10 birds/dosage.


Table 1Relative JAK2 protein abundance and phosphotyrosine activity (Tyr-P) in tissues of birds fed ad libitum or pair fed to the reduced level ofvoluntary intake of birds infused with 100 or 200 �g/kg BW/day cGH(means § SEM)a

a All birds received pulsatile intravenous infusion of vehicle for 7days during feed restriction, prior to collection of tissue data.

b Probability of signiWcant diVerence vs control group fed ad libi-tum.

c Densitometry Units. 120JAK2 in liver and 130JAK2 in skeletal(Pectoralis major) muscle were evaluated.

Ad libitum (Control)

Pair-fed group Pb

(n D 9) 100 (9) 200 (9)

JAK2 protein (DUc)Liver 2.01 § 0.17 2.34 § 0.17 2.55 § 0.18 0.80Skeletal muscle 0.86 § 0.08 0.81 § 0.06 0.65 § 0.07 0.79

JAK2 Tyr-P (DUc)Liver 1.38 § 0.14 0.87 § 0.08 0.76 § 0.04 0.38Skeletal muscle 0.88 § 0.12 0.82 § 0.09 0.65 § 0.07 0.85

at the 100 �g/kg BW dosage of GH for both tissues andfor both protein and tyrosine phosphorylation (Figs.5B and 6B). This dosage resulted in a mean peak circu-lating GH plasma concentration of 53 § 3.4 ng/ml (alsorefer to Vasilatos-Younken et al., 2000).

JAK2 protein abundance and tyrosine phosphoryla-tion of 120JAK2 in liver and 130JAK2 in breast muscleof birds pair-fed to the reduced level of voluntaryintake of 100 and 200 GH treatment groups did notdiVer from levels in the control group fed ad libitum(Table 1).

4. Discussion

Although reports of GH signaling via JAK2 in cellsystems are abundant in the literature, data presented inthe present paper on signaling both in vitro and in vivois novel, as is this Wrst report of eVects of chronic in vivoexposure to GH on JAK2 protein abundance.

Fig. 6. Skeletal (breast) muscle JAK2 protein abundance and tyrosine phosphorylation in birds chronically infused with cGH (A; upper and lowerpanel, respectively). Tissue lysates from female broilers infused with 0, 10, 50, 100, or 200 �g/kg BW/day cGH in a pulsatile pattern for 7 days begin-ning at 8 weeks of age were immunoprecipitated with anti-JAK2 (�-JAK2) antibody, separated on 7.5% SDS–PAGE, electro-transferred to nitrocel-lulose membranes, and detected with either �-JAK2 (A; upper panel) or anti-phosphotyrosine (�-PT; A; lower panel). Lane labeled +JAK2 is apositive control lysate of Cos-M9 cells transfected with pBOSHAI containing murine wild-type JAK2, generously provided by Dr. Don Wojchowski,Department of Veterinary Sciences, Penn State University. Densitometry (B) of the predominant 130 kDa band revealed a maximal response at the100 �g/kg BW/day cGH dosage for both JAK2 protein abundance and tyrosine phosphorylation. Values represent the means § SEM (N D 9–10birds/dosage).


Chicken JAK2 has been cloned (Sayed et al., 2004),however, no information on the translated protein hasbeen published. The molecular mass of rat JAK2deduced from the JAK2 sequence is 130.5 kDa (Duheet al., 1995), however, translation of rat JAK2 mRNA inrabbit reticulocytes produced a protein that migrated onSDS–PAGE at 120 kDa and was speciWcally immuno-precipitated by anti-JAK2 Ab (Duhe et al., 1995). Previ-ous electrophoretic analysis indicates that authenticJAK2 observed in NB2 cells migrated at 120 kDa (Rui etal., 1992), which coincided with the largest in vitro tran-scription/translation product immunoprecipitated byanti-JAK2 Ab. This corresponds to our observations inthe liver, but in the muscle, the dominant protein was130 kDa, which has also been reported in rat liver andadipose tissue (Hellgren et al., 2001). Jiang et al. (1998)reported a high molecular weight variant of Mr » 170kDa in 3T3 pre-adipocytes and, given the signiWcantdiVerence in Mr and the fact that treatment with degly-cosidases failed to alter migration on SDS–PAGE [thereare six glycosylation sites in the deduced amino acidsequence from the rat JAK2 cDNA (Duhe et al., 1995;Harpur et al., 1992)] , speculated it to be either the prod-uct of a unique gene with immunologic crossreactivity toJAK2, or a variant arising from alternative splicing ofthe JAK2 mRNA. Alternatively, it is possible that phos-phorylation contributes to the higher of the smallermolecular weight (130 kDa versus 120 kDa) variants.

In timecourse studies, there was no detectable JAK2activation in liver in response to a single acute exposureto GH. At 8 weeks of age, circulating (plasma) GH is vir-tually undetectable in the broiler (mean D .3 § .03 ng/mlfor control birds receiving vehicle only in the presentinfusion study), suggesting that JAK2 protein expressionmay be GH-dependent in liver but not in skeletal muscle.This diVerential expression is interesting, given that liveris a GH-responsive tissue in the chicken and at 8 weeksof age, GH elicits a number of eVects, including suppressionof hepatic Type III deiodinase activity and enhancement ofhepatic IGF-I protein and mRNA (Vasilatos-Younkenet al., 2000), whereas GH is presumed not to have directeVects on skeletal muscle in the chicken.

To determine whether circulating GH would alterJAK2 levels in vivo, we Wrst developed an in vivomodel of growth and GH enhancement in the chicken.Intravenous infusion of GH was used to re-establish ajuvenile pattern of circulating GH (high amplitude GHpeaks) in birds at an age when endogenous GH isbarely detectable (Vasilatos-Younken et al., 1992). ThesigniWcance of the pulsatile nature of GH secretion toGH signaling is currently gaining increasing attention(see Schwartz, 2001, for review). Tannenbaum et al.(2001) reported a direct link between STAT5 activationand the spontaneous, endogenous secretory pulses ofGH in rats, with STAT5 DNA binding activity closelyfollowing the upswing and downswing of GH pulses. In

addition, Gebert et al. (1999) demonstrated that inhypophysectomized rats given intermittent pulses ofGH, hepatic STAT5 was repeatedly phosphorylated,but that continuous GH treatment led to a decrease inSTAT5 phosphorylation. In the rodent, STAT5bappears to determine the expression of sexually dimor-phic body growth and gene expression, and is in turndetermined by the sexually dimorphic pattern of GHsecretion (Choi and Waxman, 2000). Thus, reinstate-ment of a juvenile GH pulse pattern [high-amplitudepeaks, with an approximate 1 h average peak durationand 1.5 h inter-peak interval (Vasilatos-Younken andZarkower, 1987)] by the use of a microprocessor-con-trolled infusion system in the present study, allowed fora more physiological pattern of exposure to the hor-mone and, presumably, less likelihood of desensitiza-tion of the signaling pathway.

In this study, we focused on the eVects of chronicGH on JAK2 protein expression and tyrosine phos-phorylation. Janus kinase 2 participates in the molecu-lar events immediately following activation of a varietyof cytokine receptors, including GH (Argetsinger et al.,1993; Wang et al., 1993), PRL (Rui et al., 1994), Epo(Witthuhn et al., 1993), and IL-l, 2, 4, 6, 7, 11, and 12(Yu et al., 1996) (see Sandberg et al., 2004 for compre-hensive review), and is phosphorylated in response toreceptor activation by these cytokines and physicallyassociates with the membrane-proximal portion oftheir receptors. Although immunodetection of tyrosinephosphorylated JAK2 in JAK2 immunoprecipitatesdoes not conclusively link the observed changes in liverand muscle with GH signaling exclusively, the demon-stration that JAK2 was associated with the GHR–GH–GHR complex in tissue lysates IP with �-cGH; theclose dose–response relationship of JAK2 tyrosinephosphorylation with dosage of cGH; and the fact thatJAK2 tyrosine phosphorylation occurred in tissuesthat were harvested within 10–15 min of a GH pulse, allsupport that this is the case.

In response to chronic pulsatile GH infusion, whichreinstated a more juvenile pattern of circulating GH,8-week-old birds exhibited increased JAK2 proteinabundance and tyrosine phosphorylation in both liverand skeletal muscle. Maximal 2- and 3-fold increasesabove control levels in JAK2 protein expression, and 3-and 4-fold increases in tyrosine phosphorylation forliver and muscle, respectively, occurred at the 100 �g/kgBW/day dosage, which equated to a mean peak circu-lating GH plasma concentration of 53 § 3.4 ng/ml. Thereduction in tyrosine phosphorylation and biologicalresponses above this concentration may reXect thethreshold above which GH binds to its receptor in a 1:1stoichiometry, and GHR dimerization and eVective sig-nal transduction are decreased. These data are alsoconsistent with decreased JAK2 activity in agingrodents, which have low levels of GH versus younger


animals (Xu et al., 1995), and collectively suggest thatJAK2 is developmentally regulated by GH.

The mechanism by which GH enhances JAK2 pro-tein abundance is not known, however, its dose-depen-dent nature would support a direct eVect, possibly atthe post-transcriptional level. Particularly signiWcant isthat the variety of biological responses previouslyreported for the same birds as used in this study (Vasi-latos-Younken et al., 2000) reXect similar dose–response relationships, including decreased hepatictype III iodothyronine deiodinase (5DIII) activity lead-ing to increased circulating T3 concentrations, andincreased hepatic IGF-I protein abundance. Althoughnot conclusive because of the in vivo nature of thisstudy, the fact that the GH dose–response curves forthese biological actions are the mirror image of theJAK2 protein/tyrosine phosphorylation curves, sug-gests the latter may constitute one mechanism by whichGH may enhance its own signaling. However, given thecomplexity of downstream GH signaling events thatrequire JAK2 for initiation (Schwartz et al., 2002),other mechanisms must also be considered in achievingWnal biological responses. Similarly, it was previouslyreported that decreased IGF-I gene expression in liverof 31-month-old rats was associated with decreasedJAK2 and GHR phosphorylation versus younger ages(Xu et al., 1995).

The lack of any diVerences in JAK2 protein and phos-photyrosine activity for birds pair-fed to the reducedlevel of voluntary feed intake of birds receiving the 100and 200 GH treatments versus ad libitum fed controls,further support that alterations in JAK2 were not sec-ondary to changes in feed intake, and again more likelydue to direct eVects of the hormone.

In summary, (1) JAK2 immunoreactive proteinsthat associate with the GHR and are tyrosinephosphorylated in response to GH were identiWed inan avian hepatoma cell line and in both GH responsive(liver) and “non-responsive” (skeletal muscle) tissues;(2) tyrosine phosphorylation of JAK2 occurred withinminutes of exposure to a single i.v. injection of GH invivo in skeletal muscle, but was not detectable in liverof 8-week-old birds; and (3) there were GH dose-dependent increases in abundance of JAK2 proteinand tyrosine phosphorylation in tissues of olderanimals when chronically exposed to GH enhancementin a physiologically relevant pattern, such as occurs inyoung, rapidly growing juvenile birds. These were asso-ciated with previously reported GH dose-dependentbiological responses, including alterations in the path-way of thyroid hormone metabolism. Enhanced JAK2suggests one possible mechanism whereby chronic,physiologically appropriate exposure to the ligandenhances GH biological action via increasedabundance of a key upstream component of GHsignaling.

Acknowledgments

This work was supported by USDA-NRI Grant No.98-35206-6411 to R. Vasilatos-Younken. Appreciation isexpressed to Dr. William Baumbach, American Cyan-amid (Princeton, NJ), for providing us with the cGH-RcDNA; Dr. Nicholas R. Staten, Monsanto (St. Louis,MO), for anti-GH-R antiserum; and Dr. Don Wojchow-ski, Department of Veterinary Science, Penn State Uni-versity, for Cos-M9 cells transfected with pBOSHAIcontaining wild-type JAK2.

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Regulation of JAK2 protein expression by chronic, pulsatile GH administration in vivo: A possible mechanism for ligand enhancement of signal transduction