help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1736
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elsasser, T. H.
Right arrow Articles by Schmidt, W. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elsasser, T. H.
Right arrow Articles by Schmidt, W. F.
Endocrinology Vol. 148, No. 8 3792-3802
Copyright © 2007 by The Endocrine Society

Growth Hormone (GH)-Associated Nitration of Janus Kinase-2 at the 1007Y-1008Y Epitope Impedes Phosphorylation at This Site: Mechanism for and Impact of a GH, AKT, and Nitric Oxide Synthase Axis on GH Signal Transduction

Ted H. Elsasser, Cong-Jun Li, Thomas J. Caperna, Stanislaw Kahl and Walter F. Schmidt

Growth Biology Laboratory (T.H.E., C.-J.L., T.J.C., S.K.) and Environmental Quality Laboratory (W.F.S.), U.S. Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705

Address all correspondence and requests for reprints to: Dr. Ted H. Elsasser, U.S. Department of Agriculture, Agricultural Research Service, Growth Biology Laboratory, B.A.R.C.-east, Beltsville, Maryland 20705. E-mail: elsasser{at}anri.barc.usda.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A generalized increase in liver protein tyrosine nitration (3'-nitrotyrosine, 3'-NT) occurs after GH injection in a time frame consistent with observed acute GH hyporesponsiveness. Here we investigated whether the GH-associated nitration process might be targeted to the 1007Y-1008Y-phosphorylation epitope of Janus kinase (JAK)-2 because of its homology to a defined peptide nitration motif. Using antibodies we developed to the 3'NT-substituted peptide analog of the 1007Y-1008Y-JAK2 site (nitro-JAK2), we demonstrated a rapid increase in membrane-associated nitro-JAK2 after GH. In vivo (bovine liver) and in vitro (porcine hepatocytes), GH-induced cellular levels of nitro-1007Y-1008Y-JAK2 persisted significantly longer after a stimulatory GH pulse than did levels of phospho-JAK2. Treatment of cultured cells with inhibitors of AKT or endothelial nitric oxide synthase prior to GH challenge attenuated the increases in nitro-JAK2 predominantly in the membrane subcellular fraction. In instances in which GH effected orthophosphorylation of 694Y-signal transducer and activator of transcription (STAT)-5b, the addition of AKT and endothelial nitric oxide synthase inhibitors prior to GH significantly increased the levels of phospho-694Y-STAT5b and phospho-1007Y-JAK2 over those arising from GH alone. Nuclear magnetic resonance molecular modeling of natural and 3'-NT- and orthophosphate-substituted peptide analogs of the 1007Y-1008Y site demonstrated significant effects of 3'-nitration on the planar orientation and intramolecular stabilizing points of the affected tyrosines. When these peptides were used as substrates for in vitro tyrosine kinase phosphorylation reactions, 3'-NT in the 1007Y and/or 1008Y positions blocked the generation of 1007Y-phosphotyrosine. The data suggest that the nitration of JAK2 may act as an inhibitory counterpart to phosphorylation activation, reflecting a very localized break on the progression of GH signal transduction processes spanning JAK-STAT-AKT interactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COORDINATED CELLULAR responses to GH, in particular those further attributable to IGF-I, are moderated through a molecular cascade involving both activation, activation-termination, and interaction between receptor-postreceptor signal transduction elements (1, 2, 3, 4). Observations indicate that complementary signal transduction elements are necessary to facilitate the efficiency of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway to achieve full expression of IGF-I in some cell types in which JAK-STAT action alone is sufficient for GH to exert control IGF-I production (5). Thus, whereas we can say that some biochemical mechanisms of GH action and regulation are certainly known, others are yet to be dissected out of the complex background and defined.

GH actions at the cellular level can be thought of in terms of the reactivity of tissues changing between GH-responsive and GH-resistant states (6, 7). These changing states of relative GH responsiveness have come to be recognized as playing a significant role in such diverse processes as the sexual dimorphic character of GH responsiveness (8), short-term GH-derived refractoriness (9), and the reprioritization of GH-directed actions on metabolism that develop during infection (10, 11). Within this concept of GH responsiveness, the deactivation or repression of signal transduction elements plays an important role to counter or terminate their activation (i.e. by phosphorylation). The temporal patterns and cycles of activation and inactivation of signal transduction elements define the intended biochemical activity. To date, the major inactivation or down-regulating mechanisms controlling the GH axis include changes in GH receptor abundance, dephosphorylation of previously phosphorylation-activated proteins (by several phosphatases), and the up-regulation of several suppressor proteins [suppressors of cytokine signaling (SOCS)] capable of binding to and actively repressing the association of other interacting transduction elements (12, 13, 14, 15). The study of disease scenarios has led directly to the identification of several GH signal transduction features that further mark GH resistance. These include ubiquitin proteosome-mediated decreases in GH receptor numbers (12, 15, 16) and signal transduction element abundance (17) as well as up-regulated activities of the SOCS protein family (18, 19, 20) to limit JAK-STAT-mediated gene processing.

In both normal and pathological situations, a specific tissue response to GH also reflects the state of balance between activities governed by the JAK-STAT pathway (i.e. liver or muscle induction of IGF-I) (18, 20, 21), in contrast to other actions, i.e. those mediated via the phosphatidylinositol 3-kinase (PI3 kinase)-AKT pathway (9, 22). At the present time, there exist gaps in knowledge regarding molecular interactions between JAK-STAT and PI3-AKT pathway interactions that acutely and with succinct localization affect the relative state of tissue responsiveness or resistance to GH in either normal or pathological situations (9, 23).

Studies from our laboratory pointed to posttranslational nitration modification of protein tyrosine residues as a factor contributing to a temporary disruption in GH regulation of IGF-I that develops at the onset of rather low-level proinflammatory stress (24). Using an antibody to nitrotyrosine (25) as a marker for generalized protein tyrosine nitration, the endogenous tyrosine nitration product generated from peroxynitrite (ONOO–) after nitric oxide (NO) and superoxide anion have combined under increased intracellular oxidative status (25, 26), we demonstrated that the increase in nitrated proteins in liver tissue after in vivo endotoxin [lipopolysaccharide (LPS)] challenge correlated with a progressive decrease in plasma IGF-I. Although daily injection of GH increased baseline circulating concentrations of IGF-I in calves before LPS challenge, the cumulative proinflammatory response after LPS overrode the capacity for GH to stimulate IGF-I, reflecting previously published accounts of the development of the GH resistance during proinflammatory stress (11, 18). In our studies, however, {alpha}-tocopherol treatment (vitamin E, 1000 IU/d, im for 5 d) minimized liver protein nitration and maintained plasma levels of IGF-I more closely to normal, with plasma concentrations of IGF-I restabilizing more rapidly in the vitamin E-treated LPS-challenged animals. To this point, {alpha}-tocopherol recently was shown to interact with and neutralize reactive nitrogen intermediates derived from NO such as ONOO– and block the series of reactions that result in the nitration of select target molecules (27). Quite relevant to the present topic, using a modified immunohistochemistry protocol with enhanced sensitivity, we quantified the increased presence of nitrated proteins in the liver of GH-treated animals, even before the administration of the LPS challenge (28). This increased basal level of generalized tissue nitration could be tracked to four critical control points affected by GH spanning such factors as the arginine cationic amino acid transporter (CAT-2), the phosphorylation activation of endothelial NO synthase (eNOS), GH-associated decreased activity of arginase in the urea cycle, and increased xanthine oxidase activity (29).

More recently we also demonstrated that nitration of tyrosine residues 1007 and 1008 of JAK2 [tyrosine residues critical to GH/cytokine receptor phosphorylation activation of JAK2 (30, 31)] could become nitrated (32). This JAK-specific nitration was present at very low levels under basal conditions and significantly increased after endotoxin challenge concomitant with a significant decrease in cell IGF-I mRNA as quantified by in situ hybridization. Central to this observation was the data indicating that this particular nitration developed in and was colocalized to membrane caveolae and their accompanying content of endothelial nitric oxide synthase and the recently characterized caveolar localization of the GH receptor (33).

Our hypothesis here is that the GH-mediated nitration of JAK2 in critical regulatory tyrosine-associated sites serves as a novel regulatory posttranslational modification, complementary but antagonistic to phosphorylation, that can, with specificity, affect and coordinate GH signal transduction processes in discrete cell and organ locations. Therefore, the purpose of the present study was to determine steps in a pathway that could explain how tyrosine residues at positions 1007 and 1008 in JAK2 are nitrated after GH injection and whether the development of GH-mediated nitration is coordinated in any temporal manner with the development of other GH signal transduction events.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and in vivo hormone challenge
All procedures in this study involving the use of live animals were approved in accordance with the regulations and guidelines set forth by the U.S. Department of Agriculture Beltsville Animal Care and Use Committee. After veterinary health evaluation and the withdrawal time for the use of recombinant bovine GH in beef cattle (2 wk, as established in the response to our submitted Investigational New Drug Application, Food and Drug Administration Center for Veterinary Medicine, Rockville, MD), all bovine test animals were returned to the Growth Biology Laboratory Research Nutrition Herd. Test animals were male Angus x Hereford calves averaging 254 kg body weight, approximately 10 months old, n = 5); calves were fed a balanced protein and energy diet ad libitum and grew at an average rate of 1.47 kg body weight per day, a growth rate previously shown to be associated with a high level of responsiveness to a single GH challenge in terms of induction of IGF-I (24).

Blood sampling, in vivo GH challenge, and liver biopsy
One day before use in the protocol, each calf was loosely restrained in a metabolism stanchion and prepared for the GH challenge and repeated blood sampling by inserting a 14-gauge Teflon indwelling cannula (Abbocath; Abbot Laboratories, North Chicago, IL) into the right jugular vein and maintaining patency with the instillation of sterile heparinized (10 U/ml) saline. GH was injected after an initial blood sample designated as time 0; additional blood samples were obtained after the GH injection at 2, 5, 10, 30, and 60 min with a follow-up blood sample at collected 24 h after GH.

Liver tissue was used obtained from each animal as previously described (34) to assess the patterns of phosphorylation and nitration of JAK2 as stimulated by the GH challenge. Liver tissue was obtained before GH administration and at 2, 5, 10, 30, and 60 min after the administration of the GH. Three liver biopsy cores (Tru-cut biopsy needle; 14 gauge; Allegiance Healthcare Corp., McGraw Park, IL) averaging approximately 30 mg wet weight each were obtained at each time point using the depth markings on the biopsy needle and previously determined pattern of sampling that minimized or eliminated complications from vascular bleeding. Samples were either frozen in liquid N and stored at –85 C or fixed overnight in 4% paraformaldehyde in PBS, transferred to 70% ethanol, and processed by paraffin embedding for sectioning at 6 µm for immunohistochemical evaluation. For a given time point, all biopsy samples were collected within 20 sec of the desired time.

Recombinant bovine GH (Monsanto Co., St. Louis, MO) was dissolved completely in 0.1 M carbonate and bicarbonate buffer (pH 8.9) using pyrogen-free water and filtered for sterilization through a low protein binding 0.2 µm membrane. Recombinant GH was injected (100 µg GH per kilogram body weight in 10 ml) as an iv bolus to initiate the signal transduction cascade. Mean circulating basal plasma concentrations of GH before GH injection were 3.4 ± 1.1 ng/ml and exceeded 400 ng/ml in all animals 5 min after the iv delivery of recombinant GH. Responsiveness to the GH challenge and the minimization of interference on the response from the biopsy procedure was confirmed by the determination of plasma IGF-I concentrations increasing from a mean of 157 ± 22 ng/ml at time 0 to 208 ± 37 ng/ml 24 h after GH.

Plasma concentrations of GH and IGF-I
Plasma concentrations of GH and IGF-I were measured by RIAs previously validated in our laboratory (35).

In vitro modeling of hepatocyte JAK2 nitration responses to GH challenge
Porcine hepatocyte primary culture.
Because bovine hepatocytes are relatively unstable in primary culture and rapidly loose hormone responsiveness, the detailed study of JAK2 nitration was further evaluated using an established protocol for metabolically active primary cultured porcine hepatocytes. Hepatocytes for a given replicate of a study were obtained from the liver of a single male pig (~60 kg body weight), and experiments were replicated at least three times. Hepatocytes were isolated by the two-step collagenase digestion procedure previously described (36, 37). Hepatocytes (4.5 x 106) were seeded into T-25 flasks, precoated with pig tail collagen, and cultured as previously described (37). In addition, for immunohistochemical studies 106 hepatocytes in 1.0 ml media were seeded onto collagen-coated chambered (2 cm2/chamber) microscope slides (Lab-Tek, Naperville, IL). After an overnight attachment period in serum-containing medium, cells in flasks and chamber slides were washed twice and the medium replaced with serum-free William’s E medium containing 10 nM dexamethasone, 100 µM ß-mercaptoethanol, 10 mM HEPES, 10 nM Na2SeO3, 1 mM carnitine, 2 mM glutamine, antibiotics, 0.01% dimethylsulfoxide, 0.1% BSA, and 1 ng/ml bovine insulin. Cells were cultured for an additional 48 h in serum-free basal medium before experimental conditions were imposed.

In vitro GH challenge and modulation of the eNOS/AKT axis
Hepatocytes were challenged with recombinant porcine GH (kindly donated by Southern Cross Biotechnology Ltd., Toorak, Victoria, Australia). The growth media were replaced with serum-free media for 6 h; the serum-free media were aspirated from the T-25 flasks or chamber slides and replaced with the same type of media containing the GH. Recombinant GH was dissolved in the serum-free culture media for hepatocytes; hepatocytes were challenged with 100 ng/ml. For time-course studies, the activity of cells cultured in T-25 flasks was arrested with the addition of M-Per buffer (Pierce, Inc., Rockford, IL) as per the manufacturer’s instructions and supplemented with protease and phosphatase inhibitors) and gentle agitation at time ‘0' with the addition of fresh serum-GH-free media) and at 5, 10, 15, 30, 60, or 75 min after the addition of GH, as different experimental protocols dictated; the activity of cells in chamber slides was terminated by the addition of fixative (4% paraformaldehyde in PBS). Dispersed cells and contents were sonicated to a uniform consistency. The protein content measured by traditional Lowry procedure, adjusted to 10 mg/ml, aliquotted, and frozen at –85 C until analyzed.

The cascade of signal transduction processes was tracked through the development of posttranslational phosphorylations of JAK2, STAT5b, and eNOS; the nitration of JAK2; and by determining the effects of specific inhibitors of AKT and eNOS on the development of phospho-STAT5b, eNOS, and nitrated JAK2. For AKT or eNOS inhibition studies, 10 µM AKT inhibitor [1L-6-OH-CH3-chiro-inositol 2-(R)-2-O-CH3-3-O-octadecylcarbonate; Calbiochem, San Diego, CA; catalog no. 124005] or eNOS inhibitor (1 mM NG-monomethyl-L-arginine; Calbiochem) were added to the culture media 30 min before GH treatment administration. Recombinant GH was added to culture media at 100 ng/ml. Cell lysates were prepared directly using either M-PER buffer (Pierce) or ProteoExtract subcellular extraction kit buffer 1 (Calbiochem) by following the manufacturers’ instructions. Protease inhibitor cocktail for use with mammalian cell extracts and phosphatase inhibitor cocktail I and cocktail II (Sigma, St. Louis, MO) were added to lysis buffers immediately before using. Extracted proteins were frozen at –86 C until assayed. Western blot analyses were performed using standard protocols for enhanced chemiluminescence detection of specific antigen bands.

Subcellular extraction and localization of hepatocyte proteins
Compartmental localization of the nitration of JAK2 was assessed by subcellular fractionation of cultured cells. After treatment with GH at the indicated time points, proteins were extracted from hepatocytes using the ProteoExtract subcellular extraction kit (Calbiochem) following the manufacturer’s instruction. Stepwise extraction resulted in the separation of proteins into cytosol, membrane, and nuclear compartments. Caveolin-1, core histone, and glyceraldehyde 3-phosphate dehydrogenase proteins were used as markers for the membrane, nuclear, and cytoplasmic fractions, respectively, as assessed by Western blots of contemporary gels for these proteins.

Western blot protocol
SDS-PAGE performed using 4–20% gradient polyacrylamide gels (Invitrogen, Carlsbad, CA) under the nonreducing conditions as suggested by the manufacturer. Prestained molecular weight standards were included (SeeBlue-plus2; Invitrogen). Separated proteins were transferred to pure nitrocellulose membrane (Protran, 0.2 µm; Schleicher & Schuell, Dassel, Germany). Western blot analyses were performed using the following antibodies: anti-acetyl histone3, antiacetyl-phospho-histone3 (Cell Signaling Technology, Beverly, MA), anticaveolin, antiphospho-STAT5b, antiphospho-eNOS, anti-JAK2, antiglyceraldehyde 3-phosphate dehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-JAK2 (Upstate Biotechnology, Charlottesville, VA), or our antinitro-JAK2. Secondary antibodies used were horseradish peroxidase-conjugated antimouse, antigoat, or antirabbit IgG antibodies. Nonspecific binding was blocked (5% of fat-free dry milk) followed by incubation with the antibodies specified. Membranes were then washed with PBS-Triton X-100 (0.1%). Secondary antibodies (1:25,000 diluted with PBS-Triton X-100) were added and incubated for 1 h. After being washed, membranes were exposed to SuperSignal West Pico stable peroxide solution with luminol/enhancer (Pierce) and exposed to x-ray film. Resolved films of the Western blots were scanned and analyzed using Image-Pro software (Media Cybernetics, Silver Spring, MD) to quantify the gray-scale density of the bands. Protein loading per lane was estimated by Coomassie blue staining of a contemporary run control gel for each blot.

Nitrated JAK2 antibody and peptide development
In light of the information developed by Feng et al. (30) wherein an important phosphorylation activation domain of JAK-2 kinase at the 1007-Y1008Y epitope was reported, we had constructed to our specifications a series of 20-amino acid peptide analogs of the JAK-2 sequence spanning residues 1001–1020 with specific substitutions of 3'-nitrotyrosine or orthophosphotyrosine at the 1007Y or 1008Y locations; peptides were synthesized by SynPep Corp. (Dublin, CA), purified by reverse-phase HPLC to greater than 95% purity and consisted of the following sequences: 1) 1001LPQDKE-Y-Y-KVKEPGESPIFW1020; 2) LPQDKE-YOPO3-Y-KVKEPGESPIFW; 3) LPQDKE-YOPO3-YOPO3-KVKEPGESPIFW; 4) LPQDKE-YONO2-Y-KVKEPGESPIFW; 5) LPQDKE-Y-YONO2-KVKEPGESPIFW; and 6) LPQDKE-YONO2-YONO2-KVKEPGESPIFW.

An antibody highly specific to the nitrated sequence LPQDKE-YONO2-YONO2-KVKEPGESPIFW (antinitro-JAK2) was developed by covalently linking this peptide to keyhole limpet hemocyanin (KLH) and immunizing white New Zealand rabbits. Detailed characterization of this antibody was presented in the citation by Elsasser et al. (32). The singular specificity of this antibody was achieved by several solid-phase affinity immunoadsorptions against relevant peptides and proteins including peptides a, b, and c as well as agarose-conjugated KLH. Absorption of this antibody against nitrated peptides d, e, and f eliminated signals positive for nitro-JAK2 in Western blot and immunohistochemical applications.

Acquisition of data describing tyrosine nitration effects on molecular interactions and conformational changes surrounding the peptide activation epitope
Sample preparation and nuclear magnetic resonance (NMR) acquisitions.
Samples (~1.5 mg/ml) were prepared for NMR spectroscopic analysis by dissolving each peptide in 0.75 ml D2O. NMR spectra were recorded with a Bruker QE spectrometer (300 MHz for 1H) with 256 acquisitions at a spectral width of 3100 Hz using water suppression by presaturation (38). The identification of the two tyrosine, nitrotyrosine, and/or tyrosine-phosphate aromatic proton sites was detected by differential spectra subtraction and confirmed by two-dimensional-correlation spectroscopy experiments. The two-dimensional-correlation spectroscopy cross peak analysis enabled the unambiguous assignment of which sites on the tyrosine aromatic peak (phenolic ring) have the additional functional groups, i.e. whether the nitrate is ortho or meta to the -OH group, whether there is one or more nitrate groups per mole, and which of the tyrosine residues has the nitrate attached. All chemical shifts are referenced to trimethylsilylproprionic acid-2,2,3,3-d4, a water-soluble structural analog of tetramethylsilane, both of which show up at 0.0 ppm in each spectrum.

Conformational molecular mechanics
The charge and bond relationships among the native, nitrated, and phosphorylated JAK2 peptides were generated in a computational model wherein side-chain substitutions were serially imparted over an ideal peptide with the resulting effect on the structure matrix calculated. A 12-mer peptide sequence beginning and ending in a proline residue (PAAAAAAAAAAP) in an initial helical conformation was virtually generated using the computational chemistry program Alchemy 2000 (Tripos, St. Louis, MO). The program performed an iterative geometric optimization of the peptide backbone using the semiempirical electrostatics model originally described by Gasteiger and Marsili (39). The correct peptide was generated then by adding the corresponding side chain group to each of the alanine residues, i.e. forming the correct 12-mer sequence relevant to each experimental JAK2 peptide. Explicit charges of +1 were added to the -NH2 of lysine residues forming -NH3+ and charges of –1 were added to -COOH of aspartic acid and glutamic acid forming -COO–. The geometric optimization for the resultant structure was performed. At the 3' position on the 1007- and the 1008-tyrosine residues of the peptides, nitrate groups were added. At the orthophenolic site of the 1007- and/or the 1008-tyrosine residues on the peptides phosphate groups were added. Each nitrate and phosphate group was assigned a charge of (–1). Each of the chemical structures generated were geometrically optimized using the same Gasteiger-Marsili electrostatics as the native peptide. Associated visualizations of the respective molecular configurations about the 1007Y-1008Y epitope were rendered using Hyper-Chem molecular imaging software (version 5.11; Hyper Cube, Gainesville, FL), as previously described (40) with the relevant nitrates, phosphates, and phenolic rings of the 1007 and 1008 tyrosines recolored using Photoshop (Adobe, San Jose, CA) for improved spatial visualization.

In vitro phosphorylation assay
To study the effect of a 3'-phenolic nitration on the capacity for the respective 1007 or 1008 tyrosine(s) to be enzymatically phosphorylated in vitro, the native and nitrated peptide analogs of the JAK21007Y-1008Y were incubated in a mixture of hepatocyte cell extract and a reaction buffer containing 60 mM HEPES-NaOH (pH 7.5), 3 mM MgCl2, 3 mM MnCl2, 1.2 mM dithiothreitol, 2.5 mg/50 ml polyethylene glycol (PEG) 20,000, 10 µM ATP, and 1 ml of phosphatase inhibitor cocktails I and II (Sigma; P-2850, P-5726 diluted 1:100 per manufacturer’s protocol). The reaction for each peptide in its own tube was stopped after 30 min at 37 C by the addition of 2x sodium dodecyl sulfate sample buffer and immersing the tube in a 90 C water bath. Aliquots of the reaction mixtures were individually loaded into lanes of 4–20% acrylamide gels with the 1007Y-phosphorylated peptide loaded into an additional lane to serve as a positive control for both molecular weight and functionality of the antiphosphotyrosine antibody used (Upstate Biotechnology, Charlottesville, VA). Antiphosphotyrosine was diluted to 1 µg/ml for Western blot use. Before use this antibody also was absorbed against solid-phase bound nitrated JAK2 peptides and agarose-KLH to eliminate a very low level of nitrotyrosine cross-reactivity present in the neat antibody preparation.

Quantitative immunohistochemistry
Biopsy tissue for immunohistochemistry was fixed in 4% paraformaldehyde for 16 h and transferred to 70% ETOH. Paraffin sections P/(6 µm) were mounted on plus-charged slides. Tissue sections were hydrated and peroxidase blocked by standard methods. Sections were treated for 10 min with 0.05% Triton X-100, and washed in Tris. Cells on chamber slides were fixing in 4% paraformaldehyde for 20 min, washed twice with Tris saline and permeabillized for 10 min with 0.05% Triton X-100 in Tris saline and rinsed in Tris saline. Immunostainings for 1007Y-phospho-JAK2 (Upstate Biotech) and phospho-STAT5b (Santa Cruz Biotechnology) were performed according to traditional avidin-biotin complex (ABC; Vecta-stain Elite; Vector Labs, Burlingame, CA) and strepavidin (Santa Cruz Biotechnology) antigen visualization protocols, respectively. After blocking for 30 min with species-appropriate control serum in 1% casein-Tris saline (Bio-Rad, Hercules, CA), primary antibody was applied (2 µg/ml) and further incubated overnight at 4 C in a humidified chamber. Antigen visualization was accomplished using 3'-diaminobenzadine tetrahydrochloride (DAB) deposition in the presence of horseradish peroxidase and the ABC method. All relevant tissue or cell samples were batch processed in a single run to eliminate interprocedural daily variation in results. Because of the low abundance of generated antigen and need to increase the resolution of the fine punctuate antigen loci, immunolocalization of nitro-JAK2 was accomplished by biotinylated tyramine enhancement as per the manufacturer’s specifications (Molecular Probes, Eugene, OR). Quantitative measures of nitration of the 1007Y-1008Y epitope of JAK2 were obtained using rabbit antinitro-JAK2 at 1:2500 or 1:5000 dilution.

The validation criteria and procedure for the quantitative immunohistochemical assessment of antigen pixel density was previously detailed and published (27). Each section was photographed at full illumination power using an BX-40 microscope (Olympus, Center Valley, PA) equipped with an Olympus DP-70 digital camera. The digitized picture files for each image were analyzed using the Image-Pro Plus Image analysis software (version 4.5.1; MediaCybernetics) through a standardized protocol (28) based on the intensity of DAB color-specific staining as defined through color cube segmentation of spectrum-specific wavelengths, hues, and intensities. The discriminators for specificity were set conservatively as per the image analysis program to ensure that false positives were not encountered. Control slides of tissues from animals previously determined to be positive or negative for the presence of nitrotyrosine (25) were used in each staining procedure in line with validation criteria outlined previously (41, 42).

Statistical analysis
Data were statistically analyzed by ANOVA using the mixed-model procedures of SAS (SAS Institute, Cary, NC) with appropriate contrast statements (43).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of a single iv administration of recombinant bovine GH on the generation of nitrated and phosphorylated JAK2 in bovine liver is shown in Fig. 1Go. Before GH challenge, tissue antigen levels of both nitro-JAK2 and phospho-JAK2 were low but detectable and were observed by immunostaining as diffuse punctate loci more localized to the perimeter of cells near the cell walls (Fig. 1AGo). After GH, tissue levels of both nitro-JAK2 and phospho-JAK2 were numerically increased by the 5-min sampling but statistically unremarkable in terms of whether one antigen or the other really dominated in the rapidity of increase. The increase in phospho-JAK2 was indicative of the fact that biochemical events associated with the initiation of the GH signal transduction cascade did, in fact, occur. Mean pixel densities (Fig. 1BGo) representative of phospho-JAK2 and nitro-JAK2 increased 3- to 4-fold (P < 0.05) within 15 min of the administration of the GH bolus. Whereas tissue levels of phospho-JAK2 antigen progressively declined from this point to near baseline levels by 30 min, the post-GH increase in nitro-JAK2 antigen continued past 15 min and was highest at the 30-min point of this cell harvest sampling (P < 0.02 vs. time 0).


Figure 1
View larger version (74K):
[in this window]
[in a new window]

  		FIG. 1. Time course for the generation of nitro-JAK2 and phospho-JAK2 in liver tissue from calves (n = 5) before and after the administration of bovine GH (100 µg/kg body weight, iv). A, Immunohistochemical identification of antigens specific for nitro-JAK2 (biotinylated tyramine enhancement method) and phospho-JAK (ABC method) at time 0, +15, and +30 min relative to the GH injection photographed through the x100 objective. Red pixels indicate the image analysis software identification of pixel color specific for the visualization reporter diaminobenzidine. B, Mean pixel density (± SEM) for nitro- and phospho-JAK2 identified counted by the Image-Pro image analysis algorithm; *, P < 0.05; **, P < 0.02 relative to the pixel density at time 0 (before GH).

 
To model more precisely the capability of GH to generate this tyrosine nitration in JAK2, we used a primary hepatocyte culture system known from previous work in our laboratory to be highly sensitive and rapidly responsive to manipulation of cellular metabolism by GH. With interpretation of responses based on the immunostaining of hepatocytes for nitro-JAK2 and phospho-JAK2, treatment of cells with GH resulted in a rapid increase in antigen-specific pixel density for both posttranslational tyrosine modifications (Fig. 2Go). The data are consistent with the concept that differences in the time course of generation and remission of phosphorylation and nitration events may reflect interactions of pathways that result in a locally defined break in the further progression of signal transduction events mediated via JAK2.


Figure 2
View larger version (95K):
[in this window]
[in a new window]

  		FIG. 2. Immunohistochemical localization of the generation of phospho-JAK2 and nitro-JAK2 as a function of time after addition of GH to primary cultured porcine hepatocytes. Monolayer cultures on slides were challenged, washed, and fixed immediately (time 0) or 10 or 30 min after addition of fresh basal medium alone or containing 100 ng/ml recombinant porcine GH. The brown color represents diaminobenzidine deposition associated with the respective antigens. Nuclei, blue, were counterstained with Carrazzi’s hematoxylin. The pictures are representative of three replications of this aspect of the study. eNOSi, eNOS inhibitor.

 
The porcine hepatocyte primary culture system was further used as a model system to examine whether the nitration of JAK2 might develop in association with the ability for GH to modulate the activation state of the NO generating enzyme eNOS. We preconditioned hepatocytes in media containing AKT and eNOS inhibitors and looked at the effect on the generation of nitro-JAK2 by GH (Fig. 3Go). Western blot data for immunoquantification of nitro-JAK2 clearly indicated that mean nitro-JAK2 band intensity increased approximately 4.5-fold at 10 min and nearly 6-fold at 30 min after the application of the GH challenge. The similar washing of cells and switching of media (excluding GH) as performed in control flasks had no effect on the nitration of JAK2 (Fig. 3Go, -GH, 0, 10, 30 min). In contrast, at both the 10-min (P < 0.05) and 30-min (P < 0.03) time points, the magnitude of the GH-specific generation of the nitro-JAK2 was decreased by the pretreatment of cells with AKT and eNOS inhibitors.


Figure 3
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 3. Effects of AKT inhibitor (AKTi) or eNOS inhibitor (eNOSi) on GH-stimulated nitration of JAK2. Monolayer cultures in T-25 flasks were washed twice in warm HEPES-buffered saline, and cells were harvested at immediately (T0) or 10 or 30 min after addition of fresh basal medium alone or one containing 100 ng/ml recombinant porcine GH. In hepatocyte cultures containing AKTi (10 µM) or eNOSi (1 mM), inhibitors were added 30 min before addition of GH. The blot represents one replicate of three independent experiments in which cells (two to three flasks per condition and time point) for each replication were obtained from a different pig. Protein loading reflects the Coomassie staining of an identical contemporary gel. The bars in the graph represent the mean (±SEM) Western blot band densities of nitro-JAK2 summated across all replications.

 
In another hepatocyte culture experiment, subcellular fractionation was used to determine whether the intracellular generation of JAK2 by GH could be localized to a given compartment. When cells were differentially processed by buffer extraction protocols to obtain subcellular fractions corresponding to cytosol, membrane, and nuclear compartments, it was clear that the nitration event was associated with the membrane and nuclear fractions (Fig. 4Go). Nitro-JAK2 was minimally detected in cytosol fractions in either the presence or absence of the GH challenge. A low level of nitrated JAK2 was observed in the membrane fraction in the absence of GH; the density of the corresponding band 30 min after GH treatment of cells increased approximately 4.5-fold. Interestingly, however, before GH the intensity of the nitro-JAK2 signal in the nuclear fraction was relatively high (~50% of the +30 min value of the membrane fraction) with only a slight additional increase in band density apparent 30 min after GH. The effectiveness of the GH challenge to launch the signal transduction cascade was again confirmed in a set of complementary Western blots wherein phospho-JAK2 and phospho-STAT5b generation was tracked. Phospho-JAK2 was mainly present in membrane fractions and increased slightly (~45%) after GH; phospho-STAT5b was not apparent before GH in either cytosol or membrane fractions, although a low level was detectable in the nuclear fraction. After GH, a small increase in phospho-STAT5b was detected in the membrane fraction with a 7-fold higher abundance found in the nuclear fraction.


Figure 4
View larger version (93K):
[in this window]
[in a new window]

  		FIG. 4. Western blot identification of nitro-JAK2 and phospho-STAT5b after the introduction of porcine GH- (100 ng/ml) containing media to primary cultures of porcine hepatocytes. Cells were treated with a sequential series of buffer treatments specific for the subcellular fractionation of the hepatocytes into cytosolic, membrane, and nuclear fractions. After PAGE and transfer to nitrocellulose, the cellular fraction proteins were blotted with antinitro-JAK2, antiphospho-JAK2, and antiphospho-Stat5b. The blot is representative of random duplicates of three culture flasks for each time and condition. The experiment shown was typical of each of three replicated experiments using cells isolated each time from a different pig. Effective subcellular fractionation was confirmed using additional Western blot determinations of markers specific for each fraction as indicated. Lanes were loaded with the same percentage of total extract from the original mixture (e.g. 5 µl of the total 750 µl). GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

 
The involvement of the AKT/eNOS component of this JAK nitration process was further investigated in a series of light microscopy studies wherein hepatocytes were challenged with GH in the combined presence of AKT and eNOS inhibitors. The patterns of nitro-JAK2 and phospho-STAT5b antigens are shown in Fig. 5Go. Microphotographs localizing the generation of antigens specific for phospho-STAT5b and nitro-JAK2 are illustrated in Fig. 5Go, A and B (DAB immunostaining). Immunostaining revealed that GH increased both nitro-JAK2 and phospho-STAT5b generation after GH by 30 min. Where the color intensity of DAB specific to phospho-STAT5b did not significantly change between 30 and 60 min after GH, nitro-JAK2 immunostaining intensity continued to increase through 60 min after GH. Where this pattern was reflected again in Fig. 5BGo from a different validation experiment, additional views indicated that the presence of AKT and eNOS inhibitors alone for 60 min had no effect on phospho-STAT5b but did decrease nitro-JAK2 immunostaining intensity from that visualized at time 0. When the red pixels generated by the image analysis algorithm (Fig. 5CGo) were statistically analyzed (Fig. 5DGo), the data indicated that the presence of AKT and eNOS inhibitors decreased the relative abundance of GH-stimulated nitro-JAK2 by 15% (P < 0.05) at the 30-min and 80% (P < 0.02) at the 60-min points after GH, respectively, further supporting a role for the AKT/eNOS component of this response. An unexpected effect of the presence of AKT and eNOS inhibitors was observed in regard to the generation of phospho-STAT5b. Antigen levels of phospho-STAT5b increased 2- to 3-fold between time 0 and time 30 with significant levels of nuclear localization; however, the presence of AKT and eNOS inhibitors dramatically augmented the presence of phospho-STAT5b at 30 min (2.5-fold, P < 0.05) and 60 min (6-fold, P < 0.02).


Figure 5
View larger version (51K):
[in this window]
[in a new window]

  		FIG. 5. The effects of AKT and eNOS inhibitors (eNOSi) on the generation of nitro-JAK2 and phosphorylation of STAT5b in primary cultures of hepatocytes challenged with porcine GH (100 ng/ml). A, Actual antigen-associated diaminobenzidine immunostaining (brown pixels) for the nitrated JAK and phosphorylated STAT5b antigens. B, A control experiment further defining the effects of AKT and eNOS inhibitors on the GH-associated change in phosphorylation of STAT5b and nitration of JAK2. Cells were fixed at 60 min after changing to media devoid of (–) or containing (+) 100 ng/ml GH and in conjunction with the indicated (+ or –) presence of inhibitors. Antigen localization indicated by brown pixels with nuclei stained blue. C, Antigen-specific nitrated JAK2 and phospho-STAT5b pixels from A representative of antigens identified for quantification by the image analysis algorithm (red pixels). D, Mean antigen pixel densities (± SEM) for nitrated JAK2 and phosphorylated STAT5b. Data represent the means of three to four determinations per observation time point; red (STAT5B) or blue (nitro-JAK2) letters represent differences from time 0: a, P < 0.05; b, P < 0.03; c, P < 0.02. Red or blue asterisks represent statistical inferences for the pixel density comparisons at 30 and 60 min after the initial exposure to GH (100 ng/ml) in the presence of AKT and eNOS inhibitors. *, P < 0.05; **, P < 0.02.

 
Whereas the presence of JAK2 nitration as a unique posttranslational modification of this important GH signal transduction element was interesting in itself, we considered it important to assess what impact the generation of this nitration had on JAK2 phosphorylation at this site. We addressed this point in two ways: 1) solution phase-high field NMR analysis coupled with molecular modeling of the native, nitrated, and phosphorylated peptide analogs of this 1007Y-1008Y activation epitope and 2) in vitro, enzyme-catalyzed phosphorylation reactivity of the different peptide analogs. NMR and molecular modeling provided data on the impact of tyrosine nitration and phosphorylation on charge and hydrophobic interactions, spatial bond distances, and planes of rotation regarding the relative change in spatial positioning of the affected tyrosine phenolic rings and associated neighboring amino acids. The addition of the nitro group to the tyrosine at the 3'-phenolic position shifted the proton-{alpha} to the nitro group down field 0.10 ppm from the same site in native tyrosine. When two tyrosines had nitro groups, the chemical shift down field was amplified (i.e. 0.407 ppm shift). The effect on the proton-{alpha} to the tyrosine phenolic OH– group was also interesting. There are two doublets of doublet centered at 7.049 and 6.809 ppm, respectively. With one nitro group present, the up field peak shifted 0.090 ppm down field, and the second shifted 0.12 ppm. When two nitro groups were present, the first peak decreases approximately 90%, whereas the second peak was correspondingly larger, again with a chemical shift of 0.12 ppm.

Computer-generated, molecular modeling images of the specific tyrosines in JAK2 located at the 1007 and 1008 positions and associated nitrations and phosphorylations in respect to other amino acids in the peptide are highlighted in Fig. 6Go. In Fig. 6Go (top panel), the significant hydrophobic bonding between the 1007 tyrosine and 1006 glutamic acid and between 1008 tyrosine and 1004 aspartic acid largely were determined to establish the relative spatial orientations of the tyrosine phenolic rings. In Fig. 6Go (bottom panel), specific rotational changes to these phenolic groups were effected with the presence of 3'-nitrations. Peptide epitope rotation in space empirically was different between nitrated and phosphorylated peptides, especially as regards the 1007 tyrosine, even when only the 1008 tyrosine was nitrated. Relative to the presented initial orientation of the phenolic groups in the native peptide, nitrate on the 1007Y caused a slight forward-clockwise rotation on the 1008 tyrosine and a backward-counterclockwise rotation of the immediate 1007 tyrosine. Interestingly, whereas nitration of the 1008 tyrosine resulted in only small apparent deviations in 1008 tyrosine orientation, the rotational effect on the neighboring 1007 tyrosine was greater than that that occurred when the 1007 tyrosine was nitrated itself. Molecular modeling algorithms indicated that nitration caused a charge destabilization between the orthohydroxyl of each tyrosine phenolic ring with a respective aspartic acid or glutamic acid residue wherein the stabilizing effect of the acidic residues was impaired. Furthermore, the presence of the nitrate at the 3' position on the ring was suggested to spatially compromise the ability for a phosphate to be transferred from ATP to the adjacent ortho position on each tyrosine.


Figure 6
View larger version (62K):
[in this window]
[in a new window]

  		FIG. 6. Molecular representation of tyrosine residue relationships within native, 3'-nitrated, and ortho-phosphorylated analogs of the 1007Y-1008Y phosphorylation activation region of JAK-2. Global energy-minimized three-dimensional structures of the peptides were constructed using the computational chemistry MOPAC (molecular orbital package) software (Alchemy 2000, version 2.0; Tripos) and visually rendered using Hyper-Chem molecular imaging software (version 5.11; Hyper Cube). In the top panel, the critical 1007 and 1008 tyrosine phenolic rings of the native peptide are depicted in purple, nitrogen in blue, oxygen in red, and hydrogen in pale green. Hydrogen bonding and electrostatic forces between 1007Y and 1006E and between 1008Y and 1004D were determined to participate in stabilizing the planar orientation of the phosphorylation epitope. In the bottom panel, nitration substitutions in the 1007Y and 1008Y effect planar rotation of the respective tyrosines and cause destabilization of orientations more favorable to phosphorylation.

 
Our interpretation of the physical chemical data predicted that the presence of a nitrate on a tyrosine largely would compromise the ability for JAK2 to be phosphorylated on the same tyrosine, the 1007Y in particular. The consequence of nitration on tyrosine kinase-facilitated peptide tyrosine phosphorylation is illustrated in Fig. 7Go. When the native peptide and its associated nitrated analogs were used as substrates for an in vitro phosphorylation reaction, it was apparent that the presence of nitration in either the 1007 or 1008 tyrosine positions completely blocked the ability for a phosphorylation event to occur (Fig. 7AGo). As can be seen in the Western blot resolved for phosphotyrosine, the native JAK2 peptide received the highest level of phosphorylation, a band that was generated at the same relative molecular weight as the phospho-JAK2 peptide added to the gel and blotted to serve as a positive control. The level of phosphotyrosine observed in the peptide containing nitrotyrosine in both the 1007 and 1008 residues amounted to less than 2% that measured in the native peptide.


Figure 7
View larger version (50K):
[in this window]
[in a new window]

  		FIG. 7. Top, Western blot of synthetic peptides representing natural sequence (Y-Y) and nitrated analogs (lanes 2–4) of a 20-amino-acid region surrounding the 1007Y-1008Y phosphorylation epitope of JAK2 subjected to an in vitro kinase phosphorylation reaction and resolved using antiphosphotyrosine antibody. The positive control, phosphotyrosine-JAK2 epitope peptide (pY-Y), was added to the gel immediately before SDS-PAGE. Bottom, Western blot for the nitrated peptides resolved using the antibody specific to nitrated JAK2 confirmed that these peptides migrated within the gel and could be localized to a relative mobility similar to that of the phosphorylated native peptide and the internal control phosphorylated peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Published information from previous experiments in our laboratory suggested a link between GH, the phosphorylation activation of eNOS and generalized liver cell protein nitration (28). This observation complemented and partially explained previous findings suggesting that proinflammatory tyrosine nitration in the liver was directly linked to loss of regulation of IGF-I, but the effect was modulatable by administering antioxidant interventions before the immune challenge (24). The present study was specifically targeted toward increasing our understanding of the biochemical steps that lead to low-level tyrosine nitration in GH-treated animals and the consequences to signal transduction that accompany such a posttranslational modification in JAK2.

With the use of a highly specific antibody generated to our specifications, we determined that one of the sites this tyrosine nitration targeted in JAK2 was 1007Y-1008Y, a site first suggested by Feng et al. (30) to be autophosphorylated and critical to the progression of the GH signal transduction cascade. In comparison with the multiplicity of JAK2 phosphorylation sites (44, 45, 46), this 1007Y-1008Y site became interesting when it was realized that protein tyrosine nitration in vivo was closely associated with a specific amino acid sequence motif that presented the phenolic ring of tyrosine to the reacting peroxynitrite in an orientation energetically quite favorable to transnitration (47). Molecular modeling studies of the peroxynitrite-based nitration of other proteins clearly indicated that specific hydrophobic and charge interactions between amino acids in the microenvironment immediately surrounding a given protein tyrosine profoundly impacts the propensity for that tyrosine to be nitrated (47). Experiments by Lanone et al. (47) indicated that tyrosine nitration in proteins was facilitated by a rather specific motif characterized as aspartate (D)/glutamate (E)/glutamine (Q)-X3–6-K-X0–1-Y-X4–7-D/E in which tyrosine was flanked by D or E or Q and lysine (K) as indicated. In their study, tyrosine nitration was targeted to this very site in the specified motif, and the nitration modification here was associated with complete loss of enzymatic activity of the type 2/inducible NOS protein studied. This concept is in close agreement with the conclusions of Souza et al. (48) and Crow et al. (49), who suggested that the propensity for a tyrosine in a peptide sequence to be nitrated resided in its proximity to acidic residues such as aspartate and glutamate. Based on the semiemperic Gasteiger-Marsili electrostatic modeling of the peptides, our analysis indicated that the aspartate and glutamate residues in positions 1004 and 1006, respectively, were critical to stabilizing that section of JAK2, at least as far as the present analysis of this truncated JAK2 region permitted. A similar effect was described by Sandberg et al. (50) in which the hydrogen bonding between 1024glutamate and 1013arginine was demonstrated as regions critical to JAK2 kinase function. Furthermore, the associated proximity of the 1003glutamine and 1015glutamate in addition to the 1005 lysine identify this region of JAK2 as a target for nitration as defined by the Lanone criteria (47). Examination of amino acid sequences flanking 10 residues to each side of the JAK2 tyrosines positioned at locations 221, 570, and 813 did not fit this profile and, as such, were less likely a nitration target than was the 1007Y-1008Y site. The fact that some tyrosines appear as favorable targets for nitration, whereas others are not further increases the likelihood that many of the discrete functions of JAK2 in specific molecular and physiological situations may be subtly modulated by the pattern of phosphorylations and nitrations that reshape the three-dimensional structure of JAK2 affecting the activity of the kinase domains as well as the presentation of binding motifs.

Two features are significant regarding the presence of nitration in the 1007 and 1008 tyrosine residues of JAK2. First, nitration is associated with the epitope existing in a spatial orientation different from that associated with the phosphorylated peptide. The implication of this finding is that the nitration-based orientation is unfavorable for enzymatic activity as inferred by the presence of orthophosphate. Second, the presence of nitration blocks phosphorylation. If we assume that the nitrated version is inactive, then the presence of nitration appears to impair further activation of the affected molecule by phosphorylation. Although still not specifically addressed in the present set of experiments, our laboratory is actively pursuing the greater issue of structure-activity relationships as regards these chemical modifications to JAK2.

Our molecular modeling data obtained with the 20 amino acid fragment of the SH1 tyrosine kinase domain compare favorably with recent data on intramolecular bond forces and 2 Å crystal structure of a recombinantly expressed JAK2-SH1 (residues 835-1132; protein database designation 2B7A) region (51). Analysis of the 297-amino acid fragment revealed that significant rotational change is present in this region when phosphorylated at position 1007 with concurrent formation of new stabilizing hydrogen bonds largely associated with lysine (positions 999, 1005, 1009, 1030), arginine (positions 897 and 971), and glutamic acid (position 1006). The result of the rotational change is to unbury the catalytic site from the depths of the more hydrophobic interior. The relative large degree of change in spatial orientation and reordered intramolecular forces observed with the recombinant JAK2 JH1 region upon phosphorylation (compared with that seen in our 20 amino acid peptide) implies that the consequences of nitration as determined with our nitrated and phosphorylated peptide analysis may even be underestimated. The lysine 1030 and 999 residues and associated hydrogen bonds needed to stabilize the 1007 and 1008 phosphotyrosines, respectively (51), were not present in our preparation. However, the presence of 3'-nitration at the 1007 and 1008 tyrosines would have compromised this intramolecular attraction and may have thwarted the needed planer rotation.

Data in this study provide the first evidence for a nitration event occurring in a significant signal transduction component of the GH axis with consequences that challenged the capacity for this site to be phosphorylated. If we focus on the violet phenolic rings of the 1007 and 1008 position tyrosines (Fig. 6Go), we immediately see that the spatial planar orientation of the rings becomes altered as a 3'-nitration occurs at either or both of the target tyrosines. More specifically, there occurs a destabilization of the interacting amino acids in the immediate region. Preliminary data (not presented) generated from Western blotting (antinitro-JAK2 blots of antiphospho-JAK2 immunoprecipitated proteins and antiphospho-JAK2 blots of antinitro-JAK2 immunoprecipitates) of GH-treated hepatocyte homogenates indicated that nitration of either the 1007 or 1008 tyrosine eliminated phosphorylation capacity. Beyond the scope of the present paper, we intend to study this further using laser dissection and matrix-assisted laser desorption ionization mass spectrometry analysis.

Four separate and independent in vitro experiments were conducted and up to three sets of observations (some redundant across experiments) obtained to define a potential mechanism through which this nitration might have evolved. Each experiment used a primary hepatocyte culture system, highly sensitive to GH effects on metabolism, and in each separate experiment the event common to all results was that the addition of media containing GH to hepatocytes resulted in the rapid generation of nitration events at the 1007Y and 1008Y JAK2 residues. We based our assumption for a molecular process for these nitration events on the following facts: 1) tyrosine nitration has been reported to occur in vivo through mechanisms involving the generation of nitric oxide and superoxide anion that interact to form peroxynitrite (36, 41); 2) these events to be closely positioned in a complementary spatial arrangement (52); 3) the localization of this GH-driven nitration event to a membrane-based subcellular fraction is consistent with associated studies from our laboratory demonstrating that membrane caveolae serve as work benches for the nitration of these same tyrosine residues to occur in association with the proinflammatory response (32); and 4) the GH receptor itself has been localized to caveolar structures (33).

The alignment of our present data with that of our associated study (32) permits the conclusion that localization of the AKT/eNOS/JAK2 components to this membrane/caveolae location facilitates the regulation of tightly coupled, proximity-based aspects of GH signal transduction involving tyrosine nitration in perhaps very localized, discrete tissue regions. This might be a mechanism through which discriminating actions of GH can be advanced or retarded among tissues with differing metabolic priorities (10). In addition, the temporal differences in the increases and decreases in cellular content of phospho-JAK, compared with nitrated-JAK might play a role in some aspects of the very acute and brief patterns of GH insensitivity that have been documented in experiments in which GH was pulsed into cell cultures. Acute self-induced GH insensitivity (as contrasted with the chronic resistance observed in stark pathology) has been recognized as early as 1994 and was associated with a variety of causes including a sudden decrease or internalization of GH receptors, increased ubiquitination-dependent GH receptor turnover, changes in protein synthesis state, and associated enzyme activities including phospholipase C (52, 53). Our measured temporal patterns of increased and decreased cell signals of JAK-STAT phosphorylation after GH closely matched the time course published by Fernandez et al. (54). In their study, repeated pulsing of cells with GH only led to resensitization of a GH response some time between 1 and 2 h after an initial GH challenge, the result of which was transient self-desensitization. To this point, however, it was apparent that where the generation of nitrated JAK2 was perturbed by pretreatment of hepatocytes with inhibitors of AKT and eNOS, the apparent responsiveness of cells to the single GH challenge was both prolonged and augmented, if the assumption can be made safely that the measured levels of phospho-JAK2 and phospho-STAT5b are indicative of maintained responsiveness.

In summary, our interpretation of the combined data from this study regarding the purpose of JAK nitration is that this posttranslational modification of JAK2 after GH serves as a modulator of phosphorylation activity by limiting aspects of the forward progression of the JAK2 signal cascade. In addition, this JAK nitration serves as a mechanism to provide cross talk between the JAK/STAT and PI3 kinase/AKT pathways. To this point we have demonstrated that the progression of the GH signal cascade via the JAK/STAT pathway is influenced by AKT and eNOS wherein the suppression of AKT and eNOS activity appears to augment the phosphorylation of STAT5b. In all likelihood the identified phosphorylated and nitrated molecules represented different populations of JAK2.

A conceptual model for the regulation of GH signal transduction progression via JAK2 tyrosine nitration is depicted in Fig. 8Go. In reference to the numbered locations in the figure, sites along the left side of the GH receptor reflect the traditional understanding of JAK/STAT/SOCS regulation of the progression of the GH signal transduction process; sites along the right side of the GH receptor depict the steps proposed in the generation of the nitrated JAK2. Literature citations and justifications for many of the components of this model have already been referenced in the introduction to this paper.


Figure 8
View larger version (64K):
[in this window]
[in a new window]

  		FIG. 8. Proposed mechanism through which GH directs the nitration of JAK2. Red and green arrows between control sites and interacting elements indicate facilitating and inhibitory pathways and influences, respectively. Please refer to text for pathway details. GH-R, GH receptor.

 
The membrane/caveolae localized GH receptor subunits dimerize when GH binds launching a cascade of auto- and transphosphorylation reactions (site 1). GH receptor functionality itself is modulated through several mechanisms involving autophosphorylation, shuttling, sequestration and internalization, dephosphorylation, and ubiquitin-associated proteosome degradation after GH binding. Numerous signal transducing events are recognized as activated after GH binding: JAK/STAT, PI3 kinase/AKT, Src, and calcium flux among others (3, 55). Key in the progression of GH regulation of IGF-I is the phosphorylation of several tyrosine residues in JAK2, which increase the kinase activity of the molecule (site 2). Activated (phosphorylated) JAK2 functions to further phosphorylate STAT5b, which facilitates its dimerization and subsequent translocation to the nucleus (site 3). Counterregulation of the JAK/STAT activation occurs when suppressor molecules such as SOCS interact with specific JAK2 domains limiting the further progression of the needed phosphorylation reactions (15, 55) (site 4) or when specific phosphatases strip phosphate off the tyrosines returning the kinase activity to a basal state. In another set of interactions, GH binding to its receptor also launches additional signal transduction pathways, in particular the PI3 kinase-based reactions.

We incorporated into our model the literature claim that AKT modulated a part of the activity of eNOS by phosphorylating a critical 1177serine residue in eNOS (56). Data from our study generated with the use of specific AKT and eNOS inhibitors indicated that GH increases the activity of AKt/phosphorylase B kinase (site 5) to phosphorylate resulting in an increase the enzymatic activity of eNOS (an NO-generating enzyme integral to caveolin within the caveolae) without a significant change in eNOS protein content (eNOS, site 6) (28). The caveolae-localized eNOS generates NO from arginine entering the hepatocyte; in the presence of superoxide anion generated by proximity-based xanthine oxidase the reactive nitrating species peroxynitrite (ONOO–) is generated (site 7). Potentially this serves as a brake on the forward progression of JAK/STAT signaling wherein nitrated JAK2 molecules would not be eligible for participation in de novo activation via phosphorylation specifically in the caveolar/membrane regions in which the events leafing to nitration develop (site 8). Finally, the fate of the nitrated JAK2 may be determined whether there occurs further ubiquitination of this JAK and therefore proteosomal degradation (57) or whether a purported denitrase is able to regenerate native JAK from the nitrated state (site 9) (58). In addition, at the present time, it was beyond the scope of the present paper to speculate on why nitrated JAK2 might be so prominently detected in the nucleus; to this point though, recent data indicate that JAK2 may itself function as a nuclear transcription factor (59), and this raises the possibility for nitrated JAK2 to function in a similar manner, perhaps with a different gene selectivity or even antagonistically to native JAK2. The GH-associated nitration processes affecting JAK2 presented here complement and extend the series of events and factors that impact the negative regulation of GH-GH receptor functions (3, 54, 55). In the future, an expanded characterization of these signal transduction nitrations may help to explain many of the tissue/time-specific effects of GH that color the spectrum of actions attributed to GH.


   Footnotes
 
This article is a U.S. Government work under the auspices of the U.S. Department of Agriculture and, as such, is in the public domain in the USA. Mention of a brand name or product does not constitute an endorsement of that product by the U.S. Government where other suitably related products are adequately functional in a similar application.

Disclosure Summary Statement: The authors have nothing to disclose.

First Published Online May 17, 2007

Abbreviations: ABC, Avidin-biotin complex; DAB, diaminobenzadine tetrahydrochloride; eNOS, endothelial NO synthase; JAK, Janus kinase; KLH, keyhole limpet hemocyanin; LPS, lipopolysaccharide; NMR, nuclear magnetic resonance; NO, nitric oxide; ONOO–, peroxynitrite; PI3 kinase, phosphatidylinositol 3-kinase; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription.

Received December 27, 2006.

Accepted for publication May 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kossiakoff AA 2004 The structural basis for biological signaling, regulation, and specificity in the growth hormone-prolactin system of hormones and receptors. Adv Protein Chem 68:147–169[Medline]
  2. Leroith D, Nissley P 2005 Knock your SOCS off! J Clin Invest 115:397–406[CrossRef][Medline]
  3. Waters MJ, Hoang HN, Fairlie DP, Pelekanos RA, Brown RJ 2006 New insights into growth hormone action. J Mol Endocrinol 36:1–7[Abstract/Free Full Text]
  4. Huang Y, Kim S, Yang N, Jiang J, Frank SJ 2004 Physical and functional interaction of growth hormone and insulin-like growth factor-1 signaling elements. Mol Endocrinol 18:1471–1485[Abstract/Free Full Text]
  5. Yoshizato H, Tanaka M, Nakai N, Nakao N, Makashima K 2004 Growth hormone (GH)-stimulated insulin-like growth factor I gene expression is mediated by a tyrosine phosphorylation pathway depending on C-terminal region of human GH receptor in human GH receptor-expressing Ba/F3 cells. Endocrinology 145:214–220[Abstract/Free Full Text]
  6. Thissen JP, Underwood, LE, Ketelslegers JM 1999 Regulation of insulin-like growth factor-1 in starvation and injury. Nutr Rev 57:167–176[Medline]
  7. Rosenfeld RG, Hwa V 2004 New molecular mechanisms of GH resistance. Eur J Endocrinol 151:11–15[CrossRef]
  8. Waxman DJ 2000 Growth hormone pulse activated STAT-5 signalling: a unique regulatory mechanism governing sexual dimorphism of liver gene expression. Novartis Found Symp 227:61–81[Medline]
  9. Ji S, Frank SH, Messina JL 2002 Growth hormone-induced differential desensitization of STAT5, ERK and AKT phosphorylation. J Biol Chem 277:28384–28392[Abstract/Free Full Text]
  10. Elsasser TH, Klasing KC, Filipov N, Thompson F 2000 The metabolic consequences of stress: targets for stress and priorities of nutrient use. In: Moberg G, Mench J, eds. The biology of animal stress: basic principles and implications for animal welfare. New York: CABI Publishing; 77–110
  11. Lang CH, Frost RA 2002 Role of growth hormone, insulin-like growth factor-I, and insulin-like growth factor binding proteins in the catabolic response to injury and infection. Curr Opin Clin Nutr Metab Care 5:271–279[CrossRef][Medline]
  12. Gebert CA, Park SH, Waxman DJ 1999 Termination of growth hormone pulse-induced STAT5b signaling. Mol Endocrinol 13:38–56[Abstract/Free Full Text]
  13. Gu F, Dube N, Kim JW, Cheng A, Iberra-Sanchez Mde J, Tremblay ML, Boisclair YR 2003 Protein phosphatase B1 attenuates growth hormone-mediated JAK-STAT signaling. Mol Cell Biol 23:3753–3762[Abstract/Free Full Text]
  14. Pasquali C, Chruchod M, Wälchli S, Espanel X, Guerrier M, Arigone F, Strous G, van Huijsduijnen R 2003 Identification of protein tyrosine phosphatases with specificity for the ligand-activated growth hormone receptor. Mol Endocrinol 17:2228–2239[Abstract/Free Full Text]
  15. Flores-Morales A, Greenhalgh CJ, Norstedt G, Rico-Bautista E 2006 Negative regulation of growth hormone receptor signaling. Mol Endocrinol 20:241–253[Abstract/Free Full Text]
  16. Strous GJ, dos Santos CA, Gent J, Govers R, Sachse M, Schantl J, van Kirkhof P 2004 Ubiquitin system-dependent regulation of growth hormone receptor signal transduction. Curr Top Microbiol Immunol 286:81–118[Medline]
  17. Fuchs SY, Fried VA, Ronai Z 1998 Stress-activated kinases regulate protein stability. Oncogene 17:1483–1490[CrossRef][Medline]
  18. Yumet G, Shumate ML, Bryant DP, Long CH, Cooney RN 2006 Hepatic growth hormone resistance during sepsis is associated with increased suppressors of cytokine signaling expression and impaired growth hormone signaling. Crit Care Med 34:1420–1427[CrossRef][Medline]
  19. Muller EE, Rigamonti AE, Cella SG 2003 Mechanisms of action of GH. J Endocrinol Invest 26:2–15[Medline]
  20. Greenhalgh CJ, Alexander WS 2004 Suppressors of cytokine signaling and regulation of growth hormone action. Growth Horm IGF Res 14:200–206[CrossRef][Medline]
  21. Frost RA, Nystron GJ, Lang CH 2002 Regulation of IGF-I mRNA and signal activators of transcription-3 and -5 (STAT-3 and -5) by GH in C2C12 myoblasts. Endocrinology 143:492–503[Abstract/Free Full Text]
  22. Dominici FP, Argentino DP, Munoz MC, Miquet JG, Sotelo AI, Turyn D 2005 Influence of the crosstalk between growth hormone and insulin signalling on the modulation of insulin sensitivity. Growth Horm IGF Res 15:324–336[CrossRef][Medline]
  23. Ridderstrale M 2005 Signaling mechanism for the insulin-like effects of growth hormone—another example of a classical hormonal negative feedback loop. Curr Drug Targets Immune Endocrin Metab Discord 5:79–92[CrossRef]
  24. Elsasser TH, Kahl S, Rumsey TS, Blum JW 2000 Modulation of growth performance in disease: reactive nitrogen compounds and their impact on cell proteins. Domest Anim Endocrinol 19:75–84[CrossRef][Medline]
  25. Greenacre SA, Ischiropoulos H 2001 Tyrosine nitration: localization, quantification, consequences for protein function and signal transduction. Free Radic Res 34:541–581[Medline]
  26. Pryor WA, Squadrito GL 1995 The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am J Physiol 268:L699–L722
  27. Pannala AS, Singh S, Rice-Evans C 1999 Interaction of carotenoids and tocopherols with peroxynitrite. Methods Enzymol 301:319–332[Medline]
  28. Elsasser TH, Kahl S, MacLeod C, Nicholson B, Sartin JL, Li C 2004 Mechanisms underlying growth hormone effects in augmenting nitric oxide production and protein tyrosine nitration during endotoxin challenge. Endocrinology 145:3413–3423[Abstract/Free Full Text]
  29. Kahl S, Elsasser TH 2004 Endotoxin challenge increases xanthine oxidase activity in cattle: effect of growth hormone and vitamin E treatment. Domest Anim Endocrinol 26:315–328[CrossRef][Medline]
  30. Feng J, Witthuhn BA, Matsuda T, Kohlhuber F, Kerr IM, Ihle JN 1997 Activation of Jak-2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop. Mol Cell Biol 17:2497–2501[Abstract]
  31. Kurzer JH, Argetsinger LS, Zhou YJ, Kouadio JL, O’Shea JJ, Carter-Su C 2004 Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-Bß. Mol Cell Biol 24:4557–4570[Abstract/Free Full Text]
  32. Elsasser TH, Kahl S, Li C, Schmidt W, Sartin J, Wesley M. Garrett WM, Rodrigo J 2007 Caveolae nitration of Janus kinase-2 at the 1007Y–1008Y site: coordinating inflammatory response and metabolic hormone readjustment within the somatotropic axis. Endocrinology, 148:3803-3813[Abstract/Free Full Text]
  33. Yang N, Huang Y, Jiang J, Frank SJ 2004 Caveolar and lipid raft localization of the growth hormone receptor and its signaling elements: impact on growth hormone signaling. J Biol Chem 279:20898–20905[Abstract/Free Full Text]
  34. Kahl S, Elsasser TH, Blum J 1997 Nutritional regulation of plasma tumor necrosis factor-{alpha} and plasma and urinary nitrite/nitrate responses to endotoxin in cattle. Proc Soc Exp Biol Med 215:370–376[CrossRef][Medline]
  35. Elsasser TH, Rumsey TS, Hammond AC, Fayer R 1988 Influence of parasitism on plasma concentrations of growth hormone, somatomedin-C and somatomedin-binding proteins in calves. J Endocrinol 116:191–200[Abstract/Free Full Text]
  36. Fernández-Fígares I, Shannon AE, Wray-Cahen D, Caperna TJ 2004 The role of insulin, glucagon, dexamethasone, and leptin in the regulation of ketogenesis and glycogen storage in primary cultures of porcine hepatocytes prepared from 60 kg pigs. Domest Anim Endocrinol 27:125–140[CrossRef][Medline]
  37. Caperna TJ, Shannon AE, Poch SM, Garrett WM, Richards MP 2005 Hormonal regulation of leptin receptor expression in primary cultures of porcine hepatocytes. Domest Anim Endocrinol 29:582–592[CrossRef][Medline]
  38. Braun S, Kalinowski HO, Berger S 1998 Water suppression by presaturation. In: Braun S, Kalinowski HO, Berger S, eds. 150 and more basic NMR experiments—a practical course. Weinheim, Germany: Wiley-Vch; 205–207
  39. Gasteiger J, Marsili M 1980 Iterative partial equalization of orbital electronegativity—a rapid access to atomic charges. Tetrahedron 36:3219–3288[CrossRef]
  40. Schmidt WF, Barone JR, Francis B, Reeves III JB 2006 Stearic acid solubility and cubic phase volume. Chem Phys Lipids 142:23-32[CrossRef][Medline]
  41. Gow AJ, McClelland M, Garner SE, Malcolm S, Ischiropoulos H 1998 The determination of nitrotyrosine residues in proteins. Methods Mol Biol 100:291–299[Medline]
  42. Viera L, Ye YZ, Estevez AG, Beckman JS 1999 Immunohistochemical methods to detect nitrotyrosine. Methods Enzymol 301:373–381[CrossRef][Medline]
  43. SAS Institute 1988 SAS system for linear models. Cary, NC: SAS Institute
  44. Matsuda T, Feng J, Witthuhn BA, Sekine Y, Ihle JN 2004 Determination of the transphosphorylation sites of JAK2 kinase. Biochem Biophys Res Commun 325:586–594[CrossRef][Medline]
  45. Argetsinger LS, Kouadio JK, Steen H, Stensballe A, Jensen ON, Carter-Su C 2004 Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity. Mol Cell Biol 24:4955–4967[Abstract/Free Full Text]
  46. Kurzer JH, Argetsinger LS, Zhou YJ, Kouadio JL, O’Shea JJ, Carter-Su C 2004 Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-Bß. Mol Cell Biol 2004 24:4557–4570[Abstract/Free Full Text]
  47. Lanone, S, Manivet P, Callebert J, Launay JM, Payden D, Aubier, M, Boczkowski J, Mebazza A 2002 Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues; implications for enzymic activity. Biochem J 366:399–404[CrossRef][Medline]
  48. Souza JM, Daikhin E, Yudkoff M, Raman C, Ischiropoulos H 1999 Factors determining the selectivity of protein tyrosine nitration. Arch Biochem Biophys 371:169–178[CrossRef][Medline]
  49. Crow J, Ye Y, Strong M, Kirk M, Barnes S, Beckman J 1997 Superoxide dismutase catalyzes nitration of tyrosines by nitrotyrosine in the rod and head domains of neurofilament-L. J Neurochem 69:1945–1953[Medline]
  50. Sandberg EM, VonDerLinden D, Ostrov DA, Sayeski PP 2004 Jak2 tyrosine kinase residues glutamic acid 1024 and arginine 1113 form a hydrogen bond interaction that is essential for Jak-STAT signal transduction. Mol Cell Biochem. 2004 265:161–169
  51. Lucet SI, Fantino E, Styles M, Bamert R, Patel O, Broughton SE, Walter M, Burns CJ, Treutlein H, Wilks AF, Rossjohn J 2006 The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor. Blood 107:176–183[Abstract/Free Full Text]
  52. Goligorsky MS, Hong L, Brodsky S, Chen J 2002 Relationships between caveolae and eNOS: everything in proximity and proximity in everything. Am J Physiol Renal Physiol 283:1–10
  53. Silva CM, Lu H, Weber MJ, Thorner MO 1994 Differential tyrosine phosphorylation of JAK1, JAK1 and STAT1 by growth hormone and interferon {gamma} in IM-9 cells. J Biol Chem 269:27532–27539[Abstract/Free Full Text]
  54. Fernandez L, Flores-Morales A, Lahuna O, Silva D, Norstedt G, Haldosen LA, Mode A, Gustafson G 1998 Desensitization of the growth hormone-induced janus kinase-2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5)-signaling pathway requires protein synthesis and phospholipase C. Endocrinology 139:1815–1824[Abstract/Free Full Text]
  55. Greenhalgh CJ, Alexander WS 2004 Suppressors of cytokine signaling and regulation of growth hormone action. Growth Horm IGF Res 14:200–206[CrossRef][Medline]
  56. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM 1999 Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399:601–605[CrossRef][Medline]
  57. Souza JM, Choi I, Chen Q, Weisse M, Daikhin E, Yudkoff M, Obin M, Ara J, Horwitz J, Ischiropoulos H 2000 Proteolytic degradation of tyrosine nitrated proteins. Arch Biochem Biophys 380:360–366[CrossRef][Medline]
  58. Kamisaki Y, Wada Y, Bian K, Balabanli B, Davis K, Martin E, Behbod F, Lee YC, Murad F 1998 An activity in rat tissues that modifies nitrotyrosine-containing proteins. Proc Natl Acad Sci USA 95:11584–11589[Abstract/Free Full Text]
  59. Nilsson J, Bjursell G, Kannius-Janson M 2006 Nuclear Jak2 and transcription factor NF1-C2: a novel mechanism of prolactin signaling in mammary epithelial cells. Mol Cell Biol 26:5663–5674[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Kurdi and G. W. Booz
JAK redux: a second look at the regulation and role of JAKs in the heart
Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1545 - H1556.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. S. Reifenberger, K. L. Arnett, C. Gatto, and M. A. Milanick
The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity
Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1191 - F1198.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
T. H. Elsasser, T. J. Caperna, C-J. Li, S. Kahl, and J. L. Sartin
Critical control points in the impact of the proinflammatory immune response on growth and metabolism
J Anim Sci, April 1, 2008; 86(14_suppl): E105 - E125.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. H. Elsasser, S. Kahl, C.-J. Li, J. L. Sartin, W. M. Garrett, and J. Rodrigo
Caveolae Nitration of Janus Kinase-2 at the 1007Y-1008Y Site: Coordinating Inflammatory Response and Metabolic Hormone Readjustment within the Somatotropic Axis
Endocrinology, August 1, 2007; 148(8): 3803 - 3813.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elsasser, T. H.
Right arrow Articles by Schmidt, W. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elsasser, T. H.
Right arrow Articles by Schmidt, W. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals