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Endocrinology Vol. 142, No. 1 147-156
Copyright © 2001 by The Endocrine Society

ARTICLES

Growth Hormone Exerts Antiapoptotic and Proliferative Effects through Two Different Pathways Involving Nuclear Factor-{kappa}B and Phosphatidylinositol 3-Kinase1

Sébastien Jeay, Gail E. Sonenshein, Paul A. Kelly, Marie-Catherine Postel-Vinay and Elena Baixeras2

INSERM, U-344, Endocrinologie Moléculaire, Faculté de Médecine Necker (S.J., P.A.K., M.C.P.V., E.B.), 75730 Paris, France; and Department of Biochemistry, Boston University School of Medicine (G.E.S.), Boston, Massachusetts 02118

Address all correspondence and requests for reprints to: Dr. Marie-Catherine Postel-Vinay, INSERM, U-344, Faculté Necker-Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France. E-mail: postel-vinay{at}necker.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dependence of murine pro-B Ba/F3 cells on interleukin-3 can be substituted by GH when cells are stably transfected with the GH receptor (GHR) complementary DNA. Recently, we demonstrated that Ba/F3 cells produce GH, which is responsible for the survival of cells expressing the GHR. This GH effect involves the activation of nuclear factor-{kappa}B (NF-{kappa}B). Here, we examined the signaling pathways mediating proliferation of growth factor-deprived Ba/F3 GHR cells. Exogenous GH stimulation of Ba/F3 GHR cells induced cyclins E and A and the cyclin-dependent kinase inhibitor p21waf1/cip1 and repressed cyclin-dependent kinase inhibitor p27kip1. The presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor Ly 294002 abolished proliferation induced by GH, arresting Ba/F3 GHR cells at the G1/S boundary, but did not promote apoptosis. Thus, the proliferative effect of GH is closely related to PI 3-kinase activation, whereas PI 3-kinase is not essential for GH-induced cell survival. Addition of Ly 294002 resulted in a moderate decrease in NF-{kappa}B activation by GH, suggesting a possible link between PI 3-kinase and NF-{kappa}B signaling by GH. Expression of c-myc was also induced by GH in Ba/F3 GHR cells, and inactivation of either PI 3-kinase or NF-{kappa}B reduced this induction. Overexpression of the dominant negative repressor mutant c-Myc-RX resulted in an inhibition of the GH proliferative effect, suggesting the involvement of c-myc in GH-induced proliferation. Taken together, these results suggest that the effects of GH on cell survival and proliferation are mediated through two different signaling pathways, NF-{kappa}B and PI 3-kinase, respectively; although cross-talk between them has not been excluded. NF-{kappa}B, which has been shown to be responsible for the antiapoptotic effect of GH, could also participate in GH-induced proliferation, as c-myc expression is promoted by PI 3-kinase, in an NF-{kappa}B-dependent and -independent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH INITIATES its wide variety of biological effects by binding to the GH receptor (GHR). After GHR dimerization, Jak-2 recruitment and activation promote its autophosphorylation and phosphorylation of GHR on tyrosine residues. In addition, a large number of other cellular proteins become tyrosyl phosphorylated in response to GH (1). Two signaling pathways have been identified to mediate the GH proliferative action: the mitogen-activated protein (MAP) kinases, designated extracellular regulated kinase-1 (Erk-1) and Erk-2, and signal transducers and activators of transcription. Molecules implicated in MAP kinase activation by GH were identified in 293 cells (2), and their involvement was confirmed in 3T3 F442A preadipocytes (3). The Shc/Grb2/SOS/Ras/Raf/MEK (MAP/Erk kinase)/MAP kinase pathway activated by GH led to the activation of several transcription factors, such as Elk-1, a member of the ternary complex factor family. GH induces phosphorylation of Elk-1 via Erk-1 and -2 and promotes the transcription of early response genes such as c-fos, egr-1, and junB (4). Furthermore, a correlation was demonstrated between insulin receptor substrate-1 (IRS-1) expression and GH-induced MAP kinase activation and proliferation in GHR-expressing 32D cells (5). Together, these data strongly suggest an important role for the MAP kinase pathway in the GH proliferative effect.

Phosphatidylinositol 3-kinase (PI 3-kinase) has also been shown to play an important role in the proliferation and cell survival induced by many cytokines through activation of the serine/threonine kinase AKT/protein kinase B (6, 7). A role for PI 3-kinase in the insulin-like effects of GH was reported in isolated rat adipocytes (8), which was related to the induction of IRS-1 (9). More recently, GH as well as PRL were reported to stimulate tyrosine phosphorylation of IRS-1, -2, and –3; their association with p85 PI 3-kinase; and PI 3-kinase activation via Jak-2 (10). Thus, PI 3-kinase seems to be involved in the insulin-like effects of GH; other possible roles for PI 3-kinase in GH signaling remain to be established. As antiapoptotic and proliferative effects of many cytokines and growth factors are under the control of the PI 3-kinase pathway in hemopoietic cells, the question of a possible involvement of PI 3-kinase in the GH proliferative effect has should be examined.

The GHR has been detected in numerous cells of the immune system, particularly thymocytes, T cells, natural killer cells, B cells, and monocytes (11, 12). Recent findings demonstrate that GH is locally produced by hemopoietic cells, suggesting that GH could act in an autocrine/paracrine mode of action in the immune system (11, 12, 13, 14). The physiological actions of GH on the immune system still remain controversial, but increasing evidence has been emerging in the literature indicating a reciprocal communication network between the endocrine and immune systems. The pro-B Ba/F3 cell line requires interleukin-3 (IL-3) and serum for growth, and their removal results in cell apoptosis. Ba/F3 cells transfected with a complementary DNA (cDNA) encoding GHR are resistant to apoptosis (14). Recently, we showed that this resistance is due to locally produced GH and is mediated through the activation of nuclear factor-{kappa}B (NF-{kappa}B) in Ba/F3 cells expressing GHR (14).

The pathway leading to NF-{kappa}B activation by GH remains to be established. NF-{kappa}B/Rel factors have been found to promote cell survival in a number of cells and growth conditions (15). Upstream proteins involved in NF-{kappa}B activation were defined in tumor necrosis factor and IL-1 signaling as MAP kinase kinase kinase (MAP3K)-related proteins (16). It was reported that activation of NF-{kappa}B-inducing kinase (NIK) and MAP/ERK kinase kinases (MEKK)-1 (17), -2, and -3 (18) led to activation of I{kappa}B kinases (IKK), which are responsible for the phosphorylation of inhibitors of NF-{kappa}B (I{kappa}B) on serine residues. We recently showed that oncogenic Raf induced NF-{kappa}B via activation of IKK-2, whereas oncogenic Ras functioned via two pathways: Raf to IKK-2 and PI 3-kinase to IKK-1 (19). This phosphorylation of serine residues of I{kappa}B{alpha} leads to ubiquitination and degradation of the inhibitory protein and thereby the release and translocation of NF-{kappa}B into the nucleus (16). Finally, NF-{kappa}B can promote cell survival by inducing the transcription of genes such as bcl-2, bcl-X (20), bfl-1/A1 (21), IEX-1 liter (22), c-IAP1, c-IAP2 (23), and c-myc (24), all reported to have antiapoptotic actions. In the case of Ba/F3 GHR cells, activation of NF-{kappa}B appeared to promote cell survival via expression of Bcl-2 and potentially Bag-1 (14).

Regulation of cell cycle by cytokines is closely related to their capacity to govern the expression levels of cyclins (25). Growth factor deprivation reduces cyclin synthesis and promotes cell cycle arrest before apoptosis. Cyclins are sequentially expressed according to the phase of the cell cycle. Indeed, cyclin D synthesis induces cell cycle entry and promotes G1/S transition. Cyclin E regulates transition through the S phase, whereas cyclin A controls the entry into S phase as well as the G2/M phase transition of the cell cycle. The activity of the cyclins is, in turn, regulated by cyclin-dependent kinase inhibitors (CKI). The Cip/Kip CKI family is composed of several members, including the p21waf1/cip1 and p27kip1 CKI proteins, which were shown to inactivate all the cyclins (25).

In an attempt to better identify the signaling molecules involved in GH cell cycle control, we used growth factor-deprived Ba/F3 GHR cells, which arrest in the G0/G1 phase of the cell cycle (14). Our results show that GH-mediated induction of proliferation correlates with the induction of cyclins E and A and c-myc expression. These actions are accompanied by induced p21waf1/cip1 and repressed p27kip1 expression. Moreover, activation of PI 3-kinase is crucial for the proliferative effect of GH, but is not essential for GH-induced cell survival. PI 3-kinase activation promotes c-myc gene expression and participates in GH-induced NF-{kappa}B activation. The use of a dominant negative mutant of c-Myc indicates that c-Myc is critical in the GH-induced proliferation of Ba/F3 GHR cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents, antibodies, and plasmids
The PI 3-kinase inhibitor Ly 294002 (Calbiochem-Novabiochem Co., San Diego, CA) was used at 20 µM. Bovine GH (bGH) was provided by William Baumbach (American Cyanamid, Princeton, NJ). Antibodies against cyclin D1 (sc-717; 1:1000), cyclin E (sc-481; 1:1000), cyclin A (sc-596; 1:1000), p21waf1/cip1 (sc-397; 1:500), and p27kip1 (sc-1641; 1:500) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA.). Antibody against cyclin D3 (1:1000) was purchased from Transduction Laboratories, Inc. (Lexington, KY). pRCßactin-containing mutant I{kappa}B{alpha} (A32/36) and pBaBE-containing mutant Myc-RX expression vectors were provided by Michael Karin (26) (University of California, San Diego, CA) and Bruno Amati (27) (Institut Suisse de Recherches Experimentales sur le Cancer, Lausanne, Switzerland), respectively. The thymidine kinase promoter-driven luciferase reporter plasmid, controlled by six reiterated {kappa}B sites (28), was a gift from Georges Rawadi (Hoescht-Marion-Roussel, Romainville, France).

Cell culture and treatment conditions
Parental Ba/F3 (Ba/F3 WT) cells and populations of stable transfectants expressing the wild-type rat GHR (Ba/F3 GHR) (29) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin, and 10% WEHI-3B cell supernatant as a source of IL-3. For experiments, cells were washed twice in RPMI medium before starvation in 2% BSA (fraction V, Sigma) and serum- and WEHI-3B-free medium (starvation medium) for 6 h. Cells were stimulated either with 1 µg/ml bGH, which was added to the starvation medium (bGH treatment) or with medium containing serum and WEHI-3B cell-conditioned medium (normal medium).

Cell cycle analyses
Cell cycle was assessed by DNA content analysis after staining with the DNA intercalator propidium iodide. Cells were harvested by centrifugation and permeabilized with 30 µl DNA-Prep LPR reagent, followed by addition of 0.5 ml DNA-Prep stain propidium iodide solution (DNA-Prep reagents, Coulter Corp., Miami, FL). After vortexing, samples were incubated at 37 C for at least 30 min, and then analyzed by flow cytometry on a FACScan (Becton Dickinson and Co., Mountain View, CA). Cell cycle distribution was determined using CellQuest software (Becton Dickinson and Co.) and manual gating.

Immunoblotting
Cells (1 x 106) were washed in PBS and lysed in sample buffer containing dithiothreitol. Concentrations of whole cell extracts were measured by Bradford assay using a reagent from Bio-Rad Laboratories, Inc. (Hercules, CA), according to the manufacturer’s directions. Samples (50 µg protein) were resolved by SDS-PAGE under reducing conditions. Proteins were transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.) and then stained with Ponceau red solution (Sigma, St. Louis, MO) to verify equal loading of proteins. Membranes were incubated overnight at 4 C in TBS-T [50 mM Tris-HCl (pH 7.6), 200 mM NaCl, and 0.1% Tween-20] with 2% BSA (fraction V, Sigma). Proteins were detected by incubation with the specific antibody in TBS-T with 2% BSA. After extensive washing in TBS-T, horseradish peroxidase-conjugated protein G (1:1000 dilution; Bio-Rad Laboratories, Inc.) was added for 1 h. The membranes were again subjected to extensive washing in TBS-T, and the specific protein bands were visualized using the enhanced chemiluminescence detection system (NEN Life Science Products, Boston, MA), according to the manufacturer’s instructions. For reprobing of the Western blots, antibodies were stripped from membranes following the instructions of the manufacturer (NEN Life Science Products). To quantify bands, densitometric analyses were performed using the Kodak Zoom digital camera model DC120 system (Eastman Kodak Co., Rochester, NY) and 1D image analysis software.

Northern blotting
Total RNA was prepared from 10 x 106 Ba/F3 WT and Ba/F3 GHR cells using the TRIzol reagent method (Life Technologies, Inc., Gaithersburg, MD). Between 10 and 15 µg total RNA were denatured, size-fractionated by formaldehyde-agarose gel electrophoresis, and blotted onto a nylon filter (Hybond-N+ membrane, Amersham Pharmacia Biotech, Aylesbury, UK). The filters were hybridized with 32P-labeled cDNA, and signal was detected using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The probes used were a 2.2-kb fragment from the mouse c-myc cDNA clone pM-c-myc54 (30) and a 1.5-kb fragment from the human glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH). Specific bands were detected by PhosphorImager (Molecular Dynamics, Inc.) and were quantified by the ImageQuant software (Molecular Dynamics, Inc.).

Gel mobility and supershift assays
For electrophoretic mobility shift assay (EMSA), Ba/F3 cells were starved for 6 h and incubated with 20 µM Ly 294002 for 1 h before stimulation with GH for an additional 1 h. The NF-{kappa}B binding site (5'-AAGTCCGGGTTTTCCCCAACC- 3', with the core NF-{kappa}B binding site underlined) from the c-myc gene, termed URE, was used as a probe (31). DNA was labeled using the Klenow fragment of Escherichia coli DNA polymerase I (Life Technologies, Inc.) and [{alpha}-32P]dCTP (Amersham Pharmacia Biotech). Nuclear extracts were prepared as previously described (14). Briefly, nuclear extracts (2–3 µg) were incubated in sample buffer [0.4 µg poly(dI-dC), 0.1% Triton X-100, 0.5% glycerol, 0.8 mM dithiothreitol, and 2 mM HEPES, pH 7.5] and adjusted to 100 mM KCl in a final volume of 25 µl. Then, 32P-labeled URE probe (40,000 cpm, ~2 ng) was added to the mixture and incubated for 30 min at room temperature. DNA-NF-{kappa}B complexes were separated on a 4.5% acrylamide gels by electrophoresis in low ionic strength Tris-borate-EDTA buffer for 2 h at 150 V. The gels were dried, and labeled complexes were visualized by autoradiography at -80 C using screens. Specific bands were quantified by densitometric analyses using the Kodak system described above.

Transient transfections and luciferase assays
Cells (10 x 106) were transfected with 30 µg of the indicated vectors by electroporation at 330 V, {infty} rad, and 1500 µF in a Bio-Rad Laboratories, Inc., apparatus. Cells were then incubated overnight in normal medium containing IL-3 and serum to allow them to recover. For luciferase assays, 30 µg NF-{kappa}B-dependent luciferase reporter plasmid was transfected. After overnight incubation in normal medium, cells were starved for 1 h and then stimulated for 16 h. Total cell extracts were prepared and used in a luciferase activity assay according to the manufacturer’s instructions (Promega Corp., Madison, WI). Results are expressed as the fold induction of luciferase activity calculated under stimulation conditions compared with luciferase activity under starvation conditions. Each point was performed in triplicate, and the results are expressed as the mean ± SD.

Statistical analyses
Results are given as the mean ± SD for the indicated numbers of independently performed experiments. A paired t test was used to calculate differences between means; differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH regulation of the cell cycle in Ba/F3 cells
The effects of GH on cell cycle proteins were investigated using parental Ba/F3 cells (Ba/F3 WT) and a population of Ba/F3 cells stably transfected with the GHR cDNA (Ba/F3 GHR) (29). Ba/F3 WT and Ba/F3 GHR cells were incubated in normal medium or in starvation medium lacking growth factors from serum- and WEHI-3B-conditioned medium for 6 h. Under these conditions, according to previous data, Ba/F3 WT cells undergo apoptosis, whereas Ba/F3 GHR cells are growth arrested (14). Cell cultures were then incubated for 24 h in the absence or presence of 1 µg/ml bGH. Protein cell lysates were isolated, and the levels of expression of cyclins D1, D3, E, and A and the cyclin-dependent kinase inhibitors (CKI) p21waf1/cip1 and p27kip1 were determined by immunoblot analyses (Fig. 1Go, A and B). Cells incubated in normal medium expressed the cyclins necessary for cell cycle progression (Fig. 1AGo, lanes 1 and 4). Expression levels of cyclins D1, D3, E, and A were down-regulated in starved Ba/F3 WT cells, and as expected, they were unaffected by the bGH treatment (Fig. 1AGo, lanes 2 and 3). FACS analysis confirmed that Ba/F3 WT cells underwent apoptosis when they were starved in either the absence or presence of bGH (Fig. 1CGo). Locally produced GH has been shown to be responsible for the survival of Ba/F3 GHR cells upon starvation conditions (14). Thus, compared with starved Ba/F3 WT cells (Fig. 1AGo, lane 2), starved Ba/F3 GHR cells maintained somewhat higher levels of cyclins D1 and D3 (Fig. 1AGo, lane 5). The levels of cyclins E and A dropped in Ba/F3 GHR cells after starvation (Fig. 1AGo, lane 5), and cells displayed a G0/G1 phase arrest (Fig. 1CGo). Addition of GH did not modify the expression levels of cyclin D1 and D3 in these cells (Fig. 1AGo, lane 6). In contrast, stimulation of starved Ba/F3 GHR cells with exogenous bGH enhanced cyclin E and A expression (Fig. 1AGo, lane 6), which was associated with the ability of bGH to promote proliferation in Ba/F3 GHR cells (Fig. 1CGo) (14).



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  		Figure 1. Effect of GH on expression levels of cyclins and cell cycle inhibitors. Ba/F3 WT and Ba/F3 GHR cells were starved for 6 h and then incubated for 24 h in normal medium (lanes 1 and 4) or in starvation medium in the absence (lanes 2 and 5) or presence of 1 µg/ml bGH (lanes 3 and 6). A and B, Cyclins D1, D3, E, and A as well as p21waf1/cip1 and p27kip1 proteins were detected by immunoblot analyses using total cell lysates, as described in Materials and Methods. Arrows indicate the positions of proteins on immunoblots. Blots from A were all obtained with the same membrane after sequential stripping and reprobing with antibodies against cyclin D1, D3, E, and A. Similarly, in B the same membrane probed with p21waf1/cip1 antibody was stripped and reprobed with p27kip1 antibody. The blots were subjected to densitometry, and the changes relative to Ba/F3 WT cells in starvation medium are expressed as fold induction or reduction. C, DNA content of cells was labeled by propidium iodide (PI), and cell cycle analyses were performed by flow cytometry. The DNA content vs. cell number is presented in each profile. Percentages correspond to cells in apoptosis (Apo; <2n content), cells in the G0/G1 phase (2n content), and cells in the S/M phase (4n content). Results are representative of four independent experiments.

 
Analysis of CKI expression is presented in Fig. 1BGo. Expression of p21waf1/cip1 was greatly reduced in Ba/F3 WT and Ba/F3 GHR cells upon starvation, whereas p27kip1 was highly induced (Fig. 1BGo, lanes 1 vs. 2 and 4 vs. 5, respectively). bGH treatment had no effect on their expression in Ba/F3 WT cells (Fig. 1BGo, lane 3). On the contrary, in Ba/F3 GHR cells stimulated by bGH, an increase in p21waf1/cip1 and a decrease in p27kip1 expression were observed (Fig. 1BGo, lanes 5 vs. 6). Thus, a drop in expression levels of cyclins D1, D3, E, and A and p21waf1/cip1 and an induction of p27kip1 correlate with growth arrest and apoptosis of Ba/F3 WT cells. In Ba/F3 GHR cells, exogenous GH induced expression of p21waf1/cip1 and cyclins E and A as well as inhibition of p27kip1 expression, and these effects were associated with the promotion of cell growth.

Involvement of the PI 3-kinase pathway in GH-induced cell proliferation
To investigate the role of PI 3-kinase, which has been implicated in the regulation of GH-induced proliferation, Ba/F3 GHR cells were incubated for 1 h in the presence of the PI 3-kinase inhibitor Ly 294002 or in DMSO, which was used as a vehicle (Fig. 2Go). DMSO treatment had no effect on cell cycle of Ba/F3 GHR cells, as 87% of starved cells were arrested in the G0/G1 phase. Cells incubated in normal medium or stimulated by bGH showed a normal cell cycle, with 53% and 56% of cells in G0/G1 phase and 46% and 43% of cells in the S/M phase, respectively (Fig. 2Go). Addition of Ly 294002 did not affect cell cycle of Ba/F3 GHR cells starved for 24 h as, again, 87% of cells were found arrested in the G0/G1 phase, and only 6% of cells were in apoptosis (Fig. 2Go). Importantly, the PI 3-kinase inhibitor essentially completely inhibited cell cycle progression induced by bGH after 24-h stimulation. In either case, the large majority of the cells (85–87%) remained in the G0/G1 phase, and 6% of cells underwent apoptosis (Fig. 2Go). These results strongly support the idea that PI 3-kinase is crucial for the proliferation of Ba/F3 cells promoted by GH. Moreover, PI 3-kinase does not seem to be involved in GH-induced cell survival.



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  		Figure 2. Inhibition of GH-mediated cell cycle progression by PI 3-kinase inhibitors. Ba/F3 GHR cells were starved for 6 h. Ly 294002 (20 µM) or DMSO was added 1 h before the end of starvation, and cells were incubated for 24 h in normal medium, starvation medium, or starvation medium plus bGH (1 µg/ml) as indicated. Cell cycle analyses were performed as described in Fig. 1CGo. Results are representative of four independent experiments.

 
Involvement of PI 3-kinase in GH-induced NF-{kappa}B activation
NF-{kappa}B was shown to be critical for GH-mediated Ba/F3 cell survival (14). We examined a possible link between PI 3-kinase- and NF-{kappa}B GH-activated pathways in Ba/F3 GHR cells. Cells were starved 6 h before treatment with the PI 3-kinase inhibitor Ly 294002 or DMSO alone for 1 h. Then, cells were incubated for 1 h in starvation medium alone or in medium containing 1 µg/ml GH. Nuclear extracts were analyzed by EMSA. As shown in Fig. 3AGo, the presence of the activated NF-{kappa}B band was not detected in Ba/F3 WT nuclear extracts under any of the conditions assayed. Consistent with our previous observations (14), nuclear extracts from starved Ba/F3 GHR cells showed a sustained activation of NF-{kappa}B (Fig. 3AGo), which has been reported to be due to locally produced GH by Ba/F3 cells (14). The presence of Ly 294002 caused a 2-fold decrease in the basal expression of NF-{kappa}B in starved Ba/F3 GHR cells. Addition of exogenous bGH enhanced NF-{kappa}B activation by 5-fold compared with that of starved Ba/F3 GHR cells, and coincubation with bGH and Ly 294002 reduced NF-{kappa}B activation by 2-fold (Fig. 3AGo). Thus, inhibition of the PI 3-kinase pathway results in a partial decrease in GH-activated NF-{kappa}B, suggesting a link between PI 3-kinase and NF-{kappa}B pathways in GH signaling.



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  		Figure 3. Effect of Ly 294002 on NF-{kappa}B activation by GH. A, EMSA. After starvation for 6 h, cells were incubated in the presence (+) or absence (-) of 20 µM Ly 294002 for 1 h and then incubated for an additional 1 h with (+) or without (-) 1 µg/ml bGH, as indicated. Nuclear extracts were prepared, and samples were subjected to EMSA for NF-{kappa}B binding using URE oligonucleotide as a probe. The fold induction of NF-{kappa}B activation compared with levels found in starved/Ly 294002-treated Ba/F3 GHR cells was determined by densitometric analyses of bands and is presented below each lane. B, Luciferase assays. Ba/F3 WT ({blacksquare}) or Ba/F3 GHR () cells (10 x 106) transfected with 30 µg NF-{kappa}B-luciferase construct and cultured in normal medium overnight for recovery. Cells were then preincubated for 1 h in the presence (+) or absence (-) of 20 µM Ly 294002 in starvation medium, followed by incubation under starvation conditions alone or in the presence of 1 µg/ml bGH for 16 h. Lysates were prepared and analyzed for luciferase activity. Results are expressed as fold induction of luciferase activity (see Materials and Methods) and are the mean ± SD of three independent experiments. Significance was calculated using paired t test: *, P < 0.05.

 
We also studied the involvement of PI 3-kinase in GH-activated NF-{kappa}B by luciferase assays. Ba/F3 WT and Ba/F3 GHR cells were both transfected with a NF-{kappa}B element-driven luciferase construct, and the effect of GH on NF-{kappa}B activity was studied in the absence or presence of Ly 294002 (Fig. 3BGo). Compared with starved Ba/F3 WT cells, a 3.4 ± 0.2-fold induction of luciferase activity was detected in starved Ba/F3 GHR cells (Fig. 3BGo), again indicating a basal activation of NF-{kappa}B in these cells (14). As expected, bGH treatment had no effect on Ba/F3 WT cells, whereas it provoked a 9.7 ± 1.1-fold induction of luciferase activity in Ba/F3 GHR cells (Fig. 3BGo). The presence of Ly 294002 did not significantly modify NF-{kappa}B activation in Ba/F3 WT cells in either starved or bGH-treated conditions (Fig. 3BGo; P > 0.05). A marked reduction in luciferase activity was found in starved Ba/F3 GHR cells incubated in presence of Ly 294002 (Fig. 3BGo; P < 0.05). Also, the bGH-induced 9.7 ± 1.1-fold induction of luciferase activity was significantly decreased to 6.2 ± 0.7-fold when Ba/F3 GHR cells were pretreated with Ly 294002 (Fig. 3BGo; P < 0.05). Together, the results obtained using EMSA and luciferase assays strongly suggest that activation of NF-{kappa}B by GH is partially dependent on the PI 3-kinase pathway in Ba/F3 GHR cells, indicating that an additional pathway is also responsible for NF-{kappa}B activation by GH.

GH effect on c-myc expression
To investigate the effect of GH on c-myc gene expression in Ba/F3 cells, Northern blot analyses were performed on total RNA prepared from Ba/F3 WT and Ba/F3 GHR cells, unstimulated (Fig. 4Go, lanes 2 and 7, respectively) or stimulated with bGH for 30 min to 3 h (Fig. 4Go, lanes 3–5 and 8–10, respectively). As a positive control, cells were also incubated in normal medium for 1 h (Fig. 4Go, lanes 1 and 6). As expected, the Northern blot showed the absence of c-myc expression in Ba/F3 WT cells treated with bGH (Fig. 4Go, lanes 3–5), and no significant effect was observed compared with c-myc expression in starved cells (Fig. 4Go, lane 2; P > 0.05). A significant increase in c-myc messenger RNA (mRNA) levels was seen in both Ba/F3 WT and Ba/F3 GHR cells cultured in normal medium with a 5.2 ± 1.0- and 4.3 ± 0.7-fold inductions compared with levels in corresponding starved cells for both cell lines (Fig. 4Go, lanes 1 vs. 2 and lanes 6 vs. 7, respectively; P < 0.05). Similarly, addition of bGH in Ba/F3 GHR cells from 30 min to 1 h induced a significant increase in c-myc mRNA levels, with a 4.4 ± 0.7-fold to a 5.0 ± 0.8-fold induction (lanes 8 and 9; P < 0.05) compared with the negative control (lane 7), which then decreased to 2.9 ± 0.4-fold after 3 h of bGH stimulation (Fig. 4Go, lanes 9 vs. 10; P < 0.05). These results indicate a transient effect of GH on c-myc gene expression in Ba/F3 GHR cells.



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  		Figure 4. Kinetics of c-myc mRNA expression under GH stimulation. Ba/F3 WT and Ba/F3 GHR cells were incubated under starvation conditions for 6 h and then were left untreated (lanes 2 and 7) or were treated with bGH for 30 min (lanes 3 and 8), 1 h (lanes 4 and 9), or 3 h (lanes 5 and 10). As a positive control, cells were also cultured in normal medium for 1 h (lanes 1 and 6). Total RNA was prepared, and 15-µg samples were analyzed by Northern blot. Membranes were hybridized with a c-myc probe and a GAPDH probe as a control (upper panel). c-myc mRNA levels were normalized to levels of GAPDH mRNA in Ba/F3 WT and Ba/F3 GHR cells. Results are expressed as the fold induction of the c-myc/GAPDH ratio compared with the ratio obtained under starvation conditions and are the mean ± SD of three independent experiments (lower panel). Statistical significance was calculated using paired t test: *, P < 0.05.

 
Regulation of GH-induced c-myc expression by PI 3-kinase and NF-{kappa}B activation pathways
We next asked whether PI 3-kinase played a role in the effects of GH on c-myc expression. RNA levels of c-myc were evaluated by Northern blot using total RNA from Ba/F3 GHR cells incubated either with DMSO as a control (Fig. 5AGo, lanes 1, 3, and 5) or with Ly 294002 (Fig. 5AGo, lanes 2, 4, and 6) for 1 h. As expected, starvation of Ba/F3 GHR cells reduced c-myc RNA levels by a factor of 3 compared with those in normal medium conditions (Fig. 5AGo, lanes 3 vs. 1; P < 0.05). No significant difference in c-myc mRNA levels were found in cells treated with DMSO or Ly 294002 and maintained under starvation conditions (Fig. 5AGo, lanes 3 vs. 4; P > 0.05). Stimulation with bGH caused a 3.9 ± 0.7-fold induction of c-myc mRNA levels (Fig. 5AGo, lanes 3 vs. 5; P < 0.05). Preincubation of Ba/F3 GHR cells with Ly 294002 markedly decreased c-myc expression in normal medium or upon bGH treatment (Fig. 5AGo, lanes 1 vs. 2 and lanes 5 vs. 6, respectively; P < 0.05). These results strongly suggest that GH acts on c-myc expression through activation of PI 3-kinase.



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  		Figure 5. Involvement of PI 3-kinase and NF-{kappa}B pathways in c-myc expression under GH stimulation. A, Effect of Ly 294002. Ba/F3 GHR cells were incubated under starvation conditions for 6 h, and either DMSO (lanes 1, 3, and 5) or 20 µM Ly 294002 (lanes 2, 4, and 6) was added 1 h before stimulation with normal medium (lanes 1 and 2), starvation medium (lanes 3 and 4), or 1 µg/ml bGH (lanes 5 and 6) for 1 h, then total RNA was prepared. Membranes were hybridized with a c-myc probe and a GAPDH probe as a control (upper panel). Results are also expressed as the fold induction of the c-myc/GAPDH ratio compared with the ratio obtained under starvation conditions and are the mean ± SD of three independent experiments (lower panel). B, Effect of overexpression of the I{kappa}B{alpha} (A32/36) mutant. Ba/F3 GHR cells (10 x 106) were transfected with control vector pRCßactin (lanes 1, 3, and 5) or the plasmid expressing the mutant I{kappa}B{alpha} (A32/36) (lanes 2, 4, and 6). After 6-h starvation, cells were incubated in normal medium for 1 h (lanes 1 and 2) or were treated with bGH for 0 h (lanes 3 and 4) and 1 h (lanes 5 and 6). Total RNA was prepared, and membranes were hybridized as described in A. Results are also expressed as the fold induction of the c-myc/GAPDH ratio compared with the ratio obtained under starvation conditions and are the mean ± SD of three independent experiments. Statistical significance in A and B was calculated using paired t test: *, P < 0.05.

 
As it has been reported that NF-{kappa}B can regulate c-myc transcription (24), the ability of GH to induce c-myc expression through NF-{kappa}B was examined using a transfection strategy. The levels of c-myc expression were visualized by Northern blot using total RNA of Ba/F3 GHR cells transiently transfected with the empty parental pRCßactin vector DNA (Fig. 5BGo, lanes 1, 3, and 5) or with the pRCßactin I{kappa}B{alpha} (A32/36) vector encoding the dominant negative I{kappa}B{alpha} (A32/36; Fig. 5BGo, lanes 2, 4, and 6). In agreement with the above results, cells grown in normal medium or stimulated with bGH showed a significant enhancement of c-myc expression of 2.8 ± 0.5- and 3.2 ± 0.4-fold, respectively, compared with cells in starvation conditions (Fig. 5BGo, lanes 1 vs. 3 and lanes 3 vs. 5, respectively; P < 0.05). The presence of the mutant I{kappa}B{alpha} (A32/36) did not significantly alter the levels of c-myc mRNA in cells grown in normal medium conditions (Fig. 5BGo, lanes 1 vs. 2; P > 0.05). Likewise, basal c-myc RNA levels in starved cells were not affected by the inactivation of NF-{kappa}B (Fig. 5BGo, lanes 3 vs. 4; P > 0.05). On the contrary, a significant 2-fold decrease in c-myc levels was found in bGH-stimulated Ba/F3 GHR cells expressing mutant I{kappa}B{alpha} (A32/36) compared with cells transfected with empty control vector (Fig. 5BGo, lanes 5 vs. 6; P < 0.05). Taken together, these results suggest that NF-{kappa}B and PI 3-kinase are both involved in the regulation of c-myc expression by GH. Moreover, c-myc may be involved in proliferative signals mediated by GH.

Requirement of c-myc in GH-induced proliferation of Ba/F3 GHR cells
To further investigate the potential role of c-myc in the effects of GH, we used the pBaBE c-Myc-RX vector, encoding a dominant negative mutant c-Myc-RX. Ba/F3 GHR cells were transiently transfected with empty vector or with the c-Myc-RX vector, and the ability of GH to induce cell cycle entry was examined (Fig. 6Go). In particular, after transfection, cultures were maintained either in normal medium or in starvation medium in the absence or presence of bGH (Fig. 6Go). Upon starvation, Ba/F3 GHR cells expressing the empty vector or the mutant c-Myc-RX protein displayed 87% of cells arrested in the G0/G1 phase and 1–3% of cells in apoptosis, respectively (Fig. 6Go). Expression of the mutant c-Myc-RX protein in cells cultured either in normal medium or with bGH for 48 h caused a reduction in the percentage of cells in the S/M phase from 50% to 12% and from 53% to 9%, respectively, compared with cells expressing empty vector (Fig. 6Go). Under these conditions, the large majority of cells were arrested in the G0/G1 phase and only 1–3% of cells were in apoptosis (Fig. 6Go). From these observations, we can conclude that the proliferation mediated by GH was lost upon introduction of the dominant negative c-Myc-RX. These results strongly suggest that c-myc is directly involved in the proliferative effect rather than in the antiapoptotic effect of GH.



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  		Figure 6. Cell cycle analyses of Ba/F3 GHR cells overexpressing the mutant c-Myc-RX protein. Ba/F3 GHR cells (10 x 106 cells) were transfected with either 30 µg control vector pBaBE or the plasmid expressing mutant c-Myc-RX protein. Cells were cultured overnight in normal medium and then incubated for 48 h in either normal medium or starvation medium in the absence or presence of 1 µg/ml bGH. DNA was stained with propidium iodide, and cell cycle analyses were performed by flow cytometry. DNA content vs. cell number is presented in each profile. Percentages represent cells in each phase of the cell cycle, as indicated in Fig. 1CGo. Results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study provides evidence that GH acts as a cytokine in Ba/F3 cells expressing GHR. We show that GH promotes the expression of cyclins E and A, c-myc, as well as p21waf1/cip1, whereas the hormone represses p27kip1 expression. PI 3- kinase is a crucial regulator of the proliferation induced by GH in Ba/F3 cells expressing GHR. In addition, PI 3-kinase participates in the activation of NF-{kappa}B and the induction of c-myc expression. Activation of c-myc mediated by GH was found to occur in NF-{kappa}B-dependent and -independent manners. Importantly, the expression of a dominant negative version of c-Myc confirmed the critical role played by this oncogene in the proliferative effects of GH, while suggesting it is not involved in GH-mediated cell survival. Our previous studies strongly support the idea that GH exerts its antiapoptotic effect through the activation of NF-{kappa}B (14). The current studies indicate that the GH proliferative effects are dependent on c-myc expression mediated via the activation of PI 3-kinase, which is partially mediated via activation of NF-{kappa}B. These pathways are summarized in Fig. 7Go.



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  		Figure 7. Schematic presentation of the signaling pathways for GH effects in Ba/F3 GHR cells. GH binding promotes Jak-2 activation, which, in turn, induces simultaneous activation of both PI 3-kinase and NF-{kappa}B activation pathways. PI 3-kinase activation accounts for the proliferative response, probably through c-Myc activation and/or expression. NF-{kappa}B activation is responsible for survival, regulating antiapoptotic proteins such as Bcl-2 (14 ). Expression of cyclins D1 and D3 is probably dependent on the NF-{kappa}B induction pathway, whereas cyclin A and E expression as well as p21waf1/cip1 probably depend on PI 3-kinase. Ly 294002, which completely inhibits PI 3-kinase function, totally abrogates GH-mediated proliferation without affecting survival. Likewise, dominant negative Myc-RX, which is known to block c-Myc activation, inhibits the GH proliferative response and could block cyclin E and A expression (55 ). Inhibition of NF-{kappa}B activation by the dominant negative I{kappa}B{alpha} (A32/36) results in down-regulation of Bcl-2 expression coupled to a decrease in cyclin D levels and subsequent apoptosis of the cell (14 ). A link between these two main pathways might exist, as NF-{kappa}B activation can be partially controlled by PI 3-kinase activation, and inhibition of NF-{kappa}B provokes a down-regulation of c-myc expression (dotted arrows).

 
The role of CKI, such as p27kip1 or p21waf1/cip1, in the cell cycle has been an area of intense investigation, as it relates to proliferation and differentiation (32). Most studies suggest that an increase in p21waf1/cip1 and/or p27kip1 levels leads to G0/G1 phase arrest before apoptosis (32, 33). Overexpression of p27kip1 has been observed to exert negative control on the G1/S transition (33, 34) and to induce apoptosis (35, 36, 37). Consistently, we have found that expression of CKI p27kip1 is increased in G0/G1-arrested Ba/F3 WT cells undergoing apoptosis to a greater extent than in growth-arrested Ba/F3 GHR cells. Furthermore, its expression dropped after GH treatment of quiescent Ba/F3 GHR cells. Our results indicate that inhibition of p27kip1 releases the arrest in G0/G1, promoting cell cycle progression and thereby the mitogenic effects of GH in Ba/F3 GHR cells. On the contrary, p21waf1/cip1 expression is decreased in arrested Ba/F3 cells undergoing apoptosis, whereas it is enhanced in cycling cells. Moreover, we found that GH is able to induce p21waf1/cip1 expression in Ba/F3 GHR cells, as reported previously for IL-2 (38). Recent studies have proposed a role for p21waf1/cip1 at the onset of mitosis (39, 40, 41). p21waf1/cip1 was shown to be synthesized in the G2 phase (39, 40, 42), and then to promote a pause in late G2 phase under normal growth conditions (40, 43, 44). The GH-mediated induction of p21waf1/cip1 expression in cycling cells may contribute to a pause in late G2 phase to ensure regular mitosis.

GH was previously reported to reduce the expression levels of cyclin D induced by insulin and epidermal growth factor in 3T3 F442A preadipocyte cells (45, 46). This effect was correlated with the ability of GH to attenuate the insulin and epidermal growth factor-induced mitogenesis in these cells (45). Here we show that, contrary to deprived Ba/F3 WT cells, starved Ba/F3 GHR cells are able to maintain levels of cyclin D1 and D3, probably induced by basal amounts of endogenous GH production by these cells (14). We have shown previously that GH is able to activate NF-{kappa}B, a crucial step for the hormonal antiapoptotic effect in Ba/F3 GHR cells (14). Among transcription factors known to control the cell cycle, NF-{kappa}B has been shown to regulate transcription of the cyclin D1 gene, which is consistent with a role for NF-{kappa}B as a regulator of cell survival (47, 48). Hence, sustained activation of NF-{kappa}B by locally produced GH (14) could be linked to the maintenance of cyclin D1 expression found in Ba/F3 GHR cells under starvation conditions.

PI 3-kinase has been shown to play an important role in mitogenic and cell survival actions of cytokines (6). For example, overexpression of a dominant negative PI 3-kinase dramatically blocked the proliferative effects of exogenous IL-3 in Ba/F3 cells without affecting cell survival (49). Similarly, we show here that inactivation of PI 3-kinase by Ly 294002 totally abolishes the ability of GH to induce proliferation, resulting in growth arrest without induction of apoptosis in Ba/F3 GHR cells. Activation of PI 3-kinase by GH has been associated with its insulin-like effects in 3T3 F442A preadipocyte cells (8). We show here that PI 3-kinase can also have an important function in the proliferative effects of GH in Ba/F3 GHR cells.

The use of the PI 3-kinase inhibitor Ly 294002 allowed us to show that GH-induced NF-{kappa}B activation is in part mediated through PI 3-kinase. A link between PI 3-kinase and NF-{kappa}B was reported previously. Indeed, in platelet-derived growth factor and tumor necrosis factor signaling, two independent pathways could lead to the activation of NF-{kappa}B; the first one involves the classical NIK pathway, and the other one implicates the activation of PI 3-kinase and its downstream target AKT/protein kinase B, which was reported to activate IKK proteins (50, 51). Based on these results, we could hypothesize that GH is able to activate NF-{kappa}B through both NIK and PI 3-kinase/IKK pathways.

The cytokine-inducible gene c-myc was previously reported to be a target of NF-{kappa}B (24), and its expression is known to be enhanced by GH (52, 53). We show that in Ba/F3 GHR cells, inhibition of either PI 3-kinase or NF-{kappa}B prevented the increase in c-myc mRNA levels upon GH treatment, which indicates that induction of c-myc expression by GH is dependent on both PI 3-kinase and NF-{kappa}B pathways. Nevertheless, basal NF-{kappa}B activation observed in starved Ba/F3 GHR cells did not appear to be sufficient to induce c-myc expression. It suggests, therefore, that the main signals responsible for c-myc induction by GH in Ba/F3 GHR cells are mediated via PI 3-kinase activation. Furthermore, inactivation of c-Myc protein by the use of the dominant negative mutant of c-Myc resulted in an inhibition of cell proliferation induced by GH. These findings are consistent with the known central role for c-myc in regulating cell proliferation (54, 55, 56).

In our previous work (14) we showed that Ba/F3 GHR cells produce GH and that the presence of endogenous hormone results in constitutive activation of the transcription factor NF-{kappa}B (14). An essential role for this signaling pathway in cell survival was demonstrated when inhibition of NF-{kappa}B activity upon expression of the mutated I{kappa}B{alpha} (A32/36) protein resulted in cell death (14). Together, our findings strongly support the idea that GH exerts its antiapoptotic effect entirely through the activation of NF-{kappa}B, whereas its proliferative effect is mediated via the activation of PI 3- kinase and the expression of c-myc.


   Acknowledgments
 
We thank M. Karin and B. Amati for generously providing I{kappa}B{alpha} (A32/36) and c-Myc-RX expression vectors, respectively. G. Rawadi is gratefully acknowledged for the gift of NF-{kappa}B-luciferase construct. We thank INSERM Unité 373 for the use of the FACS, and C. Garcia for technical assistance with the FACS analyses.


   Footnotes
 
1 This work was supported by INSERM, a grant from Association pour la Recherche sur le Cancer, and USPHS Grant CA-36355 from the NIH. Back

2 Current address: Department of Medicine and Liver Unit, Medical School, University of Navarra, 31008 Pamplona, Spain. Back

Received July 3, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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