Growth Hormone Prevents Apoptosis through Activation of Nuclear Factor-{kappa}B in Interleukin-3-Dependent Ba/F3 Cell Line

Sébastien Jeay, Gail E. Sonenshein, Marie-Catherine Postel-Vinay and Elena Baixeras1

INSERM Unité 344, Endocrinologie Moléculaire (S.J., M.C.P-V., E.B.) Faculté de Médecine Necker Paris Cedex 15, France 75730
Department of Biochemistry (G.E.S.) Boston University School of Medicine Boston, Massachusetts 02118


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pro-B Ba/F3 cell line requires interleukin-3 and serum for growth, and their removal results in cell apoptosis. Ba/F3 cells transfected with the GH receptor (GHR) cDNA become able to proliferate in response to GH. To investigate the role of GH in the control of apoptosis, Ba/F3 cells expressing either the wild-type rat GHR (Ba/F3 GHR) or a mutated rat GHR (Ba/F3 ILV/T) were used. We show that Ba/F3 GHR cells, but not parental Ba/F3 or Ba/F3 ILV/T cells, were able to survive in the absence of growth factor. Furthermore, an autocrine/paracrine mode of GH action was suggested by the demonstration that Ba/F3 cells produce GH, and that addition of GH antagonists (B2036 and G120K) promotes apoptosis of Ba/F3 GHR cells. Consistent with survival, the levels of both antiapoptotic proteins Bcl-2 and Bag-1 were maintained in Ba/F3 GHR cells, but not in parental Ba/F3 cells upon growth factor deprivation. Constitutive activation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B), which has been shown to promote cell survival, was sustained in Ba/F3 GHR cells, whereas no NF-{kappa}B activation was detected in parental Ba/F3 cells in the absence of growth factor. Furthermore, addition of GH induced NF-{kappa}B DNA binding activity in Ba/F3 GHR cells. Overexpression of the mutated I{kappa}B{alpha} (A32/36) protein, known to inhibit NF-{kappa}B activity, resulted in death of growth factor-deprived Ba/F3 GHR cells, and addition of GH was no longer able to rescue these cells from apoptosis. Together, our results provide evidence for a new GH-mediated pathway that initiates a survival signal through activation of the transcription factor NF-{kappa}B and sustained levels of the antiapoptotic proteins Bcl-2 and Bag-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The multiple actions of GH are initiated by binding of the hormone to its receptor (GHR), which belongs to the cytokine receptor superfamily (1, 2). Binding of GH is followed by receptor dimerization (3, 4) and activation of the tyrosine kinase Jak2, which then autophosphorylates itself and phosphorylates tyrosine residues within the GHR (5). The phosphorylated tyrosines in the receptor and Jak2 provide binding sites for various signaling molecules that contain SH2 (Src homologous 2) or PTB (phosphotyrosine binding) domains. Recruitment of these molecules initiates a number of signaling pathways mediating a variety of cellular responses. GH can activate the Stat (signal transducer and activator of transcription) pathway, including Stat1, Stat3, and both isoforms of Stat5 (6, 7), and thereby regulate transcription of GH-responsive genes such as serine protease inhibitor 2.1 (8) and c-fos (9). Shc (Src homologous containing) proteins that recruit Grb2 (growth factor receptor bound 2) and SOS (son of sevenless) are also activated by GH and can initiate the Ras-MAP (mitogen activated protein) kinase pathway, involved in the regulation of gene transcription and cellular growth and differentiation (10). The IRS (insulin receptor substrate) proteins, IRS-1 and -2, which regulate glucose transport and lipid synthesis (11), and protein kinase C (12) and phosphatase SHP-2 (13) have been implicated in GH signaling. Other pathways involved in various physiological actions of GH remain to be identified.

Growth factors and hormones have been shown to be regulators of cell death. For example, epidermal growth factor, nerve growth factor, platelet-derived growth factor, and insulin-like growth factor-1 act as survival factors in hematopoietic cells and neurons (14). Steroid hormones are also potent regulators of apoptosis in several tissues such as the mammary gland (14). Similarly, PRL has been reported not only to trigger proliferation of Nb2 lymphoma cells, but also to counteract the glucocorticoid-driven apoptosis (15, 16). A recent study reported a protective action of GH on Fas-mediated apoptosis in monocytes (17).

Members of the Bcl-2 and NF-{kappa}B protein families have been extensively implicated in cytokine-signaling of cell survival (18, 19). The Bcl-2 family includes pro-survival (e.g. Bcl-2 and Bcl-XL) and proapoptotic proteins (e.g. Bax and Bid) (20). Studies have demonstrated that Bcl-2 and Bcl-XL are differentially regulated during B cell development (21), and overexpression of Bcl-2 or Bcl-XL was shown not only to lengthen survival of cytokine-dependent cells upon cytokine withdrawal (18), but also to protect B cells from apoptotic signals induced by glucocorticoids and chemotherapeutic drugs (22). Bag-1, a Bcl-2 binding protein, was recently found to enhance cellular proliferation and viability during growth factor deprivation in interleukin-3 (IL-3)-dependent Ba/F3 and PRL-dependent Nb2 cells (16). Moreover, altered expression of Bcl-2 and Bax are associated with the antiapoptotic effect of PRL in Nb2 cells (23). Likewise, the effect of GH on the survival of monocytes exposed to Fas-mediated cell death is associated with up-regulation of Bcl-2 (17).

NF-{kappa}B/Rel factors have also been found to promote cell survival in a number of cells and growth conditions (24). Classical NF-{kappa}B is composed of p50 and p65 (RelA) subunits. In most cells, other than mature B lymphocytes, NF-{kappa}B/Rel proteins are expressed ubiquitously, but sequestered in inactive forms in the cytoplasm by association with inhibitory proteins, termed I{kappa}Bs, for which I{kappa}B{alpha} represents the paradigm (25). Activation of NF-{kappa}B/Rel involves I{kappa}B phosphorylation on serine residues, followed by ubiquitination and rapid degradation of the inhibitory protein by the proteasome, which allows for nuclear translocation of the NF-{kappa}B/Rel factor (25). Constitutively active NF-{kappa}B/Rel factors have been found in mature B cells and in pro-B cells, including the Ba/F3 cell line (26, 27, 28). Further, constitutive activation of NF-{kappa}B by the oncogenic TEL/platelet-derived growth factor receptor ß fusion protein was shown to protect Ba/F3 cells from apoptosis induced by IL-3 deprivation (28).

The aim of this study was to examine the role of GH in the regulation of cell death. Stable Ba/F3 transfectants expressing the wild-type rat GHR (Ba/F3 GHR) or a functionally deficient form of the rat GHR (Ba/F3 ILV/T) were used to study GH signaling pathways involved in cell survival. We have previously shown that Ba/F3 GHR cells depend on GH for their growth and can be maintained in culture in the presence of the hormone (29). Here we show that Ba/F3 GHR cells escape from death upon cytokine and serum deprivation. We found that locally produced GH is responsible for such a protective effect, which is associated with sustained expression of Bcl-2 and Bag-1 proteins. Moreover, we have identified a novel signaling pathway for GH via activation of NF-{kappa}B, which is crucial in mediating the antiapoptotic effect of GH.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Cycle Analyses of Ba/F3 Cells Expressing the GH Receptor
The potential role of GH as a growth and survival factor was studied using Ba/F3 cells stably transfected with the rat GHR cDNA (Ba/F3 GHR) (29). As negative controls, parental Ba/F3 cells (Ba/F3 WT), which do not express GHRs, or Ba/F3 cells expressing a mutated rat GHR (Ba/F3 ILV/T), which has been shown to be inactive, were used (29). Ba/F3 WT, Ba/F3 ILV/T, and Ba/F3 GHR cells were cultured in serum- and IL-3-free medium (starvation medium) for 6 h to induce maximal synchronization. Cell cycle analyses were performed after 48 h of cell treatment under three culture conditions: 1) normal medium; 2) starvation medium; 3) starvation medium plus 1 µg/ml bovine GH (bGH). When cultured in normal medium, the three cell lines showed a typical cell cycle with 56–69% of cells in G0/G1 phase and 30–42% of cells in S/M phase (Fig. 1Go). Under this condition, almost no cells appeared to be undergoing apoptosis, as judged by the DNA content (<1%) in sub-G0/G1 phase. Under starvation conditions, Ba/F3 WT and Ba/F3 ILV/T cells extensively underwent apoptosis (84% and 77%, respectively), whereas Ba/F3 GHR cells were arrested in G0/G1 phase (88%), and 5% of cells were undergoing apoptosis (Fig. 1Go). Addition of bGH did not modify the proportion of Ba/F3 WT and Ba/F3 ILV/T cells undergoing apoptosis, as expected, since these cells do not express a functional GHR. On the contrary, bGH treatment of Ba/F3 GHR cells promoted cell cycle progression, with 33% of the cells in S/M phase. Thus, Ba/F3 cells expressing GHR are resistant to apoptosis upon growth factor deprivation, and addition of exogenous GH induces cell cycle progression in these cells.



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Figure 1. Cell Cycle Analyses of Ba/F3 Cells under Various Culture Conditions and GH Treatment

Ba/F3 WT, Ba/F3 ILV/T, and Ba/F3 GHR cell types were starved for 6 h, and then cultured under three conditions: in normal culture medium, starvation medium, or in starvation medium plus 1 µg/ml bGH. Cells were harvested 48 h later and cell pellets were permeabilized and stained with PI, and cell cycle analyses were performed by flow cytometry. The DNA content vs. cell number is presented in each histogram. Percentages correspond to cells in the G0/G1 phase (2n content) or in the S/M phase (4n content) or in apoptosis phase (hypodiploid content). Results are representative of four independent experiments.

 
Production of GH by Ba/F3 Cells
To explain the survival of Ba/F3 GHR cells cultured in starvation conditions, the possibility of a local production of hormone by the cells was addressed. As Ba/F3 WT cells underwent apoptosis upon growth factor deprivation, we took advantage of the ability of Ba/F3 GHR cells to survive under starvation conditions (Fig. 1Go) to investigate the presence of GH in cell supernatant. Ba/F3 GHR cells were then maintained in starvation medium for 24, 48, and 72 h, and presence of GH was evaluated by RIA in the supernatants of cells. Presence of GH in starvation medium alone was also evaluated as a control. As shown in Table 1Go, low GH concentrations were detected in 150-fold concentrated supernatants from Ba/F3 GHR cells, and the levels increased with longer times of culture. No GH was found in starvation medium. Thus, Ba/F3 cells produce GH.


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Table 1. GH Measurement in Ba/F3 GHR Cell Supernatant

 
We next asked whether this locally produced GH was responsible for cell survival by using two human GH antagonists, B2036 and G120K, which have been shown to bind to the rat GHR with a lower affinity than to the human GHR (30). These two antagonists were tested at concentrations ranging from 0.5 to 2 µg/ml for their ability to inhibit the potential effects of endogenous GH on Ba/F3 GHR cell survival. Starvation of Ba/F3 GHR cells led to apoptosis of a small proportion of cells (11%). Addition of 0.5 µg/ml of B2036 or G120K was sufficient to enhance the level of cell death up to 30%. Maximal effect of both antagonists was reached at 1 to 2 µg/ml where about 40% of cells underwent apoptosis (Fig. 2Go). Taken together, these results strongly suggest that locally produced GH is sufficient to rescue Ba/F3 GHR cells from apoptosis.



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Figure 2. Effect of GH Antagonists, B2036 and G120K, on Ba/F3 GHR Cell Apoptosis

Ba/F3 GHR cells were starved for 48 h without ({square}) or with the indicated concentrations of B2036 ({blacksquare}) or G120K (). Cells were then permeabilized and stained with PI and subjected to cell cycle analysis, as in Fig. 1Go. Results are expressed as the percentage of DNA content in the hypodiploid peak of the cell cycle. Results represent the mean ± SD of four experiments.

 
Expression of Bcl-2 Related Proteins in Ba/F3 GHR Cells
Previous studies have shown that Ba/F3 cells are able to express the antiapoptotic Bcl-2, Bcl-XL, and Bag-1 proteins under growing conditions, and overexpression of these proteins was found to enhance cell survival (16, 31). Expression of these proteins was evaluated by immunoblot analyses of total cell lysates prepared from Ba/F3 WT and Ba/F3 GHR cells either unstimulated (Fig. 3Go, lanes 2 and 6) or stimulated with bGH (Fig. 3Go, lanes 3, 4, 7, and 8). Cells cultured in normal medium for 8 h were used as positive control for expression of Bcl-2-related proteins in Ba/F3 WT and Ba/F3 GHR cell lysates (Fig. 3Go, lanes 1 and 5). Immunoblot analysis of Bcl-2 and Bag-1 showed that expression of these proteins was down-regulated in unstimulated Ba/F3 WT cells after 6 h of culture in starvation medium (Fig. 3Go, lane 2). In contrast, only a slight decrease was observed in the levels of Bcl-2 and Bag-1 in lysates from starved Ba/F3 GHR cells (Fig. 3Go, lane 6) compared with those in cells cultured in normal medium (Fig. 3Go, lane 5). Bcl-XL expression was not detectable in lysates from either Ba/F3 WT or Ba/F3 GHR cells cultured in starvation medium (Fig. 3Go, lanes 2 and 6). As expected, treatment of Ba/F3 WT cells with bGH for 6 or 8 h (Fig. 3Go, lanes 3 and 4) did not affect the expression of Bcl-2, Bcl-XL, and Bag-1, and levels were similar to those obtained in starvation conditions (Fig. 3Go, lane 2). Also, addition of exogenous bGH to Ba/F3 GHR cell culture for 6 and 8 h caused a modest increase in the expression levels of Bcl-2 and Bag-1 proteins back to baseline values (Fig. 3Go, lanes 7 and 8). In contrast, Bcl-XL expression was induced in Ba/F3 GHR cells after 6 and 8 h of treatment with bGH (Fig. 3Go, lanes 7 and 8) to levels comparable to those obtained in cells grown in normal medium (Fig. 3Go, lane 5). Therefore, constant levels of Bcl-2 and Bag-1 are associated with survival of starved Ba/F3 GHR cells. Bcl-XL, however, is not present in starved Ba/F3 GHR cells, although it is inducible by addition of exogenous GH. Thus, our results suggest that endogenous GH is sufficient to induce constant levels of Bcl-2 and Bag-1 proteins, which are likely to be involved in the short-term survival in Ba/F3 GHR cells upon cytokine deprivation.



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Figure 3. Expression of Bcl-2, Bcl-XL, and Bag-1 Proteins in Ba/F3 Cells

Ba/F3 WT and Ba/F3 GHR cells were starved for 6 h before incubation in normal medium for 6 h (lanes 1 and 5), or in starvation medium alone for 6 h (lanes 2 and 6) or in starvation medium containing 1 µg/ml bGH for 6 h (lanes 3 and 7) or for 8 h (lanes 4 and 8). Then Bcl-2, Bcl-XL, and Bag-1 proteins were detected by immunoblot analysis of total cell lysates, as detailed in Materials and Methods.

 
NF-{kappa}B Activation by GH in Ba/F3 GHR Cells
The transcription factor NF-{kappa}B can protect a wide variety of cells from apoptosis (24). The ability of GH to induce NF-{kappa}B activation was then examined. Nuclear extracts of Ba/F3 cells have been reported to yield two NF-{kappa}B complexes, the upper and lower bands corresponding to classical p65/p50 heterodimers and p50/p50 homodimers, respectively (28). Indeed, electrophoretic mobility shift assay (EMSA) of nuclear extracts from Ba/F3 WT and Ba/F3 GHR cells grown overnight (18 h) in normal medium showed two bands (Fig. 4AGo). The specificity of these two complexes in Ba/F3 GHR nuclear extracts was verified upon successful competition with 10- and 20-fold excess of the unlabeled wild type upstream region element (URE) oligonucleotide, whereas addition of a mutant version, with two internal G to C conversions, was much less effective (Fig. 4AGo). An antibody against the p65 subunit (27), known to block the binding of p65/p50 complexes (32), was next used to test for the presence of p65 in the slower migrating complex. Incubation of Ba/F3 WT and Ba/F3 GHR nuclear extracts with the anti-p65 antibody specifically eliminated binding of the upper complex (Fig. 4AGo). These data corroborate the specificity of DNA binding and indicate that Ba/F3 GHR cells, like the parental Ba/F3 cells, contain p65/p50 heterodimers of classical NF-{kappa}B.



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Figure 4. Effect of GH on NF-{kappa}B Activity

Nuclear extracts were prepared from Ba/F3 WT or Ba/F3 GHR cells and analyzed for NF-{kappa}B binding using the URE oligonucleotide as probe in EMSA. A, Ba/F3 WT and Ba/F3 GHR cells were cultured in normal medium and harvested at 18 h. Specificity of the bands were analyzed by competition binding assays of the nuclear extracts in the presence of 10-fold (+) or 20-fold (+) excess of unlabeled wild-type (WT) or mutant URE {kappa}B probes. EMSA of nuclear extracts from cells was also done in the absence (-) or in the presence (+) of anti-p65 antibody. B, After 6 h of serum and IL-3 deprivation, cells were incubated in starvation medium in the absence (-) or in the presence (+) of 1 µg/ml bGH for 18 h. Nuclear extracts were then analyzed for NF-{kappa}B binding by EMSA. Panels A and B are from the same gel exposed for the same length of time and thus can be compared directly.

 
We next analyzed the effects of starvation conditions, in the absence and in the presence of bGH, on NF-{kappa}B DNA binding ability. Ba/F3 WT and Ba/F3 GHR cells were incubated overnight under starvation conditions and then incubated in the absence or in the presence of bGH (Fig. 4BGo). After overnight starvation, p65/p50 heterodimer complexes were barely detected in nuclear extracts of Ba/F3 WT cells. In contrast, NF-{kappa}B activity was maintained in Ba/F3 GHR cells under similar starvation conditions. Furthermore, addition of bGH to the starvation medium appeared to enhance NF-{kappa}B levels in Ba/F3 GHR, but not in Ba/F3 WT cells. Taken together, these results suggest that maintenance of NF-{kappa}B activity may play a role in survival of Ba/F3 GHR cells. Furthermore, the activation of NF-{kappa}B could be mediated via GHR signaling, presumably due to the small quantities of endogenously secreted GH in the Ba/F3 GHR cells.

To test directly the effects of GH-induced signaling on NF-{kappa}B activation, a time course experiment was performed. Nuclear extracts were prepared from Ba/F3 WT or Ba/F3 GHR cells incubated under starvation conditions for 6 h (0 time point) followed by treatment with bGH for 15 min to 16 h. NF-{kappa}B binding was analyzed by EMSA (Fig. 5Go). Very low or undetectable basal levels of p65/p50 heterodimer were found in starved Ba/F3 WT cells and they were not altered by bGH. In contrast, exogenous bGH enhanced NF-{kappa}B activation in Ba/F3 GHR cells. An increase in NF-{kappa}B activation was detected by 15 min post-bGH addition. Levels were maximal at 1 h of stimulation. The presence of p65/p50 heterodimer was maintained in Ba/F3 GHR cells even after 16 h of bGH stimulation (Fig. 5Go). Thus, stimulation by exogenous bGH increases the expression of classical NF-{kappa}B in Ba/F3 GHR cells.



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Figure 5. Kinetics of Induction of NF-{kappa}B by GH in Ba/F3 GHR Cells

Ba/F3 WT and Ba/F3 GHR cells were incubated for 6 h under serum and IL-3 deprivation conditions and stimulated for the indicated times (h) with 1 µg/ml bGH. Nuclear extracts were prepared and subjected to EMSA for NF-{kappa}B binding as in Fig. 4Go.

 
Involvement of NF-{kappa}B in GH-Induced Survival of Ba/F3 GHR Cells
To investigate the potential role of NF-{kappa}B in the GH-induced survival of Ba/F3 GHR cells, we performed analysis of cell death after inhibition of NF-{kappa}B pathway. The pRCßactin I{kappa}B{alpha} (A32/36) vector encoding the dominant negative mutant I{kappa}B{alpha} (A32/36) was transiently transfected in Ba/F3 GHR cells. Then, the capacity of GH to induce survival in Ba/F3 GHR cells in these conditions was examined. As a control, the empty parental pRCßactin vector DNA was similarly transfected to assess for effects of the vector sequences. Cell cycle analyses were performed on Ba/F3 GHR cells expressing I{kappa}B{alpha} (A32/36) protein after 48 h postelectroporation with pRCßactin I{kappa}B{alpha} (A32/36) vector. Cells sham transfected or transfected with the empty vector were maintained either in normal medium (Fig. 6Go, panels A and B) or in medium containing bGH (Fig. 6Go, panels G and H). Under these conditions, no differences in cell cycle profiles and apoptosis rate were detected as judged by DNA content. Likewise, cells expressing the mutant I{kappa}B{alpha} (A32/36) protein and maintained in normal medium showed a cell cycle similar to that found in cells transfected with the empty vector (Fig. 6Go, panels C vs. B). Sham or empty vector transfected cells maintained in starvation conditions exhibited 19% of cells in apoptosis. This observation can be attributed to the absence of growth factor-mediated survival signals that contribute to cell restoration after electroporation handling (Fig. 6Go, panels D and E). Upon the same starving conditions, cells expressing the mutant I{kappa}B{alpha} (A32/36) protein showed 4-fold increase in apoptosis (72%) (Fig. 6Go, panels F vs. D and E). Moreover, apoptosis was no longer abolished by addition of bGH to I{kappa}B{alpha} (A32/36) expressing cells, which exhibited an apoptosis rate of 83%, a similar value to that found in starvation conditions (Fig. 6Go, panels I vs. F).



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Figure 6. Cell Cycle Analyses of Ba/F3 GHR Cells Overexpressing Mutant I{kappa}B{alpha} (A32/36) Protein

Ba/F3 GHR cells were sham transfected (panels A, D, and G) or transfected with either 30 µg of empty pRCßactin vector (panels B, E, and H) or 30 µg of the mutant I{kappa}B{alpha} (A32/36) vector (panels C, F, and I). Cells were cultured overnight in serum and IL-3-containing medium. Followed by 6 h starvation, cells were then incubated for additional 48 h in either normal medium or starvation medium or in the presence of 1 µg/ml bGH as indicated in the figure. DNA content was assessed by PI staining followed by FACS analysis. Percentages correspond to cells in the hypodiploid peak (undergoing apoptosis). The results are representative of four independent experiments.

 
From these observations, we can conclude that the protection mediated by GH was lost upon introduction of the dominant negative I{kappa}B{alpha} (A32/36), which strongly suggests that NF-{kappa}B activation is directly involved in the GH survival signal. Furthermore, the survival signal involved in GHR activation pathway seems to differ from that induced by the normal medium, which contains IL-3 plus serum, a cocktail of cytokine and growth factors that does not appear to be affected by the NF-{kappa}B inactivation.

Levels of Bcl-2 Related Proteins in Ba/F3 GHR Cells Expressing the Mutant I{kappa}B{alpha} (A32/36)
As shown in Fig. 3Go, Bcl-2 and Bag-1 proteins were expressed in lysates from starved Ba/F3 GHR cells, which did not undergo apoptosis, whereas the expression of Bcl-XL was only observed in cells in proliferation. In an attempt to establish a relationship between NF-{kappa}B activation and Bcl-2, Bag-1, and Bcl-XL protein expression, we examined the presence of these proteins in Ba/F3 GHR cells transfected with the mutant I{kappa}B{alpha} (A32/36) vector. Ba/F3 GHR cells were transfected with the pRCßactin vector or pRCßactin I{kappa}B{alpha} (A32/36) vector and then cultured in either normal or starvation medium or in the presence of bGH. Nuclear extracts were prepared from these cells to first check the effect of the mutant I{kappa}B{alpha} (A32/36) on NF-{kappa}B binding activity by EMSA (Fig. 7AGo). In comparison with cells transfected with control vector (Fig. 7AGo, lanes 1, 3, and 5), a marked decrease in NF-{kappa}B activation was detected in Ba/F3 GHR cells overexpressing I{kappa}B{alpha} (A32/36) (Fig. 7AGo, lanes 2, 4, and 6). This decrease was independent of culture conditions. As expected, the results show that overexpression of I{kappa}B{alpha} (A32/36) strongly inhibits NF-{kappa}B activation in Ba/F3 GHR cells.



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Figure 7. Expression of Bcl-2 Related Proteins in Ba/F3 GHR Cells Overexpressing Mutant I{kappa}B{alpha} (A32/36) Protein

Ba/F3 GHR cells were transfected with either empty pRCßactin vector (lanes 1, 3, and 5) or with the pRCßactin I{kappa}B{alpha} (A32/36) vector (lanes 2, 4, and 6), as described in Fig. 6Go. After 24 h in normal medium, cells were starved for 2 h and placed in either normal medium again, or in starvation medium or in the presence of 1 µg/ml of bGH for an additional 6 h as indicated. Nuclear extracts were prepared and subjected to EMSA for NF-{kappa}B binding (A). Presence of I{kappa}B{alpha}, Bcl-2, Bag-1, and Bcl-XL proteins were sequentially determined in the same membrane by immunoblot analyses of total cell lysates for each treatment (B).

 
The expression of I{kappa}B{alpha}, Bcl-2, Bag-1, and Bcl-XL proteins was next assessed sequentially by immunoblot analysis of total cell lysates (Fig. 7BGo). As expected, I{kappa}B{alpha} was clearly detected in lysates from cells transfected with the pRCßactin I{kappa}B{alpha} (A32/36) vector (Fig. 7BGo, lanes 2, 4, and 6), but was present in greatly reduced amounts in lysates from parental vector-transfected cells (Fig. 7BGo, lanes 1, 3, and 5). Bcl-2 or Bag-1 protein levels were detected at equivalent levels independently of culture conditions in lysates from parental vector-transfected cells. Cells expressing the dominant negative I{kappa}B{alpha} showed a clear decrease in the levels of Bcl-2 protein in all three culture conditions (Fig. 7BGo, lanes 2, 4, and 6). In contrast, while Bag-1 levels were decreased in the presence of the dominant negative I{kappa}B{alpha} in cells incubated under normal (Fig. 7Go, lanes 1 vs. 2) or starvation conditions (Fig. 7BGo, lanes 3 vs. 4), a partial induction was noted upon bGH treatment (Fig. 7BGo, lanes 4 vs. 6). In cells transfected with control vector, bGH treatment as well as normal culture conditions induced up-regulation of Bcl-XL levels (Fig. 7BGo, lanes 1 and 5) in comparison to the very low expression of Bcl-XL in deprived cells (Fig. 7BGo, lane 3). Overexpression of the mutant I{kappa}B{alpha} (A32/36) promoted a 2-fold decrease of Bcl-XL expression under bGH treatment (Fig. 7BGo, lane 6), whereas no differences were found in cells cultured in starvation or in normal conditions (Fig. 7BGo, lanes 2 and 4). Thus, NF-{kappa}B seems able to regulate expression of Bcl-2, Bag-1, and Bcl-XL, although the regulation of Bag-1 and Bcl-XL appears to be more complex, with the possible involvement of other signaling molecules. Taken together, these results demonstrate that GH mediates activation of NF-{kappa}B and suggest that NF-{kappa}B could, in turn, promote cell survival via expression of Bcl-2, and potentially Bag-1 and Bcl-XL.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this work, we demonstrate that GH can protect pro-B Ba/F3 cells expressing the rat GHR against growth factor deprivation through an autocrine mechanism. We show that Ba/F3 GHR cells produce GH, and the presence of endogenous GH upon growth factor deprivation results in sustained Bcl-2 and Bag-1 protein levels as well as constitutive activation of the transcription factor NF-{kappa}B. An essential role of this pathway in cell survival was indicated when inhibition of NF-{kappa}B activity upon expression of the mutated I{kappa}B{alpha} (A32/36) protein resulted in cell death. Our findings provide evidence for a new pathway involved in signaling for GH antiapoptotic action.

Several lines of evidence indicate that a number of hormones, such as PRL, insulin-like growth factor-1, and GH, play a role in cell survival as well as in development and function of the immune system (14, 33). The GH receptor has been shown to be widely expressed in hematopoietic cells (34), and GH is able to stimulate the proliferation of several cell types including activated murine T cells (35, 36), and direct evidence of the GH proliferative effect on pro-B Ba/F3 cells expressing the GHR has been reported (29).

Local production of GH in lymphoid tissues has been demonstrated (33, 37). Indeed, we show that Ba/F3 GHR cells produce GH. Interestingly, the relevance for the locally produced GH is demonstrated since Ba/F3 GHR cells can survive upon serum and cytokine withdrawal from the culture medium. Further demonstration of the importance of local production of GH came from the use of B2036 and G120K hGH antagonists that prevent receptor dimerization (3, 38) and thus GH action. Addition of both antagonists resulted in enhanced mortality of Ba/F3 GHR cells cultured in starvation medium. These findings suggest that GH, in addition to its endocrine mode of action, can act in an autocrine/paracrine manner in cells of the immune system.

It is known that in a quiescent cell, survival and control of cell cycle entry predominantly depends on the expression of proteins of the Bcl-2 family (31, 39, 40). As reported, IL-3 induces cell cycle progression coupled to the expression of Bcl-XL, but not of Bcl-2, in Ba/F3 cells (31). On the contrary, Bcl-XL protein down-regulates rapidly upon serum and IL-3 deprivation, and then cell death follows (31). This observation has suggested the existence of two interdependent pathways leading to apoptosis in IL-3-deprived Ba/F3 cells: while the presence of Bcl-2 correlated with short-term survival after cytokine deprivation, de novo expression of Bcl-XL after addition of IL-3 seemed crucial for long-term survival of cells (31). Our results show that a large proportion of starved Ba/F3 GHR cells were arrested at G0/G1 phase of the cell cycle and did not undergo apoptosis. At this stage, levels of Bcl-2 and Bag-1 were sustained, while levels of Bcl-XL decreased. Addition of bGH, which induces proliferation of Ba/F3 GHR cells, restored Bcl-XL expression levels while levels of Bcl-2 and Bag-1 were only modestly increased. Therefore, as shown for IL-3 (31), Bcl-2 is likely involved in GH signaling for short-term survival. Bcl-XL, however, appears involved in GH-induced long-term cell survival coupled to cellular proliferation process.

Overexpression of the Bcl-2 binding protein, Bag-1, was previously reported to induce cell survival of cytokine-deprived Ba/F3 and Nb2 cells (16). As shown for Bcl-2, sustained levels of Bag-1 were associated with short-term survival of Ba/F3 GHR cells. Accordingly, coordinated expression of Bcl-2 and Bag-1 genes has been shown to be essential for IL-2-mediated protection from apoptosis of Ba/F3 cells (41). Altogether, it can be concluded that Bcl-2 and Bag-1 are involved in pathways for GH signaling of Ba/F3 cell survival.

We present evidence for the involvement of NF-{kappa}B activation in GH survival effects in starved Ba/F3 GHR cells. Locally produced GH appeared to be sufficient to induce constitutive NF-{kappa}B DNA binding activity, which likely contributes to Ba/F3 GHR cell survival. NF-{kappa}B DNA binding activity was enhanced by addition of bGH to Ba/F3 GHR cell culture. Cell cycle analyses on cells expressing the mutant I{kappa}B{alpha} (A32/36) protein demonstrated that the ability of GH to inhibit cell death is dependent on NF-{kappa}B activation pathway in Ba/F3 GHR cells. Therefore, we can conclude that, as described for IL-3 in Ba/F3 cells (28), NF-{kappa}B is a crucial mediator of the antiapoptotic signal delivered by GH.

We found that expression of at least Bcl-2 was dependent on the activation of NF-{kappa}B by GH in Ba/F3 GHR cells. The regulation of Bag-1 and Bcl-XL expression appears complex, mediated via NF-{kappa}B-dependent and -independent signaling pathways. Overexpression of the mutant I{kappa}B{alpha} (A32/36) provoked a decrease of NF-{kappa}B activation and reduced the expression of the three Bcl-2 family members to varying degrees, both in starved and bGH-treated Ba/F3 GHR cells (Fig. 7Go, A and B). In these conditions, a marked increase of cell mortality occurred (Fig. 6Go, panels F and I). In contrast, Ba/F3 GHR cells overexpressing I{kappa}B{alpha} (A32/36) cultured in the presence of serum and IL-3 were in cell cycle progression, although expression levels of Bcl-2 and Bag-1, but not Bcl-XL, were decreased. These results suggest that cell survival and proliferation induced by serum could occur through the sustained expression of Bcl-XL, in a NF-{kappa}B-independent manner. These observations are consistent with the existence of a link between Bcl-2-related molecules and NF-{kappa}B signaling pathways. Indeed, induction of Bcl-2 and Bcl-XL expression through NF-{kappa}B activation has been implicated in the protective effect of tumor necrosis factor against neuron-induced injury (42). Interestingly, the presence of NF-{kappa}B DNA binding sites in the promoters of Bcl-2 and Bcl-X genes has been shown and more recently the prosurvival Bcl-2 homolog Bfl-1/A1 has been described as a direct transcriptional target of NF-{kappa}B (43, 44). In addition, activation of NF-{kappa}B by Bcl-2 through degradation of I{kappa}B{alpha} correlates with the protection of rat myocytes from apoptosis (45).

Aberrant activation of NF-{kappa}B/Rel factors has now been observed in many tumors. We first reported that breast cancer cells were typified by aberrant activation of NF-{kappa}B (46). Specifically, mammary tumors induced upon carcinogen treatment of Sprague Dawley rats, human breast tumor cell lines, and primary human breast tumor tissue samples were found to constitutively express high levels of nuclear NF-{kappa}B/Rel, whereas normal rat mammary glands and untransformed breast epithelial cells contained the expected low basal levels. Importantly, inhibition of this activity in breast cancer cells in culture led to apoptosis. Other tumors recently shown to display constitutive activation of NF-{kappa}B, include the human cutaneous T cell lymphoma HuT-78 (47), Hodgkin’s lymphomas (48), melanoma (49), pancreatic adenocarcinoma (50), and primary adult T cell leukemias (51). The mechanism whereby activation of NF-{kappa}B occurs remains to be elucidated. Recently, a secreted factor has been implicated in Hodgkin’s lymphomas. It is intriguing to speculate on whether such an autocrine mechanism involves GH or other hormone or growth factor.

In conclusion, our results demonstrate that: 1) GH exerts an antiapoptotic effect; 2) GH is able to activate NF-{kappa}B; 3) the antiapoptotic effect of GH is closely mediated through NF-{kappa}B activation; 4) low concentrations of GH induce Bcl-2 and Bag-1 whereas high concentrations of GH can induce Bcl-XL expression coupled to cell cycle progression. A possible link between activation of NF-{kappa}B by GH and expression of Bcl-2 and potentially Bag-1 and Bcl-XL is also suggested.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antibodies
Mouse monoclonal anti-Bcl-2, rabbit polyclonal anti-Bag-1, and rabbit polyclonal anti-I{kappa}B{alpha} antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-Bcl-XL antibody was from Transduction Laboratories, Inc. (Lexington, KY). Anti-p65 antibody (27) was generously provided by Nancy Rice (National Cancer Institute, Frederick MD).

Cell Culture and Treatment Conditions
The Ba/F3 is a pro-B murine cell line that does not express endogenous GHR. Stable transfectants expressing either the wild-type GHR (Ba/F3 GHR) or a mutated GHR (Ba/F3 ILVT) have been prepared as previously described (29). Ba/F3 ILV/T cells express a mutant form of the rat GHR: three amino acids (I, L, and V) in the box 1 sequence of the receptor have been substituted to threonine, which results in a mutant GHR unable to initiate the phosphorylation of Jak2 and therefore to promote cell proliferation (29). Cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 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 (normal medium). Under starvation conditions, cells were extensively washed in RPMI, and then preincubated for 6 h in a serum- and IL-3-free medium containing 2% BSA (Fraction V, Sigma, St. Louis, MO), 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 10 U/ml penicillin, and 10 µg/ml streptomycin (starvation medium). For GH stimulation (bGH treatment), cells were starved for 6 h in starvation medium and then 1 µg/ml of bGH (kindly provided by William Baumbach, American Cyanamid Co., Princeton, NJ) was added to the cell culture.

Cell Cycle Analysis and Assessment of Apoptosis
Cell cycle and apoptosis were assessed by DNA content analysis after staining with the DNA intercalator propidium iodide (PI). Briefly, cells (106) were harvested by centrifugation and permeabilized with 30 µl of DNA-Prep LPR reagent, followed by addition of 0.5 ml of DNA-Prep stain PI solution (DNA-Prep reagents, Coulter Corp., Hialeah, FL). After vortexing, samples were incubated at 37 C for 30 min, and then analyzed by flow cytometry on a FACScan (Becton Dickinson and Co., Mountain View, CA) using low flow rate. Cell cycle distribution was determined using CellQuest software (Becton Dickinson and Co.) and manual gating. Apoptosis was determined as the percentage of DNA localized in the hypodiploid peak (sub-G0/G1) of the cell cycle.

GH Assays
For GH measurement in culture medium, stable clones of Ba/F3 cells (1.5 x 106 cells/ml) expressing wild-type GHR (Ba/F3 GHR) were incubated in starvation medium without BSA for 24, 48, and 72 h. Cell-conditioned media were collected and concentrated 150-fold using Centricon-plus 80 membrane (Millipore Corp., Bedford, MA). GH measurements were done using a modified RIA method, as previously described (52). Human GH antagonists B2036 and G120K were obtained from Sensus Drug Development Corp. (Austin, TX). Activity of the antagonists was assessed on Ba/F3 GHR cells incubated for 48 h in starvation medium in the absence or in the presence of increasing concentrations of B2036 or G120K ranging from 0.5 to 2 µg/ml.

Immunoblotting
Cells (106) were washed in PBS and lysed either in sample buffer containing dithiothreitol or in lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor). Protein lysate concentration was measured by Bradford assay using the Bio-Rad reagent (Bio-Rad Laboratories, Inc. Hercules, CA), according to the manufacturer’s directions. Samples (50 µg of protein) were resolved by 10% SDS-PAGE under reducing conditions. Protein lysates were transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.) and stained with red ponceau to verify the amount of protein per lane. The membranes were incubated overnight at 4 C in TBS-T (50 mM Tris-HCl, pH 7.6, 200 mM NaCl, 0.1% Tween 20) with 2% BSA. Proteins were detected by incubation with the specific antibody in TBS-T with 2% BSA. After extensive washing in TBS-T, a 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 enhanced chemiluminescence detection system (NEN Life Science Products, Boston, MA), according to the manufacturer’s instructions.

Transfections
The pRCßactin vector encoding the I{kappa}B{alpha} (A32/36) was kindly provided by Michael Karin (University of California, San Diego, CA). The mutated I{kappa}B{alpha} (A32/36) protein contains serine-to-alanine mutations in amino acids 32 and 36, preventing its phosphorylation and subsequent degradation (53). For transfection experiments, 107 Ba/F3 GHR cells were transiently transfected with 30 µg of I{kappa}B{alpha} (A32/36) encoding plasmid or 30 µg of the parental pRCßactin plasmid (53). Cells were electroporated at 330 V, 1500 µF, {infty}R in a CellJect apparatus (Eurogentec, Seraing, Belgium). Transfected cells were maintained in normal medium for 24 h before subsequent treatment.

EMSA
The double-stranded oligonucleotide containing the upstream NF-{kappa}B element from the c-myc gene, termed URE, contains the following sequence: 5'-AAGTCCGGGTTTTCCCCAACC-3' (with core NF-{kappa}B binding site underlined), as previously described (54). The DNA was labeled using the Klenow fragment of Escherichia coli DNA polymerase I (Life Technologies, Inc., Gaithersburg, MD) and [{alpha}-32P]dCTP (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Nuclear extracts used for EMSA were prepared as described by Dignam et al. (55). Nuclear extracts (2–3 µg) were incubated in sample buffer (0.4 µg of poly(dIdC), 0.1% Triton X-100, 0.5% glycerol, 0.8 mM dithiothreitol, 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, which was incubated for 30 min at room temperature. For competition analysis 10- and 20-fold molar excess of unlabeled wild-type or mutant (with conversion of internal two G-to-C residues) URE competitor oligonucleotides were added to the binding reaction. For supershift experiments, 1 µl of anti-p65 antibody (27) was added to the mixture for 1 h at room temperature before the incubation with the labeled probe. The reaction was resolved on a 4.5% acrylamide gel containing Tris-Borate-EDTA buffer for 2 h at 150 V. The gel was dried and subjected to autoradiography at -80 C using screens.


    ACKNOWLEDGMENTS
 
We thank M. Karin and N. Rice for generously providing I{kappa}B{alpha} expression vector and anti-p65 antibody, respectively. B. Bennett and W. Baumbach are gratefully acknowledged for the gifts of hGH antagonists and bGH, respectively, and M.T. Bluet-Pajot for assistance with the GH measurements. We thank INSERM Unité 373 for use of the fluorescence-activated cell sorter (FACS) and C. Garcia for technical assistance in the FACS analyses.


    FOOTNOTES
 
Address requests for reprints to: Dr. Marie-Catherine Postel-Vinay, INSERM U344, Endocrinologie Moléculaire, Faculté Necker-Enfants Malades, 156, rue de Vaugirard, 75015 Paris, France.

1 Current address: Dr. Elena Baixeras, Department of Medicine and Liver Unit, Medical School, University of Navarra, 31 008 Pamplona, Spain. Back

This work was supported by the Institut National de la Santé et de la Recherche Médicale (S.J., M.C.P-V., and E.B.), Grant 5363 from Association pour la Recherche sur le Cancer, and by Public Health Service Grant CA-36355 (G.E.S.) from the National Institute of Health.

Received for publication November 10, 1999. Revision received February 1, 2000. Accepted for publication February 10, 2000.


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