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The Journal of Immunology, 2006, 177: 6889-6895.
Copyright © 2006 by The American Association of Immunologists, Inc.

Functional Interaction of Common {gamma}-Chain and Growth Hormone Receptor Signaling Apparatus1

Marsilio Adriani*, Corrado Garbi{dagger}, Giada Amodio*, Ilaria Russo*, Marica Giovannini*, Stefania Amorosi*, Eliana Matrecano*, Elena Cosentini{ddagger}, Fabio Candotti§ and Claudio Pignata2,*

* Department of Pediatrics, {dagger} Department of Cellular and Molecular Biology and Pathology, and {ddagger} Immunohematology Unit, "Federico II" University, Naples, Italy; and § Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We previously reported on an X-linked SCID (X-SCID) patient, who also had peripheral growth hormone (GH) hyporesponsiveness and abnormalities of the protein phosphorylation events following GH receptor (GHR) stimulation. In the present study, we examined a potential role of common cytokine receptor {gamma}-chain ({gamma}c) in GHR signaling using EBV-transformed lymphocytes from healthy subjects and {gamma}c-negative X-SCID patients. We demonstrated that the proliferative response to GH stimulation of the B cell lines of {gamma}c-negative patients was impaired despite a comparable cellular expression of GHR molecules to controls. In patients, after GH stimulation, no phosphorylation of STAT5 was observed. In addition, the molecule localization through confocal microscopy revealed that in B cell lines of patients no nuclear translocation of STAT5b following GH stimulation occurred differently from controls. Biochemical analysis of the nuclear extracts of {gamma}c-negative cell lines provided further evidence that the amount of STAT5b and its phosphorylated form did not increase following GH stimulation. In patients, cells reconstituted with wild-type {gamma}c abnormal biochemical and functional events were restored resulting in nuclear translocation of STAT5. Confocal experiments revealed that GHR and {gamma}c were colocalized on the cell membrane. Our study demonstrates the existence of a previously unappreciated relationship between GHR-signaling pathway and {gamma}c, which is required for the activation of STAT5b in B cell lines. These data also confirm that growth failure in X-SCID is primarily related to the genetic alteration of the IL2RG gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Severe combined immunodeficiencies represent a wide spectrum of illnesses, which differ in either the qualitative or quantitative alterations of T, B, and NK cell (1). Most forms of SCID are associated with molecular alterations of genes selectively expressed in hemopoietic cells and implicated in the cell differentiation/activation process. Thus, classical symptoms are generally considered those related to the immunological impairment that results in increased susceptibility to infections. Because patients usually die by the first year of age without an effective treatment, the clinical phenotype is predominated by the life-threatening problems.

X-linked SCID (X-SCID)3 is the most common form of the disease accounting for approximately half of all cases (2, 3). The gene responsible for X-SCID is IL2RG that encodes for the common cytokine receptor {gamma}-chain ({gamma}c), a member of the cytokine receptor class 1 superfamily. The molecule represents a shared component of several receptors critical for the development and function of lymphocytes (3). To our knowledge, an extrahemopoietic role of {gamma}c has not yet been demonstrated, although the abundance of the protein in nonhemopoietic cells would imply additional functions for this element (4, 5).

We previously reported on a patient affected with X-SCID who received a bone marrow transplantation late at 5.2 years of age. In this patient, short stature became evident, and a peripheral growth hormone (GH) hyporesponsiveness associated with abnormalities of the protein phosphorylation events that occur following GH receptor (GHR) stimulation was demonstrated.

The GHR was the first identified member of the cytokine receptor class 1 superfamily, which includes receptors for erythropoietin, G-CSF, GM-CSF, IL-2–7, IL-9, IL-11, IL-12, and many other cytokines. Due to the lack of intrinsic kinase activity, members of the cytokine receptor superfamily recruit and/or activate cytoplasmic tyrosine kinases to relay their cellular signal. The JAK2 represents the predominant nonreceptor tyrosine kinase required for the initiation of GH signal transduction upon ligand binding to the receptor (6, 7). However, GH also stimulates tyrosine phosphorylation of JAK1 (8, 9) and JAK3 (10) in certain cell lines. Signal transduction through GHR also involves a wide array of molecules, such as STATs 1, 3, and 5, ERK 1 and 2, and PI3K-protein kinase B (7). Activation of STAT5b is considered a prominent event in GH signaling and is crucial for the regulation of transcription of GH-responsive genes, including the gene encoding for insulin-like growth factor (IGF)-I, which mediates many of the GH biological functions (11, 12, 13). In our previous study, mutational screening and expressional analysis failed to reveal any molecular alteration of GHR, JAK2, and STAT5A/B genes in the patient with X-SCID and peripheral GH hyporesponsiveness (14).

Because we hypothesized a role for the {gamma}c in GHR signaling, in this study, we evaluate the functional interaction between GHR and the common {gamma} element in either freshly isolated or EBV-transformed lymphocytes from X-SCID patients and healthy subjects. In particular, the functional response to GH stimulation, the pattern of GHR-induced protein tyrosine phosphorylation and GH-induced translocation from the cytoplasm to the nucleus of STAT5 were evaluated. We demonstrate the existence of a previously unappreciated functional interaction between {gamma}c and GHR. This interaction leads to the activation and intranuclear translocation of the STAT5b protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Recombinant human GH (rGH) was obtained from Serono (Saizer 4). The ECL kit was purchased from Amersham Biosciences. The Abs anti-STAT5b, anti-STAT5a, anti-STAT1, anti-STAT3, anti-ERK (recognizing both ERK1 and ERK2), anti-phosphotyrosine ERK, anti-GHR and anti-{gamma}c and the mAbs anti-phosphotyrosine were purchased from Santa Cruz Biotechnology. The Ab anti-JAK2 was purchased from Cell Signaling Technology. The neutralizing IgG1 anti-{gamma}c mAb was purchased from R&D Systems. An IgG1 isotype-matched anti-CD3 mAb (Leu 3, UCHT1 clone) was purchased from BD Biosciences. Epidermal growth factor was purchased from BD Biosciences and used at the concentration of 100 ng/ml. Acrylamide and bisacrylamide were obtained from Invitrogen Life Technologies. Prestained molecular mass standards were obtained from Bio-Rad. Except where noted, other reagents were of either reagent or molecular biological grade from Sigma-Aldrich.

Cells and cell cultures

Mononuclear cells (PBMC) were obtained from four X-SCID patients and normal donors and heparinized peripheral blood by Ficoll-Hypaque (Biochrom) density gradient centrifugation. Upon informed consent, lymphoblastoid cell lines (BCLs) were generated by EBV immortalization of patients and control PBMC using standard procedures (15). In all cases, {gamma}c mutations led to the absence of protein expression. Cells were maintained in RPMI 1640 (Biochrom) supplemented with 10% FBS (Invitrogen Life Technologies), 2 mM/L L-glutamine (Invitrogen Life Technologies), and 50 µg/ml gentamicin (Invitrogen Life Technologies), and cultured at 37°C, 5% CO2. In BCL transduction experiments, the pGC2R{gamma} retroviral vector (16) was used to transduce X-SCID BCLs with wild-type (WT) {gamma}c as previously described (17). Transduced cells were selected in the neomycin-analog G418 (Cellgro). NIH 3T3 fibroblasts were used in a few experiments.

Proliferative assay

BCLs (1 x 105 cell/200 µl well) were cultured triplicate in 96-well U-bottom microtiter plates (Falcon; BD Biosciences) with or without rGH at reported concentrations for 4 days. The proliferative response was evaluated by thymidine uptake from cultured cells pulsed with 0.5 µCi of [3H]thymidine (Amersham Biosciences) 8 h before harvesting (18). In neutralization experiments, control EBV cells were preincubated with the neutralizing mAb 284 at the concentration of 6 ng/ml for 3 h or with the IgG1 isotype-matched Ab (Leu 3).

Flow cytometry

The expression of GHR was detected using specific rabbit Abs (Santa Cruz Biotechnology) by indirect immunofluorescence using a second-step incubation with FITC-conjugated donkey anti-rabbit Abs (Pierce). After washing in PBS, cells were incubated for 20 min with the specific Abs and 30 min with secondary Abs. After staining, all samples were washed in PBS and acquired on the FACScan flow cytometer (BD Biosciences) using Lysis I software.

Cell stimulation and protein extraction

Before hormone treatment, the cells were made quiescent through incubation in RPMI 1640 minus serum for 8–12 h. GH was used at 37°C at a concentration of 500 ng/ml in RPMI 1640 for the reported time. Incubations were terminated by washing cells with ice-cold PBS (BioWhittaker) followed by solubilization in 100 µl of lysis solution containing 20 mM Tris (pH 8), 137 mM NaCl, 1% Nonidet P-40, 10 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadatum (Na3VO4), 5 µg/ml leupeptin, and 5 µg/ml aprotinin. The cell lysates were stored at –80°C for Western blot analysis. Nuclear extracts were prepared by the method of Andrews et al. (19) and were subsequently mixed with sample buffer.

Western blot

Immunoblotting using phosphotyrosine mAb was performed as previously reported (14). Immunoblotting using specific Ab was performed according to the vendor protocols. In brief, protein samples separated by SDS-PAGE were transferred onto Mixed Cellulose Esters membranes (Immobilon-NC Mixed Cellulose Esters 0.45 µm; Millipore). The membrane was incubated at room temperature for 1 h in blocking buffer consisting of 10% BSA in wash buffer (10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20). The membrane was then washed three times in wash buffer and incubated 1 h at room temperature or overnight at 4°C with the specific Ab. The membrane was then washed three times and an appropriate IgG HRP-conjugated secondary Ab was used for the second incubation. After further washings, the membrane was developed with ECL-developing reagents, and exposed to x-ray films according to the manufacturer’s instructions (Amersham Biosciences).

Confocal microscopy

After appropriate stimulation, quiescent cells were rinsed in ice-cold PBS and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 30 min at room temperature. After four rinses of 5 min in PBS, the cells were centrifuged in a Shandon Cytospin III (Histotronix) onto a glass slide and permeabilized by incubation in a 0.2% Triton X-100 solution for 20 min. The cells were then incubated for 1 h at room temperature with rabbit Abs against STAT5b diluted 1/100 in PBS containing 1% BSA. After four washings for 5 min in PBS, the cells were incubated for 1 h at room temperature with a 1/200 dilution of FITC-conjugated donkey anti-rabbit IgG (Pierce) in PBS. After washing in PBS, the glass slides were mounted under a coverslip in a 50% glycerol/50% PBS solution. The slides were analyzed by laser scanning confocal microscopy, using a Zeiss LSM 510 version 2.8 SP1 Confocal System.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of GH on the proliferative response of EBV-transformed cell lines from normal subjects and {gamma}c-negative X-SCID patients

It has been reported that GH enhances EBV-transformed cell line proliferation in vitro, its effect being direct and not mediated by IGF-I (18). Thus, to evaluate a biological role of {gamma}c in GHR signaling, we evaluated the response of EBV-transformed lymphocytes (BCLs) from {gamma}c-negative X-SCID patients and normal controls to GH stimulation. As shown in Fig. 1A, GH enhanced proliferation of BCLs of control subjects in a dose-dependent fashion. Significant enhancement of [3H]thymidine uptake was observed at a GH concentration of 50 ng/ml, and the maximal effect was achieved at 200 ng/ml. In contrast, {gamma}c-negative BCLs did not respond at any GH concentration. To rule out that the observed phenomena were due to different numbers of the receptor molecules on the cell membrane, GHR expression was evaluated by flow cytometry analysis of the cells of controls and patients (Fig. 1B). No difference was found in the mean fluorescence intensity (130.99 ± 28.19 vs 139.88 ± 33.49 in patients and controls, respectively; p = NS) and in the percentage of positively stained cells (99.6 vs 99.7% and 99.8 vs 99.9%, respectively).


Figure 1
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  		FIGURE 1. In vitro effect of GH stimulation on proliferation of the EBV cell lines of X-SCID patients and controls and membrane localization of GHR and {gamma}c. A, BCLs were generated by EBV immortalization of the PBMC of patients and controls using standard procedures (15 ) and cultured in the presence of various concentrations of GH for 4 days. Cultures were pulsed with [3H]thymidine for the final 8 h and radioactive incorporation counted. Results are expressed as the increase of cpm from the background. Vertical bars indicate 1 SD. B, Flow cytometry analysis indicating that the expression levels of GHR are comparable in controls and patients. C, Control BCLs were pretreated with medium alone ({blacksquare}) or with the neutralizing mAb 284 ({square}) at the concentration of 6 ng/ml for 3 h, and then cultured for 4 days in the presence of GH at the concentration of 200 ng/ml. As isotype-matched IgG1 control Ab, anti-CD3 Leu 3 was used. Cultures were processed as previously described. D, Control BCLs were pretreated for 1 h with medium alone or with wortmannin at the concentration of 100 nM, and then stimulated with 50, 100, 200, or 400 ng/ml GH, as indicated. As a positive control, fibroblasts were cultured in the presence of EGF. E, {gamma}-chain colocalizes with GHR. Normal BCL cells were double labeled with anti-{gamma}c (left) and anti-GHR (center) Abs. Confocal microscopic analysis indicates a plasma membrane localization for both molecules. The yellow color in the merge (shown on the right) indicates areas of colocalization of the two proteins.

 
Moreover, to demonstrate a link between the {gamma}c and GHR, we used a neutralizing mAb in the proliferative assay. As shown in the Fig. 1C, the neutralizing mAb inhibited by 64% the proliferative response to GH. A nonspecific effect of the Ab was ruled out, because the IgG1 anti-CD3 isotype-matched Ab was ineffective in inhibiting cell proliferation.

To define whether PI3K had a role on GH-induced cell proliferation of BCLs to GH, the kinase inhibitor wortmannin was used. As shown in Fig. 1D, no inhibitory effect was appreciable. By contrast, in the positive control wortmannin was able to inhibit fibroblast proliferation to EGF by 85%.

To ascertain whether {gamma}c was linked to GHR, we then assessed by confocal microscopy the plasma membrane expression of these two molecules. As shown in Fig. 1D, by indirect immunofluorescence using specific Abs, as previously detailed, colocalization of {gamma}c and GHR was observed on the cell surface of normal BCL cells.

Pattern of protein tyrosine phosphorylation induced through GHR engagement in patients and controls cells

We next investigated the overall signal transduction properties of patients and control BCLs following GHR ligation by analyzing the number and the timing of the proteins phosphorylated on tyrosine residues. Fig. 2 illustrates a representative immunoblot with anti-phosphotyrosine Abs of whole cell lysates from BCLs of patients and controls BCLs following stimulation with GH for 5, 15, or 30 min. In contrast to what was observed in control cells, in patients, GH stimulation failed to induce phosphorylation of proteins of ~90 kDa, presumably corresponding to STAT molecules involved in the signal transduction through GHR. This pattern of protein tyrosine phosphorylation was also observed in freshly isolated PBMC from a healthy subject and a patient stimulated with the same concentration and for the same time, thus confirming the observation on BCLs (data not shown).


Figure 2
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  		FIGURE 2. Pattern of protein tyrosine phosphorylation induced through GHR engagement. BCLs from X-SCID patients and healthy subjects were starved of serum for 8–12 h and then stimulated with GH (500 ng/ml) at 37°C for the indicated time. Stimulation was stopped with cold PBS and BCLs were resuspended in lysis buffer. After SDS-PAGE and Western blot, membranes were incubated with anti-phosphotyrosine Abs.

 
GHR signal transduction pattern

The three main signal modules by which signal transduction through GHR occurs involve MAPK/ERK1 and 2, JAK2, STAT1, STAT3 and STAT5 and the PI3K-protein kinase B signaling (7).

To evaluate whether the {gamma}c was involved in GHR-signaling events, we first focused on STAT5 molecule. As shown in Fig. 3, in the BCLs of controls, tyrosine phosphorylation of STAT5 was evident, with a peak of activity observed between 5 and 15 min after GH stimulation. By contrast, in the BCLs of patients, no phosphorylation of STAT5 was detectable after stimulation. In all cell lines examined, STAT5b and STAT5a protein expression was comparable in patients and controls.


Figure 3
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  		FIGURE 3. STAT5 phosphorylation induced through GHR stimulation. rGH stimulation failed to induce STAT5 tyrosine phosphorylation in {gamma}c-negative BCLs. BCLs from X-SCID patients and healthy subjects were starved of serum for 8–12 h and then stimulated with GH (500 ng/ml) at 37°C for the indicated time. After SDS-PAGE and Western blot, membranes were incubated with anti-pSTAT5, anti-STAT5b, or anti-STAT5a Abs.

 
To define whether the blockage in GHR signaling was specific of STAT5 or involved other molecules as well, we then studied JAK2, STAT1, ERKs, and STAT3 phosphorylation (Fig. 4). No difference in the phosphorylation events between the BCLs of patients and controls was appreciable. As shown, the expression of the molecules was comparable in control and patient cells.


Figure 4
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  		FIGURE 4. Phosphorylation events induced through GHR stimulation. BCLs from X-SCID patients and healthy subjects, starved of serum for 8–12 h, were stimulated for the indicated time with rGH at the concentration of 500 ng/ml. After SDS-PAGE and Western blot, membranes were incubated with (A) anti-pJAK2 or anti-JAK2, (B) anti-pSTAT1 or anti-STAT1, (C) anti-pERK or anti-ERK2, (D) anti-pSTAT3 or anti-STAT3.

 
STAT5 nuclear translocation after GHR triggering

Recently, it has been reported that tyrosine phosphorylation of STATs molecules was not sufficient for the activation of the protein (20, 21). Because the activated STAT5 translocates into the nuclei, confocal microscopy was initially used to test the subcellular localization of STAT5b in control and patient {gamma}c-negative cells under resting conditions and after stimulation with GH.

BCLs of patients and controls were stimulated with GH for 30 min, fixed, and incubated with antiserum against STAT5b. As shown in Fig. 4, under basal conditions all cells displayed fluorescent staining of the cytoplasm indicating the presence of STAT5b in this compartment, and only a negligible staining of nucleus indicating absence of STAT5b in this compartment. Stimulation with GH for the time indicated induced nuclear translocation of STAT5b in the BCLs of controls, as demonstrated by the marked increase in STAT5b immunoreactivity within the nucleus and not in {gamma}c-negative BCLs.

We next evaluated by immunoblot of nuclear and cytoplasmic extracts the amount of STAT5b translocation and compared it with the tyrosine phosphorylation of the molecule. As shown in Fig. 5, in control cells, GH stimulation determined a rapid increase of nuclear STAT5b amount. The translocation occurred early being evident 5 min after GH stimulation. Moreover, it still persisted 30 min after stimulation. The translocation paralleled the amount of the tyrosine-phosphorylated form of the protein into the nuclei. This was inversely correlated with the amount of the cytoplasmic form of the molecule. However, after 30 min, the reconstitution of the cytoplasmic aliquot became evident. In the patient cells, no changes were observed (Fig. 6).


Figure 5
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  		FIGURE 5. STAT5b subcellular localization. Control cells of X-SCID patients and healthy subjects were cultured in the absence or presence of 500 ng/ml rGH for 30 min at 37°C. Unstimulated or stimulated cells were analyzed by confocal microscopy for STAT5b (green) distribution in the cell, focusing particularly on whether this protein was present in the nuclei.

 

Figure 6
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  		FIGURE 6. Nuclear fraction of the overall STAT5b amount and of the phosphorylated form of STAT5 in resting or rGH-stimulated BCLs. Patient and control BCLs were stimulated with rGH (500 ng/ml) or medium alone at 37°C for the indicated time. Stimulation was stopped with cold PBS and nuclei were isolated as described in Materials and Methods. After SDS-PAGE of nuclear and cytoplasmic extracts and Western blot, membranes were incubated with anti-STAT5b or anti-pSTAT5 Abs.

 
GH-induced signaling and STAT5b nuclear translocation in X-SCID EBV cells transduced with the WT {gamma}c gene

We next evaluated whether reconstitution of X-SCID cells with WT {gamma}c led to a functional recovery. As shown in Fig. 7A, pGC2R{gamma} cells expressed {gamma}c at a normal extent. These cells proliferated in a comparable fashion to control cells following GH stimulation (Fig. 7B). Moreover, in WB experiments using an anti-phospho-STAT5 Ab, a phosphorylation of the molecule was observed in pGC2R{gamma} cells (Fig. 7C). Finally, in reconstituted cells, GH stimulation induced a normal nuclear translocation of STAT5b, as shown in Fig. 7D.


Figure 7
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  		FIGURE 7. GHR signaling in patient BCLs reconstituted with WT {gamma}c (pGC2R{gamma} cells). A, Membrane expression of {gamma}c in patient or pGC2R{gamma} cells by flow cytometry. B, Proliferative response in control, patient, or reconstituted BCLs. Cells were cultured in the presence of various concentrations of GH for 4 days and pulsed with [3H]thymidine as previously described. Results are expressed as increase of cpm from the background. Vertical bars indicate 1 SD. C, STAT5 phosphorylation induced through GHR stimulation for the indicated time in pGC2R{gamma} cells. Membranes were incubated, as indicated, with anti-pSTAT5, anti-STAT5a, or anti-STAT5b Abs. D, STAT5b subcellular localization through confocal microscopy analysis in control or pGC2R{gamma} cells unstimulated or stimulated with 500 ng/ml rGH for 30 min at 37°C.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we examined a potential role of the {gamma}c chain in GHR signaling using BCLs from healthy control subjects and {gamma}c-negative X-SCID patients. At a functional level, GH enhanced proliferation of control BCLs in a dose-dependent fashion. By contrast, the functional response to GH of the BCLs of {gamma}c-negative patients was severely impaired despite a comparable cellular expression of GHR molecules.

The overall signal transduction properties of GHR in X-SCID patients and control BCLs following GH stimulation was also examined by analyzing the pattern of protein tyrosine phosphorylation. In contrast to what was observed in control BCLs, in patients, GH stimulation failed to induce phosphorylation of proteins of 90 kDa identified as belonging to the STAT molecules family, involved in the signal transduction through GHR. In particular, after GH stimulation no phosphorylation of STAT5 protein was observed in the cell lines of {gamma}c-negative patients in contrast to the control cells, in which a prompt activation of STAT5 occurred. Of note, reconstitution of X-SCID cells with the WT {gamma}c gene corrected the functional and biochemical abnormalities resulting in an appropriate nuclear translocation of STAT5. These findings strongly support an essential role of {gamma}c in GHR signaling.

STAT-dependent pathways are generally believed to be used in cellular events such as cell proliferation, differentiation, and apoptosis (22, 23), even though the overall role of the STAT molecules in GHR signal transduction has not been fully elucidated. At least three different STAT family members (STAT1, STAT3, and STAT5) are activated following GHR perturbation (24, 25, 26, 27, 28, 29), even though STAT5 seems to play a prominent role in receptor signaling. Rodent models of STAT knockouts (30) and the recent identification of a patient with a homozygous missense mutation of the STAT5b gene indicate that STAT5b is essential for a normal postnatal linear growth (31). Furthermore, the patient with STAT5b mutation also had clinical features of immune deficiency such as chronic diarrhea and severe infections, including interstitial pneumopathy. Immunologic studies showed hypergammaglobulinemia and markedly decreased IL-2R{alpha}-chain expression in response to IL-2 stimulation, suggestive of a T cell activation defect. Thus, a few features are similar to {gamma}c-negative X-SCID patients.

Although the activation of JAKs and STATs represents a prominent biochemical event during GH-dependent proliferation of lymphoid cell lines (32), other signaling pathways also contribute to a full GHR response. GH has been shown to activate the PI3K-protein kinase B signaling (33), MAPKs, and ERKs 1 and 2 (34, 35, 36). In both STAT5 knockout mouse and in the patient with STAT5 mutation, these pathways are fully functional. In keeping with this observation, also in our experimental model, no alteration was observed in ERK 1 and 2 expression and phosphorylation events involving JAK2, ERKs, STAT1 and 3 molecules that occur following GHR triggering. Moreover, in this study, the involvement of PI3K in GH-induced proliferation of BCLs was ruled out because the kinase inhibitor wortmannin was ineffective in blocking the proliferative response. Similarly, IGF-I expression has been reported to be dependent on STAT5 b, but not on the PI3K pathway (37). Taken together, these observations imply that GHR, as well as other receptors, is able to integrate different pathways which are individually differentially regulated. In support of this, it has been recently shown that GHR signaling and the subsequent IGF-I transcription regulation are under different regulatory controls in hepatocytes, fibroblasts, and myoblasts (38). This could lead to a hypothesis of differential functions of an individual receptor exerted in different tissues. A cell type-restricted STAT activation has been reported (39, 40, 41). STAT5 is not activated following GH stimulation in human fibrosarcoma cells even though these cells express the STAT5 protein (41), thus implying that a selectivity in the involvement of specific STAT subset seems to be a general feature of GHR signal transduction.

Overall, activation of STAT5b is considered a prominent event in GHR signaling and is crucial for the regulation of transcription of GH-responsive genes, including the gene encoding for IGF-I. This process relies on an appropriate phosphorylation and nuclear translocation of the molecule (7, 42). Recently, it has been proposed that STATs tyrosine phosphorylation and nuclear translocation are two events that are regulated separately (21). In particular, Giron-Michel et al. (43) demonstrated in the hybrid receptor {gamma}c/GM-CSFRβ that the {gamma}c/JAK3 complex controls the nuclear translocation of pSTAT5 rather than STAT5 phosphorylation itself. Hence, to address the issue of defining the functional implication of {gamma}c mutation on STAT5b activation, in our study, the subcellular localization of STAT5b was investigated by analyzing cytokine-induced translocation of STAT5b from the cytoplasm to the nucleus with confocal microscopy. Stimulation with GH induced nuclear translocation of STAT5b in the control cells, whereas no efficient nuclear translocation occurred in {gamma}c-negative cells. Furthermore, immunoblot of nuclear and cytoplasmic extracts showed in control cells a rapid increase of the nuclear fraction of the STAT5 molecule after GH stimulation, which paralleled the molecule phosphorylation, differently from what was observed in patient cells. Moreover, through confocal microscopy studies, we demonstrated that GHR and {gamma}c colocalize, as expected in that both molecules are type I hemopoietin receptors. A physical interaction may be hypothesized as well, even though conclusive data are still lacking.

Our data suggest that the {gamma}c chain is a required signaling subunit of the GHR complex in B cell lines. In particular, in this cell line, it is selectively required for STAT5 phosphorylation and nuclear translocation, and not for the activation of other molecules as ERKs.

Our study demonstrates the existence of a previously unappreciated relationship between individually well-studied elements, such as GHR and {gamma}c, and signaling pathways. Cross-talk between receptor signaling systems is now emerging as an important and exciting area of signaling research. Whether the participation of {gamma}c to the GHR confers some additional properties to the receptor in hemopoietic cell differentiation and functioning remains to be elucidated. Of note, in CD34+ progenitors, {gamma}c participates in hemopoietic cell differentiation by interacting with GM-CSFRβ. This interaction does not occur in normal NK cells or nonhemopoietic cells (43). Hence, the complexity of receptor signaling relies not only on the possibility that individual receptors interact one with each other, but also on a differential array of distinct subunits that may represent a hallmark of that specific cell type.

Our current study also explains what we previously reported on an atypical X-SCID phenotype and severe short stature associated with GH hyporesponsiveness and abnormal GHR-induced protein tyrosine phosphorylation (14), and indicates that growth failure in X-SCID is directly related to the genetic alteration.


   Acknowledgment
 
We thank Emanuela Minopoli for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Ministero della Salute-Roma and Regione Campania, Legge 502 and Ministero dell’Università e della Ricerca Scìentifica e Tecnologica, Progetto di Rilevante Interesse Nazionale and by the Division of Intramural Research, National Human Genome Research Institute, National Institutes of Health. Back

2 Address correspondence and reprint request to Dr. Claudio Pignata, Department of Pediatrics, Unit of Immunology, "Federico II" University, Via S. Pansini 5-80131, Naples, Italy. E-mail address: pignata{at}unina.it Back

3 Abbreviations used in this paper: X-SCID, X-linked SCID; {gamma}c, common cytokine receptor {gamma}-chain; GH, growth hormone; GHR, GH receptor; IGF, insulin-like growth factor; rGH, recombinant human GH; BCL, lymphoblastoid cell line; WT, wild type. Back

Received for publication July 21, 2005. Accepted for publication August 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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