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Department of Medicine (S.J.F., X.W., K.H.), Division of Endocrinology, Diabetes, and Metabolism, Department of Cell Biology (S.J.F., N.Y., L.D.), Departments of Medicine, Division of Hematology and Oncology (K.R.Z.), and Radiology (T.R.C.) and the Laboratory for Multimodality Imaging (T.R.C., K.R.Z.), University of Alabama at Birmingham, Birmingham, Alabama 35294-0012; Endocrinology Section (S.J.F.), Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233; and Department of Pediatrics (P.F., R.G.R., V.H.), Oregon Health & Sciences University, Portland, Oregon 97239
Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012. E-mail: sjfrank{at}uab.edu; or Kurt R. Zinn, University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 802, Birmingham, Alabama 35294-0012. E-mail: kurtzinn{at}uab.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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The emergence of adenoviral-mediated gene delivery systems and highly sensitive noninvasive imaging techniques opens new opportunities to study in vivo signaling in small animals. GH is an important pituitary-derived regulator of growth and metabolism in vertebrates (1). Unrestricted GH receptor (GHR) knockout and knockin experiments in mice elegantly demonstrate the primacy of this cell surface receptor in GH binding and mediation of GH responsiveness (2, 3). GHR is displayed widely, but is particularly enriched in liver, where GH signaling results in hepatic growth and production of IGF-I, a systemic mediator of somatogenesis (1, 4, 5, 6, 7, 8, 9). GH activates the GHR-associated Janus kinase (JAK)2, allowing tyrosine phosphorylation and nuclear translocation of the transcription factor, signal transducer and activator of transcription-5B (STAT-5B), which is critical for GH-induced transcriptional activation of the IGF-I gene and other genes that mediate GH action (4, 5, 10, 11, 12, 13, 14, 15, 16). In this communication, we describe development of a model system to noninvasively study hepatic GH signaling in vivo.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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We first prepared an adenoviral vector, Ad-GHRE-Luc. The Luc (firefly luciferase) in this construct is driven by a basal promoter (from the pGL2 plasmid) with eight repeats of the STAT5-dependent GH response element (GHRE) from the Spi2.1 gene cloned into the 5'-untranslated region. We reasoned that the tropism of injected adenovirus for liver would target enriched expression by adenoviral gene transfer of this reporter in that organ (24, 25, 26). This construct was initially tested in vitro using the C14 cell line, a human fibrosarcoma that stably expresses GHR and JAK2 and in which GH causes GHR and JAK2 tyrosine phosphorylation and STAT5 activation (19, 20, 21, 27). After infection of C14 cells with Ad-GHRE-luc, GH treatment induced enhanced bioluminescence (Fig. 1
, A and B), verifying that the reporter was responsive.
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, A–D). Females were used in these experiments because they manifest a markedly less pulsatile pattern of endogenous GH secretion (28) and might therefore be predicted to more reliably respond to exogenously administered GH. Preliminary studies indicated an appropriate viral dose and specifics of the imaging acquisitions and ensured that administration of Ad-GHRE-Luc was unaccompanied by ill effects in these mice over a period of up to 2 wk (data not shown). Mice were then injected via tail vein with 1 x 109 plaque-forming units Ad-GHRE-Luc (n = 10 per condition). Several days later, the mice were fasted overnight before baseline bioluminescence images (time 0') were obtained. Mice were then administered GH at two doses (0.5 µg/g or 2.0 µg/g iv) and subjected to serial imaging over the next 7 h (see diagram in Fig. 2A). Data are displayed as representative images in Fig. 2, B and C, and the liver bioluminescence signals (mean ± SE) are summarized in Fig. 2D. The luminescence response in the region of the liver was evident in both groups by 1 h after GH injection and peaked after 3 h. The response to the two GH doses was statistically significantly different at each time point, indicating the ability of the system to detect graded responses. The same patterns of GH dose-dependent responsiveness and time course were observed in four other imaging sessions over a 10-d period in these mice (data not shown).
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indicated that hepatic signaling via endogenous GHR could be detected in our model system by introduction of the Ad-GHRE-Luc. We also sought to learn whether the system could ultimately be used to study in vivo signaling characteristics of exogenously introduced GHR. To facilitate such studies, we first prepared adenoviral vectors encoding the rabbit (rb) GHR (designated Ad-GHR) or an N-terminally hemagglutinin (HA)-tagged version of this GHR referred to as Ad-N-HA-GHR (Fig. 3A, left panel). In adenovirally infected human embryonic kidney (HEK)-293 cells, both GHR and N-HA-GHR were detected by immunoblotting with antiserum to the GHR cytoplasmic domain, and only the epitope-tagged version was recognized by anti-HA antibody (Fig. 3A, right panel, upper two rows). Both receptors migrated at the expected Mr, based on our previous data (18, 19, 20, 21, 29, 30, 31), and GH treatment promoted acute activation of JAK2 in cells expressing either receptor (Fig. 3A, right panel, lower row). Thus, both viruses directed in vitro cell surface expression of GHRs that bind GH and activate downstream signaling, indicating the intactness of the receptors.
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camera imaging was applied to assess the 99mTc-labeled antibody distribution over time. In the experiment shown in Fig. 3B, the control mouse was not injected with adenovirus, whereas three mice were injected with Ad-N-HA-GHR. Imaging of all four mice immediately after the injection of labeled antibody revealed its diffuse distribution throughout the body, consistent with it being in the blood pool and not concentrated in any particular organ (Fig. 3B, top row). This same pattern was observed in this representative control mouse 1 h after antibody injection, as expected. In contrast, in each of the three mice infected with Ad-N-HA-GHR, 99mTc-labeled anti-HA was markedly concentrated 1 h after injection in the region of the liver (Fig. 3B, bottom row). This finding was confirmed by analysis of the biodistribution of radioactivity in these mice. After animals were killed, radioactivity was measured among various tissues. Whether expressed as percent of total dose (data not shown) or normalized per gram of tissue (Fig. 3C), this analysis demonstrated the marked and statistically significant (P < 0.05) hepatic enrichment of 99mTc-labeled antibody in these mice.
Similar experiments were carried out for the non-epitope-tagged GHR encoded by Ad-GHR (Fig. 4). We previously reported that anti-GHRext-mAb, a monoclonal antibody against the extracellular domain of the rabbit GHR, binds to the receptor on the surface of intact cells (20). Anti-GHRext-mAb cross-reacts with human GHR, but does not interact with rodent receptors. We used 99mTc–labeled anti-GHRext-mAb to image the pattern of distribution of the non-epitope-tagged GHR encoded by Ad-GHR (Fig. 4A). Similar to N-HA-GHR, wild-type GHR was also targeted to liver by Ad-GHR infection, indicating that this GHR also reached the cell surface of liver cells in vivo by 1 h after injection of the radiolabeled antibody. Biodistribution analysis confirmed statistically higher (P < 0.05) levels of radiolabeled antibody in the liver of Ad-GHR-infected mice, when compared with the level in control mice (Fig. 4B). However, the degree of liver concentration detected was notably less for the anti-GHRext-mAb/Ad-GHR system than for the anti-HA/Ad-N-HA-GHR system. This may suggest that the affinity of anti-GHRext-mAb, a conformation-sensitive antibody (20), for GHR may be less than that of anti-HA for the epitope-tagged GHR. Nevertheless, the data in Figs. 3 and 4 confirm that adenovirally mediated expression of GHR leads to its display on the surface of liver cells in vivo.
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, we have shown in previous studies that reconstitution of GHR in GHR-deficient cells that also express JAK2 and STAT5 allowed detection of GH-induced JAK2 and STAT5 tyrosine phosphorylation (18, 19, 20, 21, 22, 32, 33). With the goal of establishing an in vivo system to evaluate GH signaling by exogenously added GHR, we tested, in cultured cells, the efficacy of coexpression of both the GHR and the STAT5-dependent luciferase reporter genes by adenoviral infection (Fig. 5A). For these studies, we used as a target 2A-JAK2, a human fibrosarcoma cell (20, 21, 35) that stably expresses transfected JAK2, but lacks GHR. Cells were either coinfected with Ad-GHR plus Ad-GHRE-Luc at a 1:1 ratio (right panel) or sequentially infected with a 1:1 ratio of the two viruses 1 d apart, either first with the reporter virus (middle panel) or first with the GHR virus (left panel). After treatment with vehicle or GH for 3 h, bioluminescence imaging demonstrated markedly enhanced GH-induced transactivation of the GHRE luciferase reporter when cells were sequentially infected compared with coinfection.
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). In a second group (n = 10), mice were injected via tail vein with 1 x 109 plaque-forming units Ad-GHRE-Luc (Fig. 5C). The third group of mice (n = 10) were first injected with 1 x 109 plaque-forming units Ad-SpiGLE-Luc, followed 1 d later by 1 x 109 plaque-forming units Ad-GHR (Fig. 5D). As with experiments shown in Fig. 2, adenoviral expression of the GHR and GHRE-Luc in liver was unaccompanied by ill effects in these mice over a period of up to 2 wk (data not shown).
Imaging experiments were performed with these groups such that animals were either fasted (as in Fig. 5, B–D) or fed overnight before baseline bioluminescence images (time 0' in Fig. 5, B–D) were obtained. Mice were then administered GH (1 µg/g iv; intermediate to the two doses used in Fig. 2) and subjected to serial imaging over the next 7 h. Data are displayed as representative images in Fig. 5, B–D, and the liver bioluminescence signals (mean ± SE) are summarized in Fig. 5 (panel E, fed mice; and panel F, fasted mice). As expected, no liver bioluminescence signal was detected in mice that did not receive Ad injections (Fig. 5B), and the background photon counts detected in the liver region were low and stable (Fig. 5, E and F). Within an imaging session the liver background signal for this group without Ad injection was not statistically different (P > 0.77) as a function of time after GH injection. However, as shown on the graphs presented in Fig. 5, E and F, this background signal was statistically (P < 0.05) less than the other two Ad-treated groups.
Mice expressing only the GHRE-Luc (via Ad-GHRE-Luc) demonstrated a statistically significant (P < 0.05) hepatic bioluminescence response to GH; as in Fig. 2, the response was detected by 1 h after GH injection (Fig. 5, panels C, E, andF). Notably, the response to GH in this group of mice was more modest than that observed in the mice tested for GH dose responsiveness in Fig. 2 (see below). The liver signal remained significantly (P < 0.05) elevated relative to time zero, and even though values approached their peak at 3 h, there was no statistically significant difference in mean liver signal for imaging points collected at 1, 3, 5, and 7 h after GH injections for this group. Further, there was no significant difference (P = 0.05) in the GH response as a function of the mice being fed or fasted. In contrast, a time-dependent and more robust GH response was detected in mice injected with both the GHR and GHRE-Luc viruses (Fig. 5, D–F), both under fed and fasting conditions. The peak liver signal at 3 h after GH injection was statistically (P < 0.001) higher than all other time points for this group and also was statistically (P < 0.001) higher compared with all other imaging time points for the remaining two groups. Of interest, the effect of coexpression of GHR with the GHRE-Luc was most marked under fasting conditions; the mean liver intensity of 293 ± 12 counts per sec at 3 h after GH injection in the fasted mice was significantly higher (P = 0.005) than 216 ± 11 counts per sec for fed mice.
In a separate imaging session and on a different day, no response was detected in mice infected with both viruses when hourly imaging was performed (with luciferin injection before each image) but GH was not given (data not shown); this rules out a spurious effect of the imaging protocol itself. Further, GH-responsive liver signaling was observed in the same animals in four separate experiments over a 1-wk period (data not shown). These data indicate that GH-responsive hepatic transactivation of a STAT5-dependent GHRE-Luc can be noninvasively detected in vivo after sequential infection with the adenoviruses encoding each gene.
It is noteworthy that in this experiment in which the effect of coexpressed Ad-GHR was directly tested, infection of nude mice with Ad-GHRE-Luc alone yielded less hepatic bioluminescence in response to either endogenous GH or administration of GH. To pursue the basis for this apparent heightened sensitivity to GH with added GHR expression, we analyzed liver GHR abundance by immunoblotting (Fig. 5, G and H). Nude mice either infected 2 wk earlier with Ad-GHR or uninfected (control) were killed and liver protein was extracted. Equal amounts of protein were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47 (Fig. 5G). This revealed abundant liver GHR (via Ad-GHR) that migrated consistent with rabbit GHR. In contrast, no GHR signal was detected with this exposure in the liver sample from the control mouse. To examine this further, we compared equal amounts of liver extract from a noninfected nude mouse with those of noninfected mice of several strains in duplicate (Fig. 5H). Interestingly, whereas a low level of GHR was detected in nude mouse liver, GHR abundance differed among four common strains tested [note that the mouse GHR migrates faster than rabbit GHR, consistent with previous observations (1)]. Low levels were also seen in C57BL/6, with progressively higher levels in BALB/c, C3H/HeJ, and CBA mice. To our knowledge, a systematic examination of strains with regard to hepatic GHR abundance by blotting has not been reported, but these results conform to our previous observations of easily detectable hepatic GHR in C3H/HeJ mice and much lower levels (most reliably detected only after immunoprecipitation) in C57BL/6 mice (36, 37). Hepatic GH responsiveness has previously been detected in C57BL/6 mice (36, 38, 39, 40) and also in nude mice (41, 42); nonetheless, these results suggest that endogenous GHR abundance (and, consequently, possibly GH responsiveness) may vary substantially between mouse strains and that these differences may be exploited in experiments that explore in vivo signaling of wild-type vs. mutated GHRs.
Impact of the Experimental System
In this report, we demonstrate that GH-induced in vivo hepatic GHR signaling can be detected noninvasively by serial bioluminescence imaging in mice infected with an adenovirus driving the expression of a STAT5-dependent luciferase gene. This establishes the first such system, of which we are aware, that allows noninvasive assessment of GH signaling. Notably, the degree of signal elicited appears to vary between experiments (i.e. between the experiments shown in Figs. 2 and 5), probably reflecting subtle differences in the groups of animals tested and the specific preparations of the reagents used in experiments done months apart and with animals derived from different litters. Importantly, however, within each experiment, there was a remarkable lack of variability between mice treated identically. Thus, we were easily able to detect GH dose-responsiveness in the experiment shown in Fig. 2 and the augmentation effect of coexpression of GHR in the experiment shown in Fig. 5, and SEMs among animals treated identically were very small.
There are several advantages of a noninvasive system such as we have developed. Because of the long-lived hepatic expression of adenoviral-delivered genes, experiments can be performed repeatedly over a period of at least 2 wk, allowing potential correlations to biologically relevant endpoints of GH action, such as growth and metabolic effects. The apparent lack of toxicity of the paradigm is also attractive. Further, previous work indicates that bioluminescence imaging can be used in both suckling mice and older mice that are shaved and that it is often not necessary to shave the mice (as with C57/BL6 mice in Ref. 26) and that luciferase has a relatively short biological half-life (
2 h) (43). Notably, the chemical reaction with luciferin as a substrate and catalyzed by firefly luciferase results in a broad peak of light emission and has longer wavelength components (>600 nm) that are more penetrating to tissue, and not absorbed by blood, etc. (44). For these studies, we feel that bioluminescence imaging is particularly useful. For example, compared with another possible imaging system in which positron emission tomography (PET) imaging is linked to expression of the herpes simplex virus thymidine kinase (TK) gene driven by the STAT5-responsive promoter, there are both theoretical and practical benefits of bioluminescence imaging. First, the short luciferase biological half-life is a major plus, whereas the half-life of TK in these circumstances is less certain. Second, the PET system would require administration of radioactive TK substrate that is trapped at the location of TK expression, but PET imaging also detects the untrapped radioactive substrate that must clear from the mouse by excretion to detect specific TK expression in the liver. In a short-term experiment like the current study, the clearance of the radiolabeled substrate would be a significant issue. This is quite different from the nonradioactive luciferin that has no detectable signal unless it encounters the luciferase that is confined to the liver. Further, trapped radioactive TK substrate does not clear, and therefore one would have to wait on radioactive decay to be eliminated. Even the relatively short radioactive half-life (108 min) of most TK substrates would present a problem, because it would create a background in the liver due to the trapped radioactive substrate. It would not be feasible to keep injecting the radioactive TK substrate every hour, as one would detect the previously administered dose (both trapped and clearing). Finally, microPET imaging is normally done one mouse at a time and requires about 15 min to acquire data and substantial time for data reduction; in contrast, bioluminescence imaging evaluates five mice at once and can be done in 5 min and at significantly lower cost per mouse.
We note with caution, however, that the effect of various physiological and pathophysiological situations themselves on the response to the particular array of multiple STAT5 response elements used in our reporter in this study cannot be always assumed to behave as would a response element in a natural STAT5-responsive gene context [see, for example, Berry et al. (45)]. This could be a disadvantage for certain applications. Thus, proper experimental controls will be critical for each new situation evaluated using this system.
Interestingly, in nude mice, it appears that responsiveness for detectable bioluminescence of the GHRE-Luc gene is enhanced by expression of the GHR; thus, GHR mutants could be evaluated without undue concern for interference of the endogenous wild-type GHR as long as their expression is compared with that of exogenous wild-type receptor and with a negative control group of animals not infected with exogenous GHR-encoding virus. Conversely, other mouse strains that contain substantial endogenous hepatic GHR might be predicted to respond to GH even more robustly in this system without exogenous GHR expression; thus, other factors that might govern GH responsiveness could be more readily studied by introduction of the GHRE-Luc reporter alone. Further, this approach could provide a means to serially evaluate the consequences of exogenous GH administration on liver GH signaling. This would be of interest from the perspective of GH therapy.
Finally, the work presented herein may serve as a blueprint to image signaling noninvasively and repeatedly using receptors expressed in liver or other tissues. (For other tissues, such targeting could be conferred by transgenic expression of reporter and/or receptor under the control of a tissue-specific element.) For liver studies, the finding that coexpression of the two adenoviruses that encode reporter and receptor is facilitated by staggering the infections should therefore be of substantial general relevance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibodies
Anti-p-JAK2 state-specific antibody reactive with JAK2 that is phosphorylated at residues Y1007 and Y1008 (reflective of JAK2 activation) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The rabbit polyclonal antisera, anti-GHRcyt-AL47, raised against a bacterially expressed N-terminally-His-tagged fusion protein incorporating human GHR residues 271–620 [the entire cytoplasmic domain (46)], has been previously described (31), as has anti-JAK2AL33 (directed at residues 746-1129 of murine JAK2) polyclonal serum (47). Anti-GHRext-mAb is a mouse monoclonal antibody (IgG1
) directed against a bacterially expressed glutathione-S-transferase fusion protein incorporating rabbit GHR residues 1–246 (20, 29, 48, 49). This monoclonal anti-GHR antibody was purified from hybridoma supernatant using protein G-sepharose (at the University of Alabama Multipurpose Arthritis Center Hybridoma Core facility). Anti-HA monoclonal antibody was purchased from BABCO, Inc. (Berkeley, CA).
Cells, Cell Culture, Transfection, and Adenoviral Infection
HEK-293 cells (a gift of Dr. C. Wu, University of Pittsburgh, Pittsburgh, PA) cells were maintained in DMEM (low glucose) (Cellgro, Inc., Herndon, VA) supplemented with 7% fetal bovine serum (Biofluids, Rockville, MD) and 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Biofluids). Transient transfection was achieved by introducing pCDNA 3.1-driven plasmids encoding murine JAK2 (1 µg per transfection), using Lipofectamine Plus (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Adenoviral infection of HEK-293 cells was accomplished using methods previously reported (50, 51). 2A-JAK2 cells were created by transfection of 2A cells (35) (gift of Dr. George Stark, Cleveland Clinic, Cleveland, OH) with a plasmid pcDNA3.1(+)/zeo-JAK2. A stable JAK2-expressing clone was selected by immunoblotting with anti-JAK2AL33. 2A-JAK2 cells were cultured in the same medium as HEK-293 cells supplemented with 200 µg/ml G418 (Cellgro) and 100 µg/ml Zeocin (Invitrogen). Adenoviral infection of 2A-JAK2 cells was accomplished in the same way as for HEK-293 cells.
Construction of Ad-N-HA-GHR, Ad-GHR, and Ad-GHRE-Luc
The rabbit (rb) GHR cDNA (a gift of W. Wood, Genentech, Inc., South San Francisco, CA) was subcloned into the eukaryotic expression vector pcDNA3.1(–) (Stratagene, La Jolla, CA), yielding pcDNA3.1(–)/Neo GHR, as previously reported for rbGHR mutants (50, 51). pcDNA3.1 (–)/Neo N-HA-GHR, encoding rbGHR with an N-terminal HA tag, was made by using the ExSite (Stratagene) PCR-based site-directed mutagenesis method (sequences of PCR oligonucleotides are available upon request). The GHR or N-HA-GHR fragment was released by Xba1 and Pme1 and cloned into the XbaI and EcoRV sites of the adenoviral shuttle vector, pAdTrack-CMV, which was used in the AdEasy system of homologous recombination to obtain a recombined adenoviral plasmid, as described elsewhere (50). The adenoviruses (Ad-GHR and Ad-N-HArbGHR) were made and purified according to standard techniques. The 8xGHRE-pGL2 plasmid (a generous gift from Dr. Peter Rotwein, Oregon Health & Science University, Portland, OR) carries eight repeats of the GH response elements (8xGHRE) in the 5'-untranslated region of the luciferase reporter cDNA. A silent mutation was introduced by PCR at Ile442 (ATT to ATA) in the luciferase coding region to remove the PacI site (forward primer, 5'-GATTACCAGGGATTTCAG-3'; reverse primer, 5'-CCACCTGATATCCTTTGTATTTTATTAAAGACTTCAAGCGGTCAACTAT-3'). The resultant construct, 8xGHRE-pGL2-PacI, was digested with SmalI/SalI and 8xGHRE-luciferase was subcloned into pShuttle transfer plasmid (AdenoVator Vector System, QBiogen, Carlsbad, CA). Final construction of the adenovirus carrying cDNA encoding 8xGHRE-luciferase (Ad-GHRE-Luc) followed the manufacturer’s protocol.
Radiolabeling of Antibodies
Antibodies were first conjugated with succinimidyl 6-hydrazinonicotinate biofunctional chelate, and on the next day the hydrazinonicotinate-antibody conjugates were radiolabeled with 99mTc, as previously reported (52, 53). [99mTcO4–] (Central Pharmacy, University of Alabama at Birmingham) was used to label anti-HA and anti-GHRext-mAb antibodies. The level of 99mTc binding to the antibodies was always greater than 95%, as measured by thin-layer chromatography using separate strips eluted with saturated saline and methyl ethyl ketone.
Animal Experiments
Animal protocols followed all regulations and were reviewed and approved in advance of the studies by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Mice were induced and maintained with isoflurane gas anesthesia for all injections and imaging studies, and monitored continuously to allow the lowest dose (typically 1–1.5%) to prevent movement. The mice were fed a nutritionally complete casein-based diet (formula 89222; Harlan Teklad, Madison, WI) beginning 4 d before imaging to reduce background phosphorescence from the abdominal region.
In Vivo Imaging and Biodistribution of 99mTc-Anti-HA and 99mTc-Anti-GHRext-mAb
Male athymic nude mice were obtained from the National Cancer Institute Frederick Research Laboratory (Frederick, MD) and two groups (n = 3/group) were injected with 1 x 109 plaque-forming units of Ad-N-HA-GHR (group 1) or Ad-GHR (group 2) iv via the tail vein. One additional group (n = 3) was not injected with Ad vector (group 3). Four days later, the mice were iv injected with 99mTc-anti-HA (group 1, mean dose = 167 µCi, 2.4 µg) or 99mTc-anti-GHRext-mAb (groups 2 and 3, mean dose = 255 µCi, 3.4 µg). Syringes were measured for activity before and after injection using an Atomlab 100 dose calibrator (Biodex Medical Systems, Shirley, NY). The mice were imaged at the indicated times after injection by static planar technique using a -camera equipped with a pinhole collimator, with at least 50,000 total counts per image. After imaging, the tissues were collected, weighed, and counted in a Minaxi Auto-Gamma 5000 series -counter (Packard Instrument Co., Downers Grove, IL). The raw data (counts per min) from the -counter were decay corrected to the injection time. Radioactivity in the tissues were normalized to the tissue weight and dose, and expressed as percent of injected dose per gram of tissue (% ID/g).
In Vitro Testing of Adenovirus-Based GH Signaling System
2A-JAK2 cells, which lack GHR, were divided into aliquots into 12-well plates and subjected to adenovirus infection according to three different schemes. One group was infected on d 1 with Ad-GHRE-Luc and on d 2 with Ad-GHR. A second group was infected on d 1 with Ad-GHR and on d 2 with Ad-GHRE-Luc. The third group was infected with both Ad-GHR and Ad-GHRE-Luc on d 1. For each group, infectious virus titers were performed at three 1:2 serial dilutions. Cells were replenished with serum-free medium at 36 h after the first infection, and human GH (500 ng/ml; kindly provided by Eli Lilly Co, Indianapolis, IN) was added at 48 h for a 6-h incubation at 37C. Thereafter, luciferin (0.1 mg/ml) was added to each well and plates were imaged for bioluminescence using the IVIS-100 Imaging System (Xenogen Corp., Alameda, CA).
In Vivo Hepatic GH Signaling via Adenovirally Expressed GHRE-Luc Reporter Alone or with GHR
Female athymic nude mice (8 wk old) were obtained from the National Cancer Institute Frederick Research Laboratory. In experiment 1 (Fig. 2), two groups of mice (n = 10/group) were injected with the Ad encoding GHRE-Luc (1 x 109), and subjected to imaging studies (5 total days) beginning 5 d later. This experiment tested the difference in response to GH, with one group receiving GH at 0.5 µg/g body weight, and the other receiving GH at 2.0 µg/g body weight. In experiment 2 (Fig. 5) there were three groups of mice (n = 10/group). One group received no Ad injection, the second group received the Ad encoding GHR followed by Ad-GHRE-Luc on the following day, whereas the third group received the Ad-GHRE-Luc only. All Ad injections were 1 x 109 plaque-forming units. After 5 d the imaging studies were initiated, with GH injected at 1.0 µg/g body weight. One imaging series was conducted in the absence of GH injection. For all bioluminescence imaging studies, the animals were either fasted or fed overnight before baseline bioluminescence images were obtained, after which GH was injected, and repeated imaging studies were collected at 1, 3, 5, and 7 h after injection of GH. Before and, at indicated time points, after GH, mice were injected with luciferin ip (2.5 mg) and imaged after 10 min with the IVIS-100 Imaging System, as detailed in the figure legend. Images were collected on anesthesized mice oriented in the same position on a heated shelf (37 C). Image acquisition time was 300 sec, and the camera binning was 8. Data acquisition software ensured that no pixels were saturated. Light emission from the liver regions (relative photons/sec) was measured by region of interest analyses with software provided by the vendor. The intensity of light emission was represented with a pseudocolor scaling of the bioluminescent images. The bioluminescent images were overlaid on black and white photographs of the mice.
Protein Extraction and Immunoblotting
Detergent extraction, electrophoresis, and immunoblotting of tissue culture cells were performed as described previously (19, 20, 34, 50, 51, 54, 55). For evaluation of liver GHR levels, frozen livers were extracted with the NE-PER system (Nuclear and Cytoplasmic Extraction Kit; Pierce Chemical Co., Rockford, IL). Cytoplasmic extracts were resolved by SDS-PAGE and immunoblotted.
Statistical Analysis
One- and two-way ANOVAs were carried out using SAS, version 8.2 (SAS Institute Inc., Cary, NC). Data presented in Fig. 3C were tested by one-way ANOVA to compare the liver retention (% ID/g) of 99mTc-labeled anti-HA antibody relative to other tissues. Data presented in Fig. 4B were tested by one-way ANOVA to determine the effect of Ad-GHR relative to control (no Ad vector) on retention (% ID/g) of 99mTc-labeled anti-GHR antibody in individual tissues. Two-way ANOVA was used to test the data presented in Fig. 2D, to compare bioluminescence signal (relative photons/sec) over time from liver for mice injected with two different doses of GH, all mice having received the same dose of Ad-GHRE-Luc. Two-way ANOVA tested data presented in Fig. 5, E and F, to compare bioluminescence signal (relative photons/sec) over time from liver for mice injected with GH, and having received no Ad injection, the Ad-GHRE-Luc alone, or Ad-GHR plus Ad-GHRE-Luc. Two-way ANOVA was also applied to compare the peak (3 h) GH-induced liver bioluminescence signal in fed vs. fasting animals, for mice that received either the Ad-GHR alone, or Ad-GHR plus Ad-GHRE-Luc.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Results of this work were presented in part at the 86th Annual Meeting of The Endocrine Society, New Orleans, LA, 2004.
Disclosure Statement: S.J.F., X.W., K.H., N.Y., P.F., V.H., T.R.C., and K.R.Z. have nothing to declare. R.G.R. has received consulting fees from Tercica, Biocritique, Diagnostic Systems Laboratories, and Eli Lilly, has equity interest in ProteoGenix, and has received lecture fees from Genentech, Novo, and Eli Lilly.
First Published Online June 27, 2006
1 S.J.F. and X.W. contributed equally to this work. 
2 This paper is dedicated to the memory of Dr. Chaudhuri, who passed away on 26 July 2006.
Abbreviations: GHR, GH receptor; GHRE, GH response element; HA, hemagglutinin; HEK, human embryonic kidney; ID, injected dose; JAK, Janus kinase; PET, positron emission tomography; STAT, signal transducer and activator of transcription; TK, thymidine kinase.
Received for publication December 27, 2005. Accepted for publication June 19, 2006.
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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and interferon responses. Mol Cell Biol 17:695–706[Abstract] camera imaging of gene transfer using an adenoviral vector encoding an epitope-tagged receptor as a reporter. Gene Ther 10:105–114[CrossRef][Medline]-secretase cleavage. J Biol Chem 280:19331–19342This article has been cited by other articles:
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  X. Wang, J. Jiang, J. Warram, G. Baumann, Y. Gan, R. K. Menon, L. A. Denson, K. R. Zinn, and S. J. Frank Endotoxin-Induced Proteolytic Reduction in Hepatic Growth Hormone (GH) Receptor: A Novel Mechanism for GH Insensitivity Mol. Endocrinol., June 1, 2008; 22(6): 1427 - 1437. [Abstract] [Full Text] [PDF]
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N. Yang, X. Wang, J. Jiang, and S. J. Frank Role of the Growth Hormone (GH) Receptor Transmembrane Domain in Receptor Predimerization and GH-Induced Activation Mol. Endocrinol., July 1, 2007; 21(7): 1642 - 1655. [Abstract] [Full Text] [PDF]
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