Endoplasmic Reticulum Stress Prolongs GH-Induced Janus Kinase (JAK2)/Signal Transducer and Activator of Transcription (STAT5) Signaling Pathway

Amilcar Flores-Morales, Leandro Fernández, Elizabeth Rico-Bautista, Adriana Umana, Ciro Negrín, Jian-Guo Zhang and Gunnar Norstedt

Department Of Molecular Medicine (A.F.-M., E.R.-B., G.N.), Karolinska Institute, 17176 Stockholm, Sweden; Health Science Center, Pharmacology Section (L.F., C.N.), Las Palmas de Gran Canaria University, 35080 Las Palmas de Gran Canaria, Spain; The Walter and Eliza Hall Institute of Medical Research and The Cooperative Research For Cellular Growth Factors, Victoria 3050, Australia (J.-G.Z.); and Department Of Chemistry, National University, Bogota, Colombia (A.U.)

Address all correspondence and requests for reprints to: Amilcar Flores Morales, Ph.D., Centrum for Molecular Medicine, CMM, L8:01, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail: Amilcar. Flores{at}molmed.ki.se


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The desensitization of the GH-induced Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) signaling pathway plays a crucial role in GH regulation of hepatic genes. Previous studies have demonstrated that the inactivation of the GH-induced JAK2/STAT5 pathway is regulated by protein translation and suppressors of cytokine signaling (SOCS). In this study we sought to explore the relationships between endoplasmic reticulum stress, GH-induced JAK2/STAT5 activity and SOCS expression. 1,2-bis(o-Aminophenoxy)ethane-N,N,N,N-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM), used to provoke endoplasmic reticulum stress, caused a drastic inhibition of protein translation that correlated with the phosphorylation of the eukaryotic translation initiation factor 2{alpha}. Both GH and BAPTA-AM caused a rapid induction of the transcription factor C/EBP homology protein (CHOP) and an additive effect was observed with combined treatment, which suggests a regulatory role of GH on endoplasmic reticulum stress. Endoplasmic reticulum stress did not interfere with the rapid GH activation of STAT5 DNA binding activity. However, BAPTA-AM prolonged the DNA binding activity of STAT5 without affecting STAT5 or JAK2 protein levels. GH-induced phosphorylation of JAK2 and STAT5 DNA binding activity were prolonged in the presence of BAPTA-AM, suggesting that endoplasmic reticulum stress prevents the inactivation of STAT5 DNA binding activity by modulating the rate of JAK2/STAT5 dephosphorylation. Like BAPTA-AM, the endoplasmic reticulum stressors dithiothreitol and A23187 also prolonged the GH-induced STAT5 DNA binding activity. We were not able to correlate BAPTA-AM effects to the GH-dependent expression of SOCS proteins or SOCS mRNA, suggesting that endoplasmic reticulum stress modulates the rate of JAK2/STAT5 dephosphorylation through mechanisms other than inhibition of SOCS expression. This study indicates that cellular stress may modulate transcription through the JAK/STAT pathway.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SIGNAL TRANSDUCERS AND activators of transcription (STATs) constitute a family of latent cytoplasmic transcription factors that migrate to the nucleus in response to extracellular signals and activate gene transcription (1). Tyrosine phosphorylation of STATs by members of the Janus kinase (JAK) family plays a pivotal role for cytokine-induced signaling pathways. In general, it appears that cytokines induce the JAK/STAT pathway in a transient manner. GH activation of the JAK2/STAT5 signaling pathway can serve as a model to study molecular mechanisms that underlie the transient activation of this pathway (2). Previously, we reported that the duration of a JAK2 and STAT5 signal in liver BRL-4 cells is markedly prolonged by cycloheximide (CHX), an inhibitor of protein translation (2). Our interpretation was that CHX blocked the production of a GH-inducible protein that turns the JAK2/STAT5 signaling off. This has been substantiated by the discovery of a new class of proteins called suppressors of cytokine signaling (SOCS). SOCS are induced by cytokines and function to silence or attenuate the JAK/STAT pathway (3). There are several different SOCS, and the relations of specific SOCS to GH actions are presently being studied (4, 5, 6).

The nature of cytokine actions is often dependent on the kinetics of JAK/STAT activation. For example, growth arrest of malignant lymphoma cells by IFN{gamma} has been related to a prolonged rather than transient activation of STAT1 (7), while continuous STAT5b activation by the male pattern of GH secretion is essential for the liver sex differentiation in rodents (8). Cytokines often exert their actions concomitantly with stressful situations to the cell. Therefore, it is important to establish whether stress-induced cellular pathways can modulate cytokine signaling, as this interaction can change the outcome of cytokine actions. For these reason we have undertaken to investigate if the cellular response to stress can modulate the JAK/STAT pathway.

Cellular stress initiates a complex cascade of stress-inducible enzymes and transcription factors in an attempt to adapt to changes in the immediate environment (9, 10). The nature of the cellular response depends on the type and dose of stress. The correct folding of proteins in the endoplasmic reticulum can be altered by certain types of stressful stimuli, and this generates a powerful cellular reaction known as the unfolded protein response (UPR). The UPR can be triggered by a variety of agents including glycosylation inhibitors, reducing agents, glucose starvation, hypoxia, viral particles, growth factor depletion, and perturbation of intracellular calcium homeostasis (11). These agents provoke the accumulation of unfolded proteins in the endoplasmic reticulum, which in turn leads to inhibition of protein translation initiation. This occurs through activation of protein kinases that specifically phosphorylate the {alpha}-subunit of eukaryotic translation initiation factor 2 (eIF-2{alpha}). A small increase in the phosphorylation of eIF-2{alpha} rapidly results in inhibition of the initiation of protein synthesis. In addition to changes in the translational machinery, the UPR selectively activates transcription of genes encoding endoplasmic reticulum resident chaperones (e.g. Bip/GRP78) that perform diverse roles to maintain endoplasmic reticulum function and facilitate protein folding. The UPR also triggers the transcriptional activation of the transcription factor C/EBP homology protein (CHOP), also known as growth arrest and DNA damage protein 153 (GADD153) (12). CHOP is not normally expressed in cells but is induced by a variety of stress insults. This factor can heterodimerize with members of the bZIP family of transcription factors and, in doing so, alters gene expression and regulates both cell death and tissue regeneration after stress (13).

Previous investigations concerning the effects of chemical agents on the duration of JAK 2/STAT5 signals have led us to hypothesize a possible relation between cellular response to stress and the kinetics of JAK2/STAT5 activation by GH. In the present study we have shown that the induction of UPR can prolong the GH-induced JAK/STAT signaling pathway. Our findings suggest that novel mechanisms to control the transient activation of JAK2 and STAT5 proteins exist. Possibly endoplasmic reticulum stress may induce effects on SOCS actions other than the inhibition of its expression. These findings imply that cellular responses to endoplasmic reticulum stress may affect the outcome of cytokine stimulation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Endoplasmic Reticulum Stress Prolongs the GH-activated STAT5 DNA Binding Activity
We have previously reported that D609, a xanthate derivative with antiviral properties and inhibitory activity on PLC, prolongs the GH-induced JAK2/STAT5 signaling pathway (2). Thus, it was reasonable to test whether pathways downstream of PLC were involved in down-regulation of the GH-induced STAT5 DNA binding activity. A pharmacological approach was used to test whether alterations in the intracellular calcium concentration or the activity of PKC were involved in this process. We tested the nonspecific serine/threonine kinase inhibitor H7 (14), the specific PKC inhibitors calphostin C (15) and bisindolylmaleimide I (16), and the kinase inhibitor 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), on GH-induced STAT5 DNA binding activity. As shown in Fig. 1AGo, bisindolylmaleimide I and calphostin C failed to prolong the duration of GH-induced STAT5 DNA binding activity. These findings suggest that members of the PKC family of serine/threonine kinases sensitive to either inhibitor (e.g., PKC{alpha}, ßI, ßII, {gamma}, {delta}, {epsilon}) are not involved in the desensitization process. However, H7 as well as DRB prevented the negative regulation of GH-induced STAT5 DNA binding activity. These serine kinase inhibitors have well documented inhibitory effects on RNA polymerase II phosphorylation and activity (14). These data, together with our previous finding that actinomycin D prolongs GH-activated STAT5 signaling (2), suggest that H7 and DRB prevent the negative regulation of GH-induced STAT5 DNA binding activity through inhibition of transcription. Members of the SOCS family are transcriptionally regulated by GH (Figs. 4Go and 5Go) and have a negative effect on the GH-activated STAT pathway (5). Thus, if SOCS mRNA has a rapid turnover, the agents that reduce SOCS mRNA synthesis/stability might prolong JAK/STAT signaling pathway.



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Figure 1. The Endoplasmic Reticulum Stressor BAPTA-AM Prevents Desensitization of GH-Stimulated STAT5 DNA Binding Activity

Nuclear extracts were prepared from BRL-4 cells treated with hGH (50 nM) for various times in the presence or absence of (A) H7 (100 µM), calphostin C (2 µM), bisindolylmaleimide I (2 µM), or DRB (10 µM), (B) BAPTA-AM (25 µM), nifedipine (100 µM), or calmidazolium (10 µM), and (C) DTT (200 µM) or A23187 (2 µM). The inhibitors or appropriate vehicle was added 30 min before initiation of the GH treatment. EMSA of the extracts were performed using the SPIGLE1 probe. The EMSA shown is representative of three independent experiments. The levels measured after 15 min of GH treatment were used as the reference values. Results are expressed as percent of reference ± SD of three independent experiments. **, P < 0.01 (inhibitors + GH vs. GH). *, P< 0.05 (inhibitors + GH vs. GH).

 


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Figure 4. The Effects of GH on SOCS mRNA in BRL-4 Cells

A, Total RNA was prepared from BRL-4 cells treated for various times in the presence or absence of hGH (50 nM). mRNA was determined in a solution hybridization assay specific for SOCS mRNA as described in Materials and Methods. B, Cells were treated with GH (50 nM) for 1 h and then washed twice with serum-free media and cultured for the indicated times in the presence or absence of hGH. At the indicated times, cells were harvested, total RNA was isolated, and SOCS mRNA was analyzed by solution hybridization as described in Materials and Methods. Results are expressed as picograms mRNA per µg total RNA. Values are the mean ± SD of three independent experiments. **, P < 0.01(+GH vs. -GH). *, P < 0.05(+GH vs. -GH).

 


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Figure 5. The Effects of GH on SOCS mRNA in the Presence of Endoplasmic Reticulum Stress

A, Total RNA was prepared from BRL-4 cells treated with hGH (50 nM) for various times in the absence (DMSO) or presence of BAPTA-AM (25 µM). The inhibitor or DMSO was added 30 min before initiation of the GH treatment. mRNA was determined in a solution hybridization assay specific for SOCS mRNA as described in Materials and Methods. Results are expressed as picograms mRNA per µg total RNA. Values are the mean ± SD of three independent experiments. **, P < 0.01 (BAPTA+GH vs. GH). *, P < 0.05 (BAPTA+GH vs. GH).

 
We tested whether or not GH activation of STAT5 DNA binding activity could be prolonged by alteration of intracellular calcium homeostasis. In the presence of the intracellular calcium chelator BAPTA-AM (Fig. 1BGo), GH treatment of BRL-4 cells resulted in prolonged activation of STAT5 DNA binding activity, whereas the drug alone had no effect. Neither calmidazolium, an inhibitor of calcium/calmodulin-regulated enzymes, nor nifedipine, an inhibitor of L-type calcium channels, affected the kinetics of GH-induced STAT5 DNA binding activity (Fig. 1BGo). Calcium levels in the lumen of endoplasmic reticulum are tightly controlled, and changes in endoplasmic reticulum calcium concentrations can cause an accumulation of unfolded proteins in the endoplasmic reticulum leading to the activation of the UPR (11). It is likely that activation of the UPR is associated with the effects of 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM) on GH-induced STAT5 DNA binding activity (Fig. 1BGo). To further investigate this hypothesis, the inducers of UPR [A23187, a calcium ionophore, and dithiothreitol (DTT), which interferes with disulfide bond formations], were tested for their capacity to prolong GH-induced STAT5 DNA binding activity. As shown in Fig. 1CGo, both A23187 and DTT prevented the negative regulation of GH-induced STAT5 DNA binding activity.

To substantiate that BAPTA-AM activates an UPR response in BRL cells, experiments were performed to analyze the rate of protein translation and the expression of the transcription factor CHOP (11). As shown in Fig. 2Go, BAPTA-AM caused a rapid inhibition of protein synthesis within the first hour after treatment. GH added in combination with BAPTA-AM caused no effect compared with the drug alone. Changes in protein translation during stress are, in many cases, related to phosphorylation of eIF-2{alpha}, which inhibits binding of Met-tRNA to the initiation complex. As shown in Fig. 2BGo, treatment with BAPTA-AM induced the phosphorylation of eIF-2{alpha} at serine 51. Analogous with the protein synthesis results, GH did not have any marked influence on the BAPTA-AM effect on eIF-2{alpha} phosphorylation. Interestingly, BAPTA-AM as well as GH, caused a rapid increase in CHOP mRNA (Fig. 2CGo), and an additive effect was observed at the 30- and 45-min time point with the combined treatment. The effect of GH is greater than the effect of BAPTA plus GH at the 60- and 90-min time point. Taken together, these findings show that BAPTA-AM induces endoplasmic reticulum stress, and that GH might regulate the cellular responses downstream of CHOP.



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Figure 2. The Endoplasmic Reticulum Stressor BAPTA-AM Inhibits Protein Translation in BRL-4 Cells

A, Cells were grown to 90% confluence, serum-starved for 16 h, and incubated for 30 min with 25 µM BAPTA-AM or dimethylsulfoxide (DMSO). Then, hGH (50 nM) was added for the indicated times in the presence or absence of 25 µM BAPTA-AM or DMSO. The cells where washed with methionine-free media and 35S-Methionine (50 µCi/ml) was added for an additional 30 min. Whole-cell extracts were prepared, and the radioactivity incorporated into newly synthesized proteins was determined as described in Materials and Methods. Values are the mean ± SD of three experiments. **, P < 0.01 (inhibitor +GH vs. GH). B, Whole-cell extracts were prepared from cells treated as described above, and phosphorylated eIF-2{alpha} was estimated by Western blot with antibodies against the phosphorylated form of the protein. This blot is representative of three independent experiments. Values are expressed as arbitrary units (AU) in which the unit refers to the levels found in untreated cells. The values represent the mean ± SD of three independent experiments. **, P < 0.01 (treatment vs. time 0). *, P < 0.05 (treatment vs. time 0). C, Total RNA was prepared from BRL-4 cells treated with hGH (50 nM) for various times in the presence or absence of BAPTA-AM (25 µM). The inhibitor or DMSO was added 30 min before initiation of the GH treatment. mRNA was determined in solution hybridization assay specific for CHOP mRNA as described in Materials and Methods. The arrow indicates when BAPTA-AM and GH were added to the cells. Results are expressed as counts per min per µg total RNA. Values are the mean ± SD of three independent experiments. **, P < 0.01 (BAPTA-AM + GH vs. GH). ++, P < 0.01 (BAPTA-AM + GH vs. BAPTA-AM).

 
In subsequent experiments, we used BAPTA-AM-treated BRL-4 cells to investigate how cellular stress influences the GH-induced JAK2/STAT5 signaling pathway. It is believed that the negative regulation of STAT activation is exerted at the level of GH receptor (GHR)/JAK2 complex (2, 6). As shown in Fig. 3Go, we demonstrate that this is the case with BAPTA-AM. Treatment with GH alone induced a rapid and transient peak of JAK2 tyrosine phosphorylation. Maximal phosphorylation was seen after 5 min and was dramatically reduced after 2 h of treatment. When BAPTA-AM was added together with GH, no major effect was observed on GH-induced tyrosine phosphorylation of JAK2 (Fig. 3Go, upper panel). However, when tyrosine phosphorylation was measured after a 2-h treatment period, a stronger signal was observed in cells treated with BAPTA-AM plus GH when compared with cells treated with GH alone, indicating a shift in the kinetics of JAK2 tyrosine phosphorylation (Fig. 3Go, upper panel). Since treatment with BAPTA-AM alone fails to prolong tyrosine phosphorylation of JAK2 (Fig. 3Go, upper panel), this suggests that BAPTA-AM disrupts the negative regulatory mechanism dependent on GH. Interestingly, even though BAPTA-AM induced a rapid translational arrest (Fig. 2AGo), no major differences were seen in protein levels of JAK2 (Fig. 3Go, middle panel) and STAT5 (Fig. 3Go, lower panel), suggesting that JAK2 and STAT5 have a relatively slow turnover rate.



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Figure 3. The Endoplasmic Reticulum Stressor BAPTA-AM Prevents Desensitization of GH-Stimulated JAK2 Phosphorylation

Cells cultured in the absence (vehicle) or presence of BAPTA-AM (25 µM) were stimulated or not with GH (50 nM) for the indicated times. BAPTA-AM was added 30 min before GH treatment. Whole cellular extracts were subjected to immunoprecipitation with anti-JAK2. The immunoprecipitates were analyzed by Western blotting for phosphotyrosine immunoreactivity (upper panel) or JAK2 protein (middle panel). Whole cellular extracts were prepared and analyzed for STAT5 protein by Western blotting; 60 µg proteins were applied in each line (lower panel). Blots are representative of at least three independent experiments. The levels measured at the earliest treatment with GH were used as the reference values. Results are expressed as percent of reference ± SD of three independent experiments. **, P < 0.01(BAPTA-AM + GH vs. GH).

 
Endoplasmic Reticulum Stress Does Not Inhibit GH-Dependent Expression of SOCS mRNA
The above findings suggest the existence of multiple mechanisms whereby GH-induced JAK2/STAT5 pathway can be prolonged. It is plausible that reduction in SOCS expression is behind the effects of endoplasmic reticulum stress on the duration of JAK2/STAT5 activation. Figure 4AGo shows the effects of GH on the expression of SOCS-1, -2, -3, and cytokine-induced SH2 protein (CIS) in BRL-4 cells. GH increased all four SOCS transcripts. Increased levels of SOCS-1, -2, -3, and CIS mRNA were evident 1 h after GH stimulation. The expression of SOCS-1 and -3, and CIS mRNA were maximal 1 h after GH treatment. After treatment for 4 h the CIS mRNA level returned to the one found in unstimulated cells, while SOCS-1 and -2 mRNA levels were kept elevated. In contrast, GH stimulation caused SOCS-2 gene expression to steadily increase over time. The expression level of glyceraldehyde-3-phosphate-dehydrogenase mRNA was constant during the experiment (not shown). To study the turnover of SOCS mRNA, GH was removed after 1 h of GH treatment, and the levels of SOCS transcripts were measured (Fig. 4BGo). Within 3 h SOCS-1, -2, and -3 mRNA levels were reduced in comparison to those found in cells treated with GH continuously. Thus it appears to be the case that GH-induced SOCS mRNAs have a rapid turnover, which explains their sensitivity to transcriptional inhibition.

We next studied the effects of BAPTA-AM on GH-induced SOCS mRNA. SOCS mRNA levels were increased in the presence of BAPTA-AM (Fig. 5Go). GH in combination with BAPTA-AM caused higher SOCS induction than GH alone. The most likely interpretation is that BAPTA-AM causes a translational arrest that blocks the production of SOCS proteins normally destined to attenuate GH signals. A failure in SOCS synthesis would result in the accumulation of GH-induced transcripts including SOCS mRNAs. Alternatively, the mechanism that actively degrades SOCS mRNA may be sensitive to stress interference.

Endoplasmic Reticulum Stress Does Not Inhibit GH-Dependent Expression of SOCS Proteins but Regulates Its Turnover
We wanted to test the hypothesis that cellular stress-induced inhibition of protein translation would result in the reduction of SOCS proteins in response to GH and, consequently, in the prolongation of GH signaling. Therefore, the levels of SOCS-1, -2, and -3 proteins were analyzed after BAPTA-induced stress in the presence or absence of GH. As shown in Fig. 6AGo, GH treatment of BRL-4 cells induced the expression of SOCS-1 and -2. SOCS-1 induction was maximal after 30 min with GH treatment while SOCS-2 showed maximal induction after 1 h of GH stimulation. When BAPTA-AM was added in combination with GH, it did not modify the expression of SOCS-1 protein. BAPTA-AM blocked the GH effects on SOCS-2 at 2 h of treatment, which correlates with the effects of this endoplasmic reticulum stressor on GH-induced JAK2/STAT5 activity. Interestingly, despite the finding that BAPTA-AM rapidly inhibits protein synthesis (see Fig. 2AGo), the de novo GH induction of SOCS proteins was not affected. This suggests that translation inhibition is not enough to prolong GH signaling and that other signals activated by BAPTA-AM-induced endoplasmic reticulum stress are necessary. This observation is supported by experiments in which the negative regulation of GH-induced STAT5 DNA binding activity is not affected by short-term inhibition of protein translation by CHX (Fig. 6BGo). Despite the fact that treatment of BRL-4 cells with CHX for 1 h resulted in a 95% inhibition of protein translation (data not shown), there is no evidence for the prolongation of GH-induced STAT5 DNA binding activity by GH (Fig. 6BGo). As previously reported (2), long-term inhibition of protein translation (4 h) was necessary to prevent down- regulation of GH-induced JAK2/STAT5 activity.



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Figure 6. The Effects of GH on SOCS Proteins in the Presence of Endoplasmic Reticulum Stress

A, Cells cultured in the absence (vehicle) or presence of BAPTA-AM (25 µM) were stimulated or not with GH (50 nM) for the indicated times. BAPTA-AM was initiated 30 min before GH treatment. Whole cellular extracts were subjected to immunoprecipitation with anti-SOCS antibodies. The immunoprecipitates were analyzed by Western blotting for SOCS-1 (upper panel), SOCS-2 (middle panel), or SOCS-3 (lower panel) proteins. Values are expressed as arbitrary units (AU) where the unit refers to the levels found in untreated cells. The values represent the mean ± SEM of three independent experiments. B, Nuclear extracts were prepared from BRL-4 cells treated with hGH (50 nM) for various times in the absence or presence of CHX (2 µg/ml) or MG132 (10 µM). The inhibitors or appropriate vehicle was added 60 min before initiation of the hGH treatment. GEMSA of the nuclear extracts was performed using the SPIGLE1 probe as described in Materials and Methods. Blots are representative of at least three independent experiments. The levels measured at the earliest treatment with GH were used as the reference values. Results are expressed as percent of reference ± SD of three independent experiments. **, P < 0.01(inhibitor + GH vs. GH), *, P < 0.05 (inhibitor + GH vs. GH).

 
The nature of the stress signals that influence the rate of inactivation of the GHR/JAK2 complex can only be speculated upon. It is possible that molecules downstream of SOCS are sensitive to stress signals. Based on the homology of the SOCS domain to the Von Hippel-Lindau protein, it has been suggested that SOCS proteins may be part of an ubiquitin-ligase complex that target the GHR for degradation (17). As shown in Fig. 6CGo, the proteasome inhibitor MG132 prevented the negative long-term regulation of GH-induced STAT5 DNA binding activity when measured 2 h after GH stimulation. It is worthwhile to note that the partial (60% of control values) down-regulation observed at 30 min is not affected by the treatment with MG132. Furthermore, unlike the endoplasmic reticulum stress inducers, proteosome inhibition caused activation of STAT5 DNA binding activity in the absence of GH. This ligand-independent effect indicates that the ubiquitin-proteasome system plays a crucial role in the negative regulation of the GH-activated JAK2/STAT5 signaling pathway.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we show that cellular stress prolongs the duration of the JAK2/STAT5 signaling pathway activated by GH. Experimentally, this was demonstrated using the intracellular calcium chelator BAPTA-AM. Treatment of BRL-4 hepatocytes with BAPTA-AM causes a cellular stress response that is evidenced by a reduction in the rate of protein synthesis, the phosphorylation of eIF-2{alpha}, and the induction of CHOP mRNA. This evidence also suggests a link between the stress-induced prolongation of JAK/STAT signaling and the actions of SOCS.

The discovery of the SOCS family has provided a new aspect of cytokine receptor regulation that we believe is especially relevant in the present study (3). SOCS proteins function in novel ways to suppress signal transduction pathways. SOCS are SH2 and SOCS box-containing proteins that bind either JAK or cytokine receptors and serve as negative regulators. Transcription of SOCS genes rapidly increases in response to cytokines, both in vitro and in vivo. This suggests that they may act in a classic negative feedback loop to regulate cytokine signal transduction (3). Overexpression experiments have shown that SOCS-1, -2, and -3 and CIS are able to inhibit GH-induced STAT5 activation (5). SOCS-1 has been shown to directly bind and inhibit JAK2, while the SOCS-3 and -2 and CIS mechanism of action remains to be determined (18). The N-terminal and SH2 domains, but not the C-terminal (SOCS homology domain) (6, 18, 19) domain of SOCS proteins, seem to be required for their inhibitory action, which suggests that binding to phosphotyrosine residues in the GHR/JAK2 complex is important. Bound SOCS may act in various ways that are not yet defined, e.g. as a direct kinase inhibitor (20), as an adaptor protein to promote association to phosphotyrosine phosphatases (21, 29), or to target the activated receptor for internalization for protein degradation through the ubiquitin/proteasome system (28). The potential role of SOCS in the regulation of GH responsiveness encouraged us to study SOCS expression in relation to pharmacological agents that induce cellular stress or interfere with a variety of signaling pathways.

Our finding that GH induces SOCS-1, -2, and -3 and CIS gene expression in BRL-4 cells, as well as in primary rat hepatocytes (4), is consistent with the notion that SOCS proteins serve as molecules that turn off GH signals. SOCS-2 mRNA increased steadily in the presence of GH, while SOCS-1 and -3 and CIS mRNA levels were transiently induced by GH with a maximal level at 1 h after GH treatment. The similar kinetics between SOCS-1 and -3 and CIS regulation may reflect the presence of similar types of GH response elements in the promoters of these genes. It has previously been shown that CIS and SOCS-3 gene promoters contain STAT response elements that are responsible for induction of these genes by erythropoietin (21) and leukemia inhibitory factor, respectively (22). STAT-mediated regulation of CIS and SOCS-3 gene expression would be in agreement with the transient time course of STAT activation by GH. The transient kinetics of GH-induced SOCS-1 and -3 and CIS expression speaks against their involvement in the long-term negative regulation of GHR signaling. In this sense it would be more reasonable if SOCS-2 were involved since its mRNA level is steadily elevated in the presence of GH (Fig. 4AGo). In this context, it is also relevant to note that GH-induced SOCS mRNAs fall rapidly in the absence of GH (Fig. 4BGo), indicating that SOCS mRNAs have a rapid turnover.

We have previously shown that transcriptional inhibition results in the prolongation of GH-induced STAT5 DNA binding activity (2). Since H7 blocks a broad spectrum of rapidly induced mRNAs by diverse stimuli, including those induced by cytokine receptor activation (14), it is likely that the mechanism of action of H7 is related to the inhibition of the GH-induced transcriptional activation of SOCS genes. A mechanistic explanation for this has recently been described; H7 is a strong inhibitor of phosphorylation of the tandem repeats found in the C-terminal domain of RNA polymerase II (23). Furthermore, GH-stimulated STAT5 DNA binding activity is prolonged by DRB, which is a more specific inhibitor of RNA polymerase II phosphorylation. Phosphorylation of RNA polymerase II C-terminal domain by a kinase activity associated with the transcription initiation complex is considered a necessary step in the establishment of a progressive, rather than an abortive, transcription complex (24). The PLC inhibitor D609 is also able to prolong the activation of GH-induced JAK2/STAT5 pathway (2). Nevertheless, it is unlikely that the effects of D609 are related to PLC inhibition as we were not able to detect GH-dependent induction of PLC activity in BRL-4 cells (data not shown). D609 inhibits the transcription of SOCS1, -2, and -3 and CIS genes (data not shown), which further supports the notion that GH-induced SOCS mRNA is necessary for the long-term down-regulation of the JAK2/STAT5 signaling pathway in the continued presence of GH.

The endoplasmic reticulum stressors, BAPTA-AM, DTT, and A23187, were able to prolong the duration of JAK2/STAT5 activation in the presence of GH. In the case of BAPTA-AM, it prolongs GH signaling independently of the levels of SOCS mRNAs. Furthermore, BAPTA-AM increased, in combination with GH, the levels of SOCS-1, -2, and -3 and CIS mRNA to a level higher than that by GH alone. On the premise that GH-activated JAK2/STAT5 activity is down-regulated by SOCS, one may postulate that endoplasmic reticulum stress blocks SOCS actions. The absence of a negative feedback regulation to the GHR and the resultant continued activation of JAK2 will result in a prolonged activation of transcription, which would increase the levels of GH-regulated transcripts, including levels of SOCS and CHOP mRNAs. Interestingly, the stress-induced phosphorylation of eIF-2{alpha} and the translation arrest are independent of GH. We have also noticed that CHOP mRNA, in addition to being increased by BAPTA-induced stress, is also GH-regulated. The functional significance of these findings merits further evaluation as CHOP has been shown to have important regulatory actions on the transcriptional activity of stress-regulated transcription factors of the bZIP family, such as JUNs and C/EBPs (25, 26).

The use of BAPTA-AM in BRL-4 cells can be regarded as a model for the UPR. A variety of signals can trigger this response, including oxidative and osmotic stress, viral infection, hypoxia, and amino acid starvation. In consideration of the putatively short life span of SOCS mRNA, the UPR signal could influence the cellular level of SOCS by preventing SOCS translation. Thus, stress-induced translational arrest, if sufficiently long, will reduce SOCS protein levels, resulting in a prolonged duration of JAK/STAT activation. Accordingly, we have previously shown that long-term incubation with CHX (up to 4 h) prevents desensitization of GH-induced JAK2 and STAT5 signaling (2).

There seems to be an additional and more rapid mechanism whereby stress influences the deactivation of the JAK2/STAT5 pathway. Blocking protein synthesis by treating BRL-4 cells with CHX during 1 h before GH stimulation does not prolong GH-induced STAT5 DNA binding activity within the time frame studied here. In contrast, BAPTA-AM, which also induced rapid translational arrest, was able to prolong GH-induced JAK2/STAT5 signaling pathway. When we performed immunodetection of SOCS proteins in BAPTA-AM-stressed cells, no major effects were observed regarding the GH induction of SOCS-1 and SOCS-2 proteins. Interestingly, a reduction in the level of SOCS-2 was evident at 2 h of combined treatment of GH with BAPTA-AM. To assign the effects observed in the activity of the JAK2/STAT5 pathway to the changes in SOCS-2 levels is speculative. It may be possible that other members of the SOCS family, such as CIS, are specifically targeted by signals from endoplasmic reticulum stress and are responsible for the prolongation of JAK/STAT signal. Another alternative is that components downstream of the SOCS pathway are sensitive to the UPR. A mechanism of interference between the UPR and the proteasome degradation of ER-associated proteins has recently been described in yeast (27). Based on its homology with the Von Hippel-Lindau {alpha} domain (17), it has been suggested, but not proven, that SOCS may act as part of an ubiquitin-ligase complex promoting the ubiquitination of cytokine receptor/JAK complexes. Ubiquitination of the phenylalanine 346 in the intracellular domain is necessary for GHR internalization (28). Thus, it may be that SOCS negatively regulates GHR signaling by promoting ubiquitination, internalization, and proteasome degradation of the GHR/JAK2 complex. Accordingly, the inhibition of proteasome activity by MG132 blocks the long-term negative regulation of STAT5 DNA binding activity in the continuous presence of GH. Although the complex nature of the UPR offers a large number of putative targets that needs to be further investigated (27), it may be speculated that endoplasmic reticulum stress acts to interfere with the SOCS actions on the ubiquitination, internalization, or the proteosomal degradation of the GHR/JAK2 complex. However, there is evidence against the involvement of receptor internalization for the negative regulation of the JAK/STAT pathway. Mutation of phenylalanine 346 inhibits the GHR internalization but has no effect on the GH- induced STAT5-mediated transcriptional activity (28). It is also worth noting that the rapid and partial (60% of control) down-regulation of GH-stimulated STAT5 binding activity (Fig. 6CGo) was not affected by proteasome inhibition. Alternatively, the stress signal may target a mechanism unrelated to the SOCS actions, such as the deactivation of the GHR/JAK2 complex by a phosphotyrosine phosphatase. It has been recently reported that the tyrosine phosphatase SHP-2 is a target for the GHR/JAK2 complex, and mutations in the receptor that inhibit this association prolong the GHR signal (29). The sensitivity of this mechanism to the UPR requires further analysis. The results of the treatments with BAPTA-AM and MG132 show that partial short-term down-regulation can be dissociated from long-term resistance to JAK/STAT activation induced by continued treatment with GH. Both short-term down-regulation and long-term resistance may represent different phenomenon and therefore distinctive mechanisms (e.g. dephosphorylation or ubiquitination) may exist to govern both processes.

The duration of JAK/STAT signaling is likely to be an important factor in regulating the biological outcome of cytokine stimulation. Transient activation of liver STATs has been linked to physiologically sex-specific actions of GH (8), while growth arrest of malignant lymphoma cells by IFN-{gamma} has been related to a prolonged, rather than transient, activation of STAT1 (7). STATs are known to interact with several transcription factors such as nuclear factor-{kappa}B (30), nuclear receptors (31), and SMA and MAD-related proteins (SMADs) (32), interactions that may be of relevance for the regulation of the cellular stress response. A stress-induced reduction in the negative regulation the JAK/STAT signaling pathway, thought to be dependent on a disrupted SOCS action, may be of functional significance. The regulation of the JAK/STAT pathway by cellular stress could play a role in apoptosis (33), and such a mechanism may be related to the pathological consequences of stress (34).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Recombinant human GH (hGH) was kindly provided by Pharmacia-Upjohn AB (Stockholm, Sweden) from whom protein G-Sepharose was also purchased. Enhanced chemiluminescence (ECL) reagents, CHX, and sodium orthovanadate were obtained from Sigma (St. Louis, MO). Xhantogenate tricyclodecan-9-yl (D609), BAPTA-AM, H7, calphostin C, bisindolylmaleimide I, nifedipine, and calmidozolium were from Calbiochem-Novabiochem (San Diego, CA). Proteinase K was purchased from Merck & Co., Inc. (Darmstadt, Germany), RNase-A and RNase-T1 from Boehringer Ingelheim GmbH Bioproducts Partnership (Heidelberg, Germany), and glass-fiber filters (Whatman GF/C) from Whatman Ltd. (Madison, Kent, UK). Reagents for in vitro transcription were obtained from Promega Corp. (Madison, WI).

Cell Culture
Buffalo rat liver cells (BRL) stably transfected with the rat GHR cDNA, designated BRL-4 cells and previously shown to respond to hGH (2), were cultured in DMEM supplemented with 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). Treatment of the cells with hGH and/or inhibitors is specified in the figure legends and in Results. All experiments were performed at least three times.

Analysis of mRNA Expression
Total RNA was isolated using TRIZOL Reagent (Life Technologies, Inc.), according to the protocol supplied by the manufacturer. mRNA levels corresponding to SOCS1, -2, -3, CIS, and glyceraldehyde-3-phosphate-dehydrogenase (35) gene expression were measured in total RNA samples. This was done using a solution hybridization/RNase protection assay. Transcript-specific 35S-labeled cRNA probes were transcribed in vitro from the respective cDNA vector construct, as described previously (4). Specific mRNA was quantitated by comparison with a standard curve obtained from hybridizations to known amounts of in vitro synthesized mRNA. The concentration of nucleic acid samples was measured spectrophotometrically. Samples were analyzed in triplicate, and the results are expressed as picograms of specific mRNA per µg total RNA. To permit accurate comparison of specific mRNA levels between different treatments, the samples of interest were always analyzed at the same time.

Preparation of Nuclear Extracts
BRL-4 cells were grown to confluence in 100-mm culture dishes and serum starved for 16 h before the addition of inhibitors and/or hGH. After treatment, the cells were washed twice with cold PBS, and nuclear extracts were prepared essentially as described (2). In brief, the cells from 100 mm-diameter dishes were collected by centrifugation, resuspended in hypotonic buffer [10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 6 mM MgCl2, 1 mM DTT, 0.4 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF, 1 mM Na3VO4, and 1 µg/ml pepstatin, aprotinin, and leupeptin] equal to approximately 3 times the packed cell volume and incubated on ice for 10 min. The cells were lysed with 20 strokes in a Dounce homogenizer (pestle B), and the nuclei were collected by centrifugation and resuspended in 3 volumes of high salt buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM Na3VO4, and protease inhibitors). After 30 min at 4 C, the pellets were removed by centrifugation, and supernatants containing the nuclear proteins were stored at -70 C until use. The protein concentration was measured using Bradford assay (Bio-Rad Laboratories, Inc., Richmond, CA) using BSA as standard.

EMSA
EMSA was performed according to standard protocols (13). The binding reactions were performed by preincubating 8 µg nuclear extract with 2 µg of poly (dI-dC) in 20 µl buffer containing 20% Ficoll, 60 mM HEPES, pH 7.9, 20 mM Tris, pH 7.9, 0.5 mM EDTA, and 5 mM DTT for 10 min at room temperature. 32P-end-labeled double-stranded SPI.GLE1 (TGTTCTGAGAAATA) (36) was added, and the mixture was incubated for 10 min at room temperature. The samples were electrophoresed on 4.5% nondenaturing polyacrylamide gels in 0.25 x TBE (1 x TBE; 0.09 M Tris-borate, 2 mM EDTA) at 150 V at room temperature. The radioactive pattern was visualized by autoradiography and quantified by PhosphorImager scanning (Fuji Photo Film Co., Ltd., Stamford, CT).

Metabolic Labeling
Exponentially growing cells were treated with GH and BAPTA-AM as described in Fig. 2Go, rinsed with methionine-free DMEM, and incubated for 30 min with methionine-free medium supplemented with trans-35S-methionine (50 µCi/ml) (NEN Life Science Products, Boston MA) for 30 min at 37 C. After washing three times with PBS, cells were harvested in lysis buffer (150 mM NaCl, 50 mM Tris·HCl, pH 7.5, 0.05% SDS, 1% Nonidet P-40, 1 mM benzamidine, 1 mM EDTA, 1 mM PMSF). The protein concentration of the extracts was measured using Bradford assay (Bio-Rad Laboratories, Inc.) with BSA as standard. An aliquot of lysate containing equal amounts of protein was precipitated with trichloroacetic acid and counted in a scintillation counter.

Western Blot and Immunoprecipitation
BRL-4 cells were grown to near confluence in 100-mm cultured dishes and treated as described in the figure legends. Thereafter, cells were washed three times with ice-cold PBS and harvested in 1 ml RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM DTT, and protease inhibitors). Aliquots from cleared lysates containing 30 µg protein were subjected to electrophoresis on SDS-PAGE. Separated proteins were electroblotted to polyvinylidene difluoride membranes (Millipore Corp. Bedford, MA). Detection of JAK2 and STAT5 was performed by Western blotting with anti-JAK2 antibody directed against murine JAK2 (Upstate Biotechnology, Inc. Lake Placid, NY) and monoclonal anti-STAT5 antibody (Transduction Laboratories, Inc., Lexington, KY) as described (2), respectively. JAK2 phosphorylation was determined by immunoprecipitation with antiphosphotyrosine monoclonal antibody (PY20, Transduction Laboratories, Inc.), followed by Western Blot with anti-JAK2. Phosphorylation of eIF-2{alpha} at Serine 51 was detected by Western blotting with a specific antibody against the phosphorylated form of the protein (Research Genetics, Inc., Huntsville, AL), as previously described (37). SOCS-1 levels were determined by immunoprecipitation of 800 µg of cell extracts with monoclonal 4H1 raised against the N-terminal domain of SOCS-1, followed by Western blot with the same antibody. SOCS-2 levels were estimated by immunoprecipitation of 1 mg of whole-cell extracts with 1 µg monoclonal antibody (4H8) raised against SOCS-2, followed by Western blot with monoclonal antibody 2D6 anti-SOCS-2. SOCS-3 levels were determined by inmunoprecipitation of 800 µg of cell extracts with rabbit anti-SOCS-3 polyclonal antiserum raised against the full-length murine SOCS-3, followed by Western blot with 1B2 anti-SOCS-3 monoclonal antibody raised against N-terminal domain of murine SOCS-3. All antibodies against SOCS proteins were a kind gift from Dr. Douglas J. Hilton.

Statistics
The data are presented as the mean ± SD (standard deviation of the mean). Statistical comparisons for each of the treated samples with the control sample (vehicle) were tested using the paired t test. The significance of differences among groups was determined using ANOVA with multiple comparisons among means, computed by using the Student-Newman-Keuls test. Statistical significance was reported if P < 0.05 was achieved.


    FOOTNOTES
 
This work was supported by Grant 13X-08556 from the Swedish Medical Research Council (to A.F.-M. and G. N.), Consejería de Educación del Gobierno Autónomo de Canarias 98–074 and MCYT PM98–033 (to L.F.), and the Swedish Institute (to E.R.-B.).

Abbreviations: BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (acetoxymethyl)ester; CHOP, C/EBP homology protein; CHX, cycloheximide; CIS, cytokine-induced SH2 protein; DRB, 5,6-dichloro-1-ß-D-ribofuranosylbenzimadole; DMSO, dimethylsulfoxide; DTT, dithiothreitol; eIF-2{alpha}, eukaryotic translation initiation factor-2{alpha}; GHR, GH receptor; JAK, Janus kinase; IFN, interferon; PMSF, phenylmethylsulfonyl fluoride; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription; UPR, unfolded protein response.

Received for publication April 19, 2001. Accepted for publication June 4, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ihle JN 1996 STATs: signal transducers and activators of transcription. Cell 84:331–334[Medline]
  2. Fernandez L, Flores-Morales A, Lahuna O, et al. 1998 Desensitization of the growth hormone-induced Janus kinase 2 (Jak 2)/signal transducer and activator of transcription 5 (Stat5)-signaling pathway requires protein synthesis and phospholipase C. Endocrinology 139:1815–1824[Abstract/Free Full Text]
  3. Starr R, Willson TA, Viney EM, et al. 1997 A family of cytokine-inducible inhibitors of signaling. Nature 387:917–921[CrossRef][Medline]
  4. Tollet-Egnell P, Flores-Morales A, Stavreus-Evers A, Sahlin L, Norstedt G 1999 Growth hormone regulation of SOCS-2, SOCS-3, and CIS messenger ribonucleic acid expression in the rat. Endocrinology 140:3693–3704[Abstract/Free Full Text]
  5. Ram PA, Waxman DJ 1999 SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms. J Biol Chem 274:35553–35561[Abstract/Free Full Text]
  6. Hansen JA, Lindberg K, Hilton DJ, Nielsen JH, Billestrup N 1999 Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins. Mol Endocrinol 13:1832–1843[Abstract/Free Full Text]
  7. Grimley PM, Fang H, Rui H, et al. 1998 Prolonged STAT1 activation related to the growth arrest of malignant lymphoma cells by interferon-{alpha}. Blood 91:3017–3024[Abstract/Free Full Text]
  8. Gebert CA, Park SH, Waxman DJ 1999 Down-regulation of liver JAK2-STAT5b signaling by the female plasma pattern of continuous growth hormone stimulation. Mol Endocrinol 13:213–227[Abstract/Free Full Text]
  9. Ferrigno P, Silver PA 1999 Regulated nuclear localization of stress-responsive factors: how the nuclear trafficking of protein kinases and transcription factors contributes to cell survival. Oncogene 18:6129–6134[CrossRef][Medline]
  10. Adler V, Yin Z, Tew KD, Ronai Z 1999 Role of redox potential and reactive oxygen species in stress signaling. Oncogene 18:6104–6111[CrossRef][Medline]
  11. Kaufman RJ 1999 Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 13:1211–1233[Free Full Text]
  12. Wang XZ, Lawson B, Brewer JW, et al. 1996 Signals from the stressed endoplasmic reticulum induce C/EBP- homologous protein (CHOP/GADD153). Mol Cell Biol 16:4273–4280[Abstract]
  13. Zinszner H, Kuroda M, Wang X, et al. 1998 CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev 12:982–995[Abstract/Free Full Text]
  14. Kumahara E, Ebihara T, Saffen D 1999 Protein kinase inhibitor H7 blocks the induction of immediate-early genes zif268 and c-fos by a mechanism unrelated to inhibition of protein kinase C but possibly related to inhibition of phosphorylation of RNA polymerase II. J Biol Chem 274:10430–10438[Abstract/Free Full Text]
  15. Kobayashi E, Nakano H, Morimoto M, Tamaoki T 1989 Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 159:548–553[Medline]
  16. Toullec D, Pianetti P, Coste H, et al. 1991 The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266:15771–15781[Abstract/Free Full Text]
  17. Stebbins CE, Kaelin Jr WG, Pavletich NP 1999 Structure of the VHL-ElonginC-ElonginB complex: implications for VHL tumor suppressor function. Science 284:455–4561[Abstract/Free Full Text]
  18. Narazaki M, Fujimoto M, Matsumoto T, et al. 1998 Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling. Proc Natl Acad Sci USA 95:13130–13134[Abstract/Free Full Text]
  19. Nicholson SE, Willson TA, Farley A, et al. 1999 Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. EMBO J 18:375–385[Abstract/Free Full Text]
  20. Yasukawa H, Misawa H, Sakamoto H, et al. 1999 The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J 18:1309–1320[Abstract/Free Full Text]
  21. Matsumoto A, Masuhara M, Mitsui K, et al. 1997 CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation. Blood 89:3148–3154[Abstract/Free Full Text]
  22. Auernhammer CJ, Bousquet C, Melmed S 1999 Autoregulation of pituitary corticotroph SOCS-3 expression: characterization of the murine SOCS-3 promoter. Proc Natl Acad Sci USA 96:6964–6969[Abstract/Free Full Text]
  23. Yankulov K, Yamashita K, Roy R, Egly JM, Bentley DL 1995 The transcriptional elongation inhibitor 5,6- dichloro-1-ß-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase. J Biol Chem 270:23922–23925[Abstract/Free Full Text]
  24. Dahmus ME 1996 Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J Biol Chem 271:19009–19012[Free Full Text]
  25. Ron D, Habener JF 1992 CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev 6:439–453[Abstract]
  26. Ubeda M, Vallejo M, Habener JF 1999 CHOP enhancement of gene transcription by interactions with Jun/Fos AP-1 complex proteins. Mol Cell Biol 19:7589–7599[Abstract/Free Full Text]
  27. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P 2000 Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258[Medline]
  28. Allevato G, Billestrup N, Goujon L, et al. 1995 Identification of phenylalanine 346 in the rat growth hormone receptor as being critical for ligand-mediated internalization and down-regulation. J Biol Chem 270:17210–17214[Abstract/Free Full Text]
  29. Stofega MR, Herrington J, Billestrup N, Carter-Su C 2000 Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol Endocrinol 14:1338–1350[Abstract/Free Full Text]
  30. Luo G, Yu-Lee L 2000 Stat5b inhibits NF{kappa}B-mediated signaling. Mol Endocrinol 14:114–123[Abstract/Free Full Text]
  31. Stoecklin E, Wissler M, Schaetzle D, Pfitzner E, Groner B 1999 Interactions in the transcriptional regulation exerted by Stat5 and by members of the steroid hormone receptor family. J Steroid Biochem Mol Biol 69:195–204[CrossRef][Medline]
  32. Ulloa L, Doody J, Massague J 1999 Inhibition of transforming growth factor-ß/SMAD signaling by the interferon-{gamma}/STAT pathway. Nature 397:710–713[CrossRef][Medline]
  33. Costoya JA, Finidori J, Moutoussamy S, Searis R, Devesa J, Arce VM 1999 Activation of growth hormone receptor delivers an antiapoptotic signal: evidence for a role of Akt in this pathway. Endocrinology 140:5937–5943[Abstract/Free Full Text]
  34. Osterziel KJ, Dietz R, Ranke MB 2000 Increased mortality associated with growth hormone treatment in critically ill adults. N Engl J Med 342:134–135[Free Full Text]
  35. Fort P, Marty L, Piechaczyk M, et al. 1985 Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res 13:1431–1442[Abstract]
  36. Sliva D, Wood TJ, Schindler C, Lobie PE, Norstedt G 1994 Growth hormone specifically regulates serine protease inhibitor gene transcription via {gamma}-activated sequence- like DNA elements. J Biol Chem 269:26208–26214[Abstract/Free Full Text]
  37. Frerichs KU, Smith CB, Brenner M, et al. 1998 Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation. Proc Natl Acad Sci USA 95:14511–14516[Abstract/Free Full Text]