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Activation of Growth Hormone Receptor Delivers an Antiapoptotic Signal: Evidence for a Role of Akt in This Pathway¹

Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela (J.A.C., R.S., J.D., V.M.A.), San Francisco s/n. 15705, Santiago de Compostela, Spain; and INSERM U-344, Endocrinologie Moléculaire, Faculté de Medicine Necker (J.F., S.M.), 75730 Paris Cedex 15, France

	Abstract

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A signaling pathway was delineated by which GH promotes cell survival. Experiments were performed in human leukemic cells (HL-60) and Chinese hamster ovary (CHO) cells. In HL-60 cells, GH treatment reduced starvation-induced cell death. In contrast, when HL-60 cells were treated with an anti-GH antibody, cell survival was sharply reduced. In CHO cells stably expressing either the wild-type (wtGHR) or a truncated form (454GHR) of the GH receptor in which GH induces a sustained activation of the receptor-associated tyrosine kinase JAK2, we found that GH stimulation inhibited programmed cell death induced by withdrawal of survival factors. This effect was enhanced in cells expressing the truncated form. In contrast, GH did not affect cell survival in CHO cells transfected with either the empty vector or a mutated GHR unable to transduce the signal (4P/AGHR). We also showed that the inhibitory action of GH on apoptosis is probably mediated via stimulation of the serine-threonine kinase Akt, as 1) GH treatment induces a prompt phosphorylation of Akt; and 2) GH effects on cell survival are abolished by transfection of an Akt mutant that exhibits dominant negative function. Experiments with pharmacological inhibitors demonstrated that GH-induced Akt phosphorylation is dependent on phosphoinositide 3-kinase activation. In contrast, we found no changes in Bcl-2 levels secondary to GHR activation.

	Introduction

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GROWTH IS A complex process that mainly reflects a continuous increase in cell number, which, in turn, depends on the balance between cell proliferation and cell death (1). For each organ, the right number of cells is regulated by a wealth of local factors as well as signals produced by other tissues. In addition, systemic controls help coordinate the growth of different organs during development (1). One of the best recognized factors involved in systemic control of body growth is GH, a polypeptide hormone that is mainly produced by the somatotropic cells of the adenohypophysis (2), although GH gene expression has been also documented in other tissues, including hemopoietic and lymphoid cells (3, 4, 5, 6, 7), where the transcription factor Pit-1 has also been detected (5, 8).

GH stimulates growth principally by increasing the synthesis and release of insulin-like growth factor I (IGF-I) by the liver and other organs. IGF-I, in turn, promotes cell survival in many tissues and cell proliferation in some. In addition, GH can also promote growth by acting in a direct way, inducing the proliferation of different cell types (1, 2). Surprisingly, a direct effect of GH on cell survival has not been investigated to date.

We show here that GH acts as a survival factor in HL-60 cells, a human leukemic cell line that express both GH and GH receptor (GHR) (7, 9). To better characterize the signaling pathway involved in this effect, we used Chinese hamster ovary (CHO) cells stably expressing either the wild-type or two different mutated forms of the GH receptor: the 4P/AGHR, which bears a quadruple substitution of the four prolines in box 1 that renders the receptor inactive (10); and the 454GHR, a truncated form of the receptor that lacks residues beyond position 454, resulting in persistent activation of the GHR-associated tyrosine kinase JAK2 (11). Our data support the idea that GH also acts as a survival factor in CHO cells expressing the GHR. Moreover, the antiapoptotic pathways induced by GH appear to be to some extent constitutively activated in cells expressing the carboxyl-terminal-deleted form of the receptor.

As phosphoinositol 3-kinase (PI-3K) and Akt have been recently shown to play a major role in the regulation of survival mechanisms in several cell types, including fibroblasts, epithelial cells, hemopoietic cells, and primary neurons (12, 13, 14, 15, 16, 17), we investigated the potential activation of this pathway by GH, and its role in the regulation of GH-induced cell survival. We show for the first time that GH activates Akt and that the GH effect is blocked by using specific inhibitors of PI-3K activity and by an Akt mutant (PKB-CAAX) that exhibits a dominant negative function (18). Together, our results demonstrate that GH inhibits apoptosis and suggest that the PI-3K/Akt pathway could play a major role in this effect.

	Materials and Methods

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Cell culture and treatments
HL-60 cells were grown in Nunc polystyrene culture flasks containing 2 ml RPMI 1640 medium (Life Technologies, Inc., Poole, UK) supplemented with 10% FCS (Life Technologies, Inc.) and 2 mM L-glutamine (Sigma Chemical Co., St. Louis, MO) at 37 C in 5% humidified CO₂. Cells were grown in serum-free medium for 9 days in the presence of 300 ng/ml recombinant human GH or vehicle or for 6 days in the presence of 50 µl rabbit polyclonal antihuman GH serum (Sigma Chemical Co.; dilution, 1:100) or 50 µl rabbit preimmune serum (dilution, 1:100).

CHO, CHO-4P/AGHR, CHO-wtGHR, and CHO-454GHR stable clones, respectively, carrying an empty vector with selectable marker (pcDNA3/Flag), a 4P/AGHR construct (10), a Flag-tagged wtGHR construct, or a Flag-tagged 454GHR construct (19) were grown in Ham’s F-12 medium (Life Technologies, Inc.) supplemented with 10% FCS (Life Technologies, Inc.) and 2 mM L-glutamine (Sigma Chemical Co.) at 37 C in 5% humidified CO₂. PI-3K inhibitors (wortmannin and LY294002) were purchased from Sigma and used at 10 nM and 10 µM, respectively. Human recombinant GH (Saizen, Serono, Madrid, Spain) was used at 1 µg/ml.

GH binding analysis
For binding studies, cells were seeded in six-well plates (10⁶ cells/well). After 4-h serum starvation, cells were incubated overnight at 4 C with [¹²⁵I]GH (10⁵ cpm/ml) in the absence or presence of increasing concentrations of unlabeled GH at 4 C in PBS-0.1% BSA. After incubation, the cells were washed with cold PBS and lysed in 0.1 N NaOH. Total radioactivity associated with the cells was measured in a -counter. Scatchard analyses were performed with the Ligand program (20).

Survival assays
Cells were harvested in lysis buffer [10 mM Tris, 1 mM EDTA (pH 8), and 0.2% Triton X-100], incubated for 20 min on ice, and centrifuged. The supernatants were then precipitated overnight at -20 C in 50% isopropanol and 0.5 M NaCl, and precipitated DNA was pelleted by centrifugation. Pellets were then resuspended, treated with ribonuclease A (100 µg/ml, 1 h, 37 C) and proteinase K (300 µg/ml, 2 h, 50 C), and resolved in 1.5% agarose gel. Ethidium bromide-stained DNA laddering was visualized with a Gel Doc System (Bio-Rad Laboratories, Inc., Hercules, CA).

Immunoblot analysis
For Akt stimulation experiments, cells were maintained in 1% FBS for 48 h, followed by serum starvation for 2 h. Cells were then lysed by adding SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue]. Cell extracts were resolved in 12% SDS-PAGE and electrotransferred onto nitrocellulose paper (Protran, Schleicher & Schuell, Inc., Dassel, Germany). Membranes were then probed with a commercial kit (Phosphoplus Akt Ser₄₇₃ Antibody Kit, New England Biolabs, Inc., Beverley, MA) that allows specific recognition of both nonphosphorylated and serine-phosphorylated Akt.

For Bcl-2 levels determination, cells were processed as described above, and membranes were probed with a polyclonal anti-Bcl-2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Transient transfections
Both CHO-wt and CHO-454 cells were transfected with the Fugene 6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, cells were plated in chamber slides the day before the transfection experiment and grown until 70% confluent. Afterward, cells were serum starved, and transient transfections were performed with 2 µg green fluorescent protein (GFP) expression construct (pEGFP-C1, CLONTECH Laboratories, Inc., Palo Alto, CA) or 2 µg PKB-CAAX (provided by B. M. T. Burgering, Utrecht University, Utrecht, The Netherlands) and 2 µg pEGFP-C1. Cells were given either recombinant GH (as indicated above) or vehicle. After 24 h, nuclei were stained with the DNA dye bisbenzimide (Hoeschst 33258, Sigma Chemical Co.). Transfected cells were identified by GFP fluorescence and scored for apoptosis by nuclear morphology.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
GH acts as an antiapoptotic factor in HL-60 cells and in CHO cells expressing the GHR
After 6 days of incubation of HL-60 cells with anti-GH antibody, chromatin cleavage, assessed by DNA fragmentation, was clearly increased compared with that in cells grown in the presence of rabbit preimmune serum (Fig. 1A). In contrast, GH treatment increased cell survival, although this effect was delayed. As shown in Fig. 1A, when serum-starved HL-60 cells were grown for 9 days in the presence of GH, DNA laddering was reduced compared with that in cells treated with vehicle (Fig. 1A).

View larger version (63K): [in this window] [in a new window]   Figure 1. Induction of apoptosis by serum deprivation in HL-60 cells or in CHO cells expressing wtGHR or different GHR mutants. A, Analysis of DNA fragmentation of serum-starved HL-60 cells. When HL-60 cells were grown in the presence of GH-blocking antibodies, extensive DNA laddering was present on day 6, whereas in cells given GH, cell death was prevented even after 9 days of treatment. PI, Preimmune serum; GH(-), vehicle-treated cells. B, Monitoring of apoptosis by analysis of DNA fragmentation in CHO cells transfected with an empty vector (control) or with 4P/AGHR, wtGHR, or 454GHR constructs. Cells were deprived of serum and treated, or not, with GH. C, Hoffman modulation contrast photomicrographs after 32 h of serum starvation in GH or vehicle-treated CHO cells. Arrows indicate characteristic morphological changes in apoptotic cells. Lens objective, x10.

In CHO cells expressing the wtGHR, we observed that serum withdrawal resulted in rapid apoptosis (Fig. 1B). The dying cells showed characteristic features of apoptosis, including nuclear condensation and membrane blebbing (Fig. 1C). Similar results were obtained in control CHO cells transfected with the empty vector (Fig. 1B). When serum-starved wtGHR cells were incubated with GH, cell death was reduced, although some apoptotic cells and chromatin cleavage were still present. In contrast, GH treatment did not induce any effect in control cells (Fig. 1B) or in cells expressing the inactive receptor (4P/A GHR). In both cases, serum starvation induced extensive cell death after 32 h that was not counteracted by GH treatment (Fig. 1, B and C).

We also tested the viability of CHO cells expressing the GHR mutant, which allows the sustained activation of JAK2 (454GHR). Interestingly, after 32 h of serum deprivation, we observed abundant healthy 454GHR-expressing cells. These cells showed well defined nuclei and large cytoplasms, whereas DNA laddering was almost absent, indicating increased survival. However, cell viability under serum starvation was barely affected by GH treatment in these cells (Fig. 1, B and C).

The GH antiapoptotic effect in CHO cells depends on Akt activation
Akt, a serine-threonine kinase that is the cellular homolog of the v-akt oncogene (21) is one of the key factors in the regulation of cell survival. As this kinase conveys survival signals from various cell surface receptors, including members of the cytokine receptor superfamily, we then tested the possible activation of Akt by GH in cells expressing either the wt or the truncated form of the GHR (454GHR). We observed that GH induced a rapid phosphorylation of Akt in both types of cells (Fig. 2A). This activation of Akt appears to be dependent on PI-3K activity, as suggested by the fact that GH was ineffective in promoting Akt phosphorylation in the presence of two unrelated PI-3K inhibitors: wortmannin and LY294002 (Fig. 2B).

View larger version (27K): [in this window] [in a new window]   Figure 2. A, Activation of Akt by GH in CHO cells expressing the wtGHR or the 454GHR. Before stimulation, cells were placed in 1% serum for 48 h, starved for 2 h, and then treated with GH for increasing times. Akt and serine-phosphorylated Akt immunoreactivity were determined in whole cellular extracts, which were resolved in 12% SDS-PAGE and electrotransferred onto nitrocellulose paper. Membranes were then probed using specific antibodies that allowed recognition of either nonphosphorylated or serine-phosphorylated Akt. The same membrane was reprobed. B, Effect of inhibition of PI-3K activation on GH-induced phosphorylation of Akt in CHO cells transfected with wtGHR or 454GHR. Cells were serum starved as in A and treated with GH for 15 min in the presence of two specific inhibitors of PI-3K activity: wortmannin and LY 294002. Akt phosphorylation was analyzed as described above.

View larger version (46K): [in this window] [in a new window]   Figure 3. Effect of the expression of a dominant negative mutant of Akt (PKB-CAAX) on cell survival in CHO cells expressing wt or truncated GHR. Cells were cells were plated in chamber slides the day before the transfection experiment and grown until 70% confluence. Afterward, cells were serum starved, and transient transfections were performed with 2 µg GFP (A and B, control cells) or with both 2 µg PKB-CAAX (an Akt mutant that exhibits dominant negative function) and 2 µg GFP (C–F). Cells were treated with either GH (1 µg/ml; E and F) or vehicle (C and D), and apoptosis was evaluated after 24 h. GFP expression is shown in A, C, and E, and nuclear morphology, visualized by bisbenzamide staining, is shown in B, D, and F. White arrows point to apoptotic nuclei of transfected cells. Lens objective, x40.

View larger version (25K): [in this window] [in a new window]   Figure 4. Quantification of apoptotic cells in transfected cells based upon nuclear morphology of positive cells identified by GFP expression. Cells were transfected with expression constructs encoding GFP alone or GFP and PKB-CAAX in the presence or absence of GH (1 µg/ml). Each bar represents the mean ± SEM of at least 120 cells scored by 2 different observers.

View larger version (21K): [in this window] [in a new window]   Figure 5. Bcl-2 content in CHO cells bearing wtGHR or 454GHR after GH treatment. Cells were maintained in 1% FBS for 48 h, followed by serum starvation for 2 h, and were treated with GH (1 µg/ml) for the indicated times. Bcl-2 immunoreactivity was demonstrated in whole cellular extracts, which were resolved in 12% SDS-PAGE and electrotransferred onto nitrocellulose paper. Membranes were then probed using a specific polyclonal anti-Bcl-2 antibody.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We first used a human leukemic cell line (HL-60) to show that GH can act as a survival factor. It has been reported that GH treatment of these cells is able to increase cell growth (9, 27). As this effect could be mediated by an increase in cell survival, we first analyzed the ability of GH to reduce serum starvation-induced apoptosis in these cells. Our results clearly support this hypothesis. Moreover, as these cells are known to express and release GH (5, 7), we used anti-GH antibodies to test the role of the endogenously expressed hormone. We demonstrate that the addition of GH antibodies results in a significant increase in chromatin cleavage. Therefore, with this cellular model we can show that either endogenously produced hormone or exogenous addition of GH can affect cell viability through the endogenous GH receptors.

To characterize more easily the signaling pathways responsible for this effect, we used CHO cells transfected with either the wtGHR or different mutated forms. The early steps in GHR signaling involve ligand-induced receptor homodimerization, which results in the association and activation of JAK2, the GHR-associated tyrosine kinase. Once activated, JAK2 promotes the phosphorylation of a cascade of target molecules, including the kinase itself and the cytoplasmic tail of the receptor (2). In turn, tyrosine phosphorylation of both JAK2 and the receptor provide docking sites for SH2 domain-containing molecules, including the Stat (signal transducer and activator of transcription) proteins, phosphatases, insulin receptor substrate-1 and -2 (IRS1 and -2), and PI-3K, as well as molecules involved in the activation of the Ras/mitogen-activated protein kinase pathway (28).

We and others previously reported that truncation of the cytoplasmic tail of the receptor (454GHR) results in prolongation and enhancement of the early steps of GHR signaling, including JAK2, Stat3, and IRS1 phosphorylation (11, 29, 30). The mechanisms responsible for this sustained activation are poorly understood, but they could involve the inactivation of JAK2 by SH2 domain containing tyrosine phosphatase-1, a phosphotyrosine phosphatase that has been shown to inactive GHR signaling. However, it has been shown that SHP-1 does not associate directly with the C-terminus of the GHR, although this region is necessary for JAK2 dephosphorylation by the phosphatase (30). Alternatively, the SOCS (suppressors of cytokine signaling) molecules could also play a role in this down-regulation. GH has been shown to induce SOCS1, -2, and -3 in 3T3F442A cells, and SOCS1 and -3 overexpression appears to inhibit GHR activation of a Stat5-dependent reporter gene (31).

In this report we show that cell survival upon serum deprivation was remarkably enhanced in CHO cells expressing 454GHR compared with that in cells expressing the wtGHR. The partial effect of GH in wtGHR cells could be explained by the fact that optimal cell survival in vitro is often best supported by a combination of survival factors, rather than a single factor (32, 33). On the other hand, it is possible that the acute stimulation of GHR in wtGHR induced a transient activation of GHR signaling secondary to the inactivation of JAK2. Alternatively, GHR signaling may be terminated by internalization and degradation of the GH-GHR complex (34, 35). Although the molecular mechanisms for this down-regulation appear complex (involving protein synthesis, proteasome-mediated protein degradation, tyrosine phosphatase activation, and serine threonine kinase activation), it is likely that one or several of them may be absent in the cells expressing the truncated mutant, thus explaining why the GH survival effect might be enhanced in these cells.

As we observed that activation of the GHR can inhibit the programmed cell death induced by serum starvation, we investigated whether this effect was due to a direct stimulation of survival pathways by GH. We first checked whether this effect was dependent on JAK2 activation, using a GHR with mutations in box 1 (4P/AGHR) that prevent interaction of this domain with JAK2, thus inactivating the transduction of the signal (10, 36). As expected, when activation of JAK2 was impeded, GH treatment was ineffective in reducing cell death induced by serum starvation.

Another molecule recruited and activated by GHR is PI-3K (37). Recruitment of PI-3K can occur through tyrosine-phosphorylated IRS1, and its activation results in the generation of phosphoinositide products that act as second messenger molecules and activate a number of intracellular targets (21). Among them, Akt was recently shown to convey survival signals from various cell surface receptors (19, 38), including members of the cytokine receptor superfamily (24, 25). Activated Akt phosphorylates and thus inactivates a number of targets, such as the proapoptotic protein Bad (38, 39, 40), glycogen synthase kinase-3 (41), caspase-9 (42), and a forkhead transcription factor (43, 44). Although PI-3K is the major kinase involved in Akt phosphorylation, other pathways of Akt activation, independent of PI-3K, have been described. Hence, it has recently been reported that in NG108 neuroblastoma cells, calcium promotes phosphorylation of Akt through a PI-3K-independent mechanism that involves Ca²⁺-calmodulin-kinase activation (45). Our results suggest that not only is GH-dependent Akt stimulation involved in GHR-induced cell survival, but it is also mandatory for this effect. Evidence for this is 2-fold: 1) GHR stimulation induces a prompt rise in Akt phosphorylation; and 2) the expression of the dominant-negative Akt produces deleterious effects that are not reversed by GH treatment. Moreover, the existence of a PI-3K-independent mechanism of Akt activation does not appear to be relevant in our experimental model, as the blockade of PI-3K pathways with wortmannin or LY294002 prevented Akt phosphorylation. In any case, we cannot discard that GH may induce the activation of other survival pathways, independent of Akt phosphorylation.

The fact that the truncated GHR mutant (454GHR) is able to fully activate PI-3K/Akt, indicates that the amino acidic residues beyond position 454 are not necessary for this signaling pathway. In fact, we observed an increased activation of PI-3K/Akt in these cells that could be related to the increased IRS1 phosphorylation by the truncated receptor, as discussed above. Interestingly, this membrane-proximal region of the cytoplasmic domain of the GHR is also sufficient for activation of mitogenic pathways depending on Stat3 or mitogen-activated protein kinase (46, 47, 48), thus supporting its key role in GHR signaling.

Akt activation induced by cytokine treatment also promotes expression of Bcl-2 in certain cell types, although this is not a constant finding (12, 24, 25). These changes occur very quickly after stimulation, as in the case of IL-2, where increased expression of Bcl-2 appeared within 10 min (25). In contrast, we did not find any change in Bcl-2 levels induced by GH treatment, thus suggesting that in our experimental model, the effects of Akt on cell survival are not dependent on changes in Bcl-2.

Together, our results show that GHR stimulation delivers an antiapoptotic signal in both HL-60 cells and CHO cells transfected with active forms of GHR. This effect appears to be mediated by PI-3K activation, with subsequent Akt phosphorylation, but is independent of changes in Bcl-2 levels. We propose, on the basis of our results, that besides stimulating IGF-I synthesis and exhibiting a direct mitogenic effect on certain cell types, GH promotes growth by increasing cell survival. In view of these findings, GH could be considered a new member of the growing family of survival factors.

	Acknowledgments

 
We are grateful to Dr. B. M. T. Burgering for the PKB-CAAX plasmid.

	Footnotes

 
Address all correspondence and requests for reprints to: Víctor M. Arce, M.D., Ph.D., Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, 15705 Santiago de Compostela, Spain.

¹ This work was supported by grants from Fondo de Investigaciones Sanitarias (94/1104 and 96/1522) and Xunta de Galicia (XUGA20803B96 and XUGA20810B97).

² Recipient of a fellowship from Universidade de Santiago/Xunta de Galicia.

Received June 24, 1999.

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