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Originally published In Press as doi:10.1074/jbc.M513612200 on May 22, 2006

J. Biol. Chem., Vol. 281, Issue 31, 21594-21606, August 4, 2006
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Insulin Reverses Growth Hormone-induced Homologous Desensitization*

Jie Xu, Zhongyu Liu, Thomas L. Clemens, and Joseph L. Messina1

From the Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019

Received for publication, December 21, 2005 , and in revised form, April 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth hormone (GH) is secreted in a pulsatile pattern to promote body growth and metabolism. GH exerts its function by activating several signaling pathways, including JAK2/STAT and MEK/ERK. ERK1/2 activation by GH plays important roles in gene expression, cell proliferation, and growth. We previously reported that in rat H4IIE hepatoma cells after an initial GH exposure, a second GH exposure induces STAT5 phosphorylation but not ERK1/2 phosphorylation (Ji, S., Frank, S. J., and Messina, J. L. (2002) J. Biol. Chem. 277, 28384–28393). In this study the mechanisms underlying GH-induced homologous desensitization were investigated. A second GH exposure activated the signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. This correlated with recovery of GH receptor levels, but was insufficient for GH-induced phosphorylation of MEK1/2 and ERK1/2. Insulin restored the ability of a second GH exposure to induce phosphorylation of MEK1/2 and ERK1/2 without altering GH receptor levels or GH-induced phosphorylation/activation of JAK2 and Raf-1. GH and insulin synergized in promoting cell proliferation. Further investigation suggested that insulin increased the amount of MEK bound to KSR (kinase suppressor of Ras) and restored GH-induced tyrosine phosphorylation of KSR. Previous GH exposure also induced desensitization of STAT1 and STAT3 phosphorylation, but this desensitization was not reversed by insulin. Thus, insulin-regulated resensitization of GH signaling may be necessary to reset the complete response to GH after a normal, physiologic pulse of GH.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Binding of GH to its receptor (GHR)2 results in tyrosine phosphorylation of Janus-activating kinase (JAK) 2 and GHR (1, 2), leading to the activation of signal transducer and activator of transcription (STAT) proteins (2–4). GH also activates ERK1/2 via the Ras/Raf-1/MEK/ERK cascade (5–8). An activated GHR·JAK2 complex recruits and phosphorylates Shc, bringing Grb2 and Son of Sevenless (SOS) to the cell membrane, resulting in the formation of active GTP-bound Ras. Ras activation initiates the activation of Raf-1 kinase, which leads to the phosphorylation and activation of MEK1/2 and then ERK1/2. Activation of ERK1/2 plays an important role in GH-induced gene expression, cell proliferation and growth, and cross-talk between GH and other growth factor signaling pathways (9–13).

GH is secreted in a pulsatile fashion (14, 15). In young adult male rats, GH is released in ~1-h pulses with peak serum concentrations of 150–400 ng/ml and interpulse intervals of 2 h or more, where serum GH concentrations are negligible (16). In female rats, GH is secreted more frequently, resulting in the continuous presence of GH in the circulation, which peaks at 50–150 ng/ml (15–18). After stimulation by a GH pulse, an obligatory recovery period is required for a succeeding GH pulse to induce STAT5 phosphorylation (19–21). Reactivation of STAT5b by the masculine, but not the feminine, GH secretory pattern is important in transducing the sexually dimorphic pattern of GH secretion into sex-specific differences of liver gene expression and body growth (22–24).

We previously reported that, in rat H4IIE hepatoma cells, desensitization and resensitization of GH-induced STAT5 phosphorylation were strongly correlated with the reduction and recovery of GHR levels, respectively (25). Recovery of GHR, however, was insufficient for recovery of GH signaling via ERK1/2, suggesting that post-receptor mechanisms are involved. In the present study, the post-GHR mechanisms by which GH induced desensitization of ERK1/2 phosphorylation were investigated. After an initial GH treatment, activation of JAK2, Ras, and Raf-1 was induced by a second GH treatment and correlated with recovery of GHR levels. However, this was insufficient to activate MEK1/2 and ERK1/2. Because insulin plays an important role in regulating GH signaling and action, the effects of insulin on a second GH-induced signaling pathway were also investigated. Prior exposure to insulin restored the ability of a second GH treatment to induce MEK/ERK phosphorylation without changing GHR levels or GH-induced activation of JAK2 and Raf-1. GH and insulin synergized in promoting cell proliferation. Further investigation suggested that insulin increased the amount of MEK bound to KSR (kinase suppressor of Ras) and restored the second GH-induced tyrosine phosphorylation of KSR. A previous GH exposure also resulted in desensitized phosphorylation of STAT1 and STAT3. However, insulin did not restore a second GH-induced phosphorylation of STAT1 and STAT3. Our study suggests that in the liver signaling via the MEK/ERK pathway may become resensitized to repeated GH pulses in the presence of insulin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Bovine GH was purchased from the National Institutes of Health NIDDK National Hormone and Pituitary Program. Fetal bovine serum, calf serum, and horse serum were purchased from Invitrogen. Rat leukemia inhibitory factor was purchased from Chemicon (Temecula, CA). Other materials were purchased from Sigma and Fisher unless otherwise noted.

Antibodies—Antibodies for Ras, Raf-1, MEK1/2, KSR, and STAT5B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for phosphothreonine 202 and phosphotyrosine (Tyr(P)) 204-ERK1/2, phosphoserine (Ser(P)) 218/222 MEK1/2, Ser(P)-473-Akt, and Tyr(P)-694-STAT5B were purchased from Cell Signaling Technology (Beverly, MA). Antibodies for Tyr(P)-1007/1008-JAK2 and Ser(P)-338-Raf-1 and anti-phosphotyrosine antibody 4G10 were purchased from Upstate%20Biotechnology">Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse Ras antibody was purchased from Oncogene Research Products (Cambridge, MA). Anti-GHR (AL47) and anti-JAK2AL33 polyclonal serum were kindly provided by Dr. Stuart J. Frank (University of Alabama at Birmingham). Secondary antibodies, including horseradish peroxidase-linked anti-mouse antibody and anti-rabbit antibody were purchased from Cell Signaling Technology (Beverly, MA).

Cell Culture and Treatments—Rat H4IIE hepatoma cells (from ATCC) were cultured in Swim's medium (United States Biological, Swampscott, MA) supplemented with 10% serum (5% horse serum, 3% newborn calf serum, and 2% fetal calf serum) and 100 units/ml penicillin and 100 µg/ml streptomycin. At ~50% confluence cells were removed from serum and maintained in serum-free (and bovine serum albumin-free) medium for 48 h before the start of experimental treatments. For the washing experiments, medium was removed by aspiration after the first exposure to GH, and the cells were gently rinsed twice with phosphate-buffered saline followed by the addition of GH-free, serum-free medium.

Protein Extraction—Cells were collected in 0.5 ml of lysis buffer containing 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 20 mM KCl, 20% glycerol, 0.2 mM EDTA, 2 mM Na3VO4, 10 mM NaF, 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Cell lysates were gently rocked at 4 °C for 1 h and then centrifuged at 10,000 x g for 10 min, and the supernatants were stored at –80 °C until use. Cell lysate protein concentrations were measured by the Bio-Rad Protein Assay.

Immunoprecipitation and Immunoblotting—For immunoprecipitation, 1 mg of protein in lysis buffer was incubated with primary antibody overnight at 4 °C. Protein G-agarose (Fast flow, Amersham Biosciences) was then added, and incubations were continued for 4 h at 4°C. Both immunoprecipitated proteins and whole cell lysate protein (50 µg) were resolved by SDS-PAGE (Bio-Rad MiniProtean II system) and transferred to S&S Protran BA85 membranes (Keene, NH). The Western blots were blocked in Tris-buffered saline containing 0.05% Tween (TBST) and 5% milk, rinsed in TBST, incubated with the primary antibody for 2 h at room temperature or overnight at 4 °C, washed in TBST, and then incubated with horseradish peroxidase-labeled conventional secondary antibody for 1 h at room temperature. Proteins were visualized by ECL Plus (Amersham Biosciences) or Supersignal West Femto Maximum Sensitivity Substrate reagents (Pierce) and autoradiography.


Figure 1
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  		FIGURE 1.
GH-induced Tyr(P)-JAK2 after 1 h of GH pretreatment plus a wash-out period. H4IIE cells were treated with a first GH exposure (500 ng/ml; GH1, solid line) for 1 h or a second GH exposure (500 ng/ml; GH2, dashed line) after the first exposure to GH and 5 h in GH-free, serum-free medium, or vehicle for the indicated times. Western blot analysis was performed with antibodies for phosphotyrosine (PY)-JAK2. The blot was reprobed with total JAK2 antibody as a loading control. A, representative Western blots. The Tyr(P)-JAK2 band is indicated by the arrow. B, densitometric analysis of autoradiographs from three separate experiments to quantify Tyr(P)-JAK2 levels. The data are expressed as the mean ± S.E. The Tyr(P)-JAK2 levels after 7.5 min of 500 ng/ml GH1 were arbitrarily set to 100%.

 
Determination of GTP-bound Ras—According to the manufacturer's instructions, cells were lysed with magnesium-containing lysis buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% IGEPAL CA-630, 10% glycerol, and 25 mM NaF. 10 µg of Raf-1 Ras-binding domain-agarose (Upstate%20Biotechnology">Upstate Biotechnology) was added to 1 mg of cell lysate and gently rocked at 4 °C for 30 min. The agarose beads were washed 3 times with magnesium-containing lysis buffer and boiled in Laemmli sample buffer for 5 min, and the supernatant was then resolved by SDS-PAGE and transferred to Protran BA85 membrane. GTP-bound Ras was determined by immunoblotting with total Ras antibody.

BrdUrd Incorporation Assay—H4IIE cells (8 x 104) were seeded and cultured in 6-well plates. Cells were then treated as described in "Cell Culture and Treatment" (above), followed by the addition of BrdUrd (1 µM) to the medium. After 12 h, cells were stained using a BrdUrd assay kit (BD Pharmingen) according to the manufacturer's instructions. Samples were analyzed by flow cytometry for fluorescence on a FACScan.

Densitometric and Statistical Analysis—Chemiluminescent images of immunoblots were analyzed by scanning densitometry. Multiple exposures of each blot were used to obtain gray-scale images of each chemiluminescent band and were quantified with the Fluorchem FC imager system (Alpha Innotech, San Leandro, CA). All data were analyzed by analysis of variance using the InStat statistical program (version 3) by GraphPad Software, Inc. (San Diego, CA).


Figure 2
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  		FIGURE 2.
GH induced GTP-bound Ras after GH pretreatment and GH-free incubation. H4IIE cells were treated with GH1 or GH2 or vehicle for the indicated times as described in the legend for Fig. 1. Whole cell lysates were affinity-precipitated with Raf-1 Ras-binding domain-agarose and then subjected to Western blot analysis by total Ras antibody to detect the activated Ras (upper panel), or whole cell lysate was directly resolved to SDS-PAGE and transferred to the membrane followed by probing with total Ras antibody as a loading control (lower panel). A, representative Western blots. B, densitometric analysis of autoradiographs from three separate experiments to quantify activated Ras (Ras-GTP) levels. The data are expressed as mean ± S.E. The Ras-GTP levels after 7.5 min of 500 ng/ml GH1 were arbitrarily set to 100%.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GH Induced Tyrosine Phosphorylation of JAK2 after 1-h GH Pretreatment Plus a Wash-out Period—In young adult male rats, GH is secreted in ~1-h pulses with ~3 h of inter-pulse intervals of non-detectable GH (16). We previously reported that 1) a short exposure to GH rapidly reduced GHR levels, which resulted in desensitization of STAT5 and ERK1/2 phosphorylation, 2) in the absence of GH for 3–6 h, GHR levels and GH-induced tyrosine phosphorylation of STAT5 (Tyr(P)-STAT5) recovered to 65–75% that in cells not pretreated with GH, and 3) unlike STAT5 phosphorylation, the ability of GH to activate the MEK/ERK pathway did not recover even after prolonged (16 h) incubation in the absence of GH (25). To allow the cells to maximally recover GHR levels and GH sensitivity and to mimic a minimum 3–4-h interpulse interval in vivo, we chose 5 h as the GH-free, serum-free incubation time. Therefore, to investigate the mechanisms by which GH induced desensitization of the ERK1/2 phosphorylation, H4IIE cells were pretreated with GH for 1 h, then incubated in GH-free, serum-free medium for 5 h followed by a second GH treatment.

After GH binding to GHR, JAK2 is phosphorylated and transphosphorylates GHR. We first characterized the effects of GH pretreatment on GH-induced tyrosine phosphorylation of JAK2 (Tyr(P)-JAK2). GH significantly induced Tyr(P)-JAK2 by 5 min, reaching a peak by 7.5 min, and decreasing after 10 min (Fig. 1, A and B). When H4IIE cells were pretreated with GH for 1 h followed by 5 h in GH-free, serum-free medium, the second GH-induced Tyr(P)-JAK2 was maximally 70–75% that induced by GH before any pretreatment (Fig. 1, A and B), correlating well with the recovery of GHR levels that we previously reported (25). This suggests that, after the initial exposure to GH, the second GH exposure was able to induce Tyr(P)-JAK2. Total JAK2 protein levels were not altered after any of the GH exposures or the washing regimens (Fig. 1A).


Figure 3
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  		FIGURE 3.
A second GH treatment induced serine 338 phosphorylation of Raf-1. H4IIE cells were treated with GH1 or GH2 or vehicle for the indicated times as described in the legend for Fig. 1. Western blot analysis was performed with phosphoserine (PS) 338-Raf-1 antibody followed by stripping and reprobing with total Raf-1 antibody. A, representative Western blots. B, densitometric analysis of autoradiographs from three separate experiments to quantify Ser(P)-338-Raf-1 levels. The data are expressed as the mean ± S.E. The Ser(P)-338 levels after 10 min of 500 ng/ml GH1 were arbitrarily set to 100%.

 
GH Induced GTP-bound Ras after GH Pretreatment and GH-free Incubation—Activation of the GHR·JAK2 complex activates Shc, which recruits Grb2·SOS to the cell membrane, where SOS can convert Ras from the GDP-bound (inactive) to the GTP-bound (active) form. Whether a second GH exposure induced Ras activation was next examined. GTP-bound Ras was induced by GH at 5 min, with a maximal increase by 7.5 min and decreased significantly by 30 min (Fig. 2, A and B). After GH pretreatment and GH-free incubation for 5 h, GH-induced GTP-bound Ras was 70–75% that induced by the initial GH treatment (Fig. 2, A and B), suggesting that the ability of the second GH treatment to activate Ras was not altered by previous GH treatment and correlated with the recovery of GHR levels and GH-induced Tyr(P)-JAK2.

To control for equal loading and to determine whether the effects on GTP-bound Ras were dependent on changes in the total amounts of Ras protein, whole cell lysates were directly subjected to Western blot analysis by total Ras antibody. The total cellular amount of Ras protein was not significantly affected by GH pretreatment (Fig. 2A).


Figure 4
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  		FIGURE 4.
Phosphorylation of MEK1/2 and ERK1/2 induced by a second GH treatment was not correlated with activation of the JAK2/Ras/Raf-1 pathway. H4IIE cells were treated with GH1 or GH2 or vehicle for the indicated times as described in the legend for Fig. 1. Western blot analysis was performed with antibodies for phosphoserine (PS)-MEK1/2 or P-ERK1/2 followed by stripping and reprobing with antibodies for total MEK1 or ERK1/2. A and C, representative Western blots. The Ser(P)-MEK band is indicated by the arrow. B and D, densitometric analysis of autoradiographs from three separate experiments to quantify Ser(P)-MEK1/2 and P-ERK1/2 levels. The data are expressed as the mean ± S.E. The Ser(P)-MEK1/2 (C) or P-ERK1/2 (D) levels after 10 min of 500 ng/ml GH1 were arbitrarily set to 100%. E, maximal activation of signaling intermediates induced by GH2. The data are expressed as the mean ± S.E. The Tyr(P)-JAK2 or Ras-GTP levels after 7.5 min of 500 ng/ml GH1 were arbitrarily set to 100%. The Ser(P)-Raf-1, Ser(P)-MEK1/2, or P-ERK1/2 levels after 10 min of 500 ng/ml GH1 were arbitrarily set to 100%. The GHR levels and GH-induced Tyr(P)-STAT5 were obtained from our previous report (25).

 
A Second GH Treatment Induced Serine 338 Phosphorylation of Raf-1—We next examined the ability of a second GH treatment to activate Raf-1 immediately downstream of Ras. Phosphorylation of serine 338 on Raf-1 (Ser(P)-338-Raf-1) is required for Raf-1 activation (26). In H4IIE cells, the first GH treatment induced Ser(P)-338-Raf-1 maximally by 10 min and decreased thereafter (Fig. 3, A and B). The peak of Ser(P)-338-Raf-1 induced by the second GH exposure at 10 min was about 55% that induced by the first GH (Fig. 3B). However, before the second GH treatment, the basal levels of Ser(P)-338-Raf-1 were significantly reduced and were only about 70% that of the basal values before the first GH application (Fig. 3B, 0 time point, dashed line versus solid line). Thus, when expressing the induction of Ser(P)-338-Raf-1 compared with the reduced basal value, there was approximately a 1.6-fold induction by the second GH treatment. This 1.6-fold induction of Ser(P)-338-Raf-1 is less than the ~2.1-fold induction by the first GH treatment. Therefore, the -fold induction of Ser(P)-338-Raf-1 by the second GH treatment was only 76% that of the first GH treatment. Total Raf-1 protein abundance was not changed after the second versus the first GH exposure (Fig. 3A). Collectively, the data from Figs. 1, 2, 3 suggest that after the previous GH treatment the second GH treatment was able to activate JAK2, Ras, and Raf-1, and their activation was correlated well with the recovery of GHR levels (25).

Phosphorylation of MEK1/2 and ERK1/2 Induced by a Second GH Treatment Was Not Correlated with Activation of the JAK2/Ras/Raf-1 Pathway—When H4IIE cells were pretreated with GH for 1 h followed by 5 h in GH-free, serum-free media, the GH-induced phosphorylation of MEK1/2 (Fig. 4, A and B) and ERK1/2 (Fig. 4, C and D) was greatly diminished compared with that induced by GH before any pretreatment. The first GH treatment induced serine phosphorylation of MEK1/2 (Ser(P)-MEK1/2) maximally by 10 min and decreased thereafter (Fig. 4, A and B). The Ser(P)-MEK1/2 induced by the second GH treatment was 27% or less than that induced by the first exposure (Fig. 4, A and B). The second GH treatment did not achieve greater than 10–15% of the P-ERK1/2 induced by the first GH treatment (Fig. 4, C and D), consistent with our previous findings (25).

Consistent with our previous study, GHR levels and GH-induced Tyr(P)-STAT5 recovered by 65–70% (Fig. 4E (25)). In the present study the maximal activation of signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1, induced by the second GH treatment was about 70–75% that induced by the first GH treatment (Fig. 4E), possibly resulting from the recovered GHR levels and similar to GH-induced Tyr(P)-STAT5. However, the ability of the second GH treatment to activate MEK/ERK was only 15–25% that induced by the first GH treatment, suggesting a disconnect of signal transduction from Raf-1 to MEK1/2 (Fig. 4E).


Figure 5
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  		FIGURE 5.
GH induced desensitization of STAT1 and STAT3 phosphorylation. H4IIE cells were treated with GH1, GH2, leukemia inhibitory factor ((LIF); 50 ng/ml), or vehicle for the indicated times as described in the legend for Fig. 1. Western blot analysis was performed with antibodies for Tyr(P) (PY)-STAT1 or Tyr(P)-STAT3 followed by stripping and reprobing with antibodies for total STAT1 or STAT3. A and C, representative Western blots. B and D, densitometric analysis of autoradiographs from three separate experiments to quantify Tyr(P)-STAT1 and Tyr(P)-STAT3 levels. The data are expressed as the mean ± S.E. The Tyr(P)-STAT1 (B) or P-STAT3 (D) levels were shown as -fold change compared with control (untreated), which were arbitrarily set to 1.

 
GH Induced Desensitization of STAT1 and STAT3 Phosphorylation—GH can activate two other STATs, STAT1 and STAT3, in addition to STAT5 in H4IIE cells (27). A second GH treatment can induce Tyr(P)-STAT5 in H4IIE cells (25), but it was not known whether STAT1 and STAT3 can be activated by a second GH treatment. Therefore, the ability of a second GH treatment to activate/phosphorylate these two STATs was also examined. The first GH treatment induced a maximal 15-fold tyrosine phosphorylation of STAT1 (Tyr(P)-STAT1) (Fig. 5, A and B) and 6-fold of Tyr(P)-STAT3 (Fig. 5, C and D). Consistent with our previous report (25), Tyr(P)-STAT5 was induced by the second GH treatment (Fig. 5A, row 2) but to a lesser degree due to the recovery of GHR by only ~65–75% at this time point. Unlike Tyr(P)-STAT5, little Tyr(P)-STAT1 was induced by the second GH treatment (Fig. 5, A, row 1, and B). Basal Tyr(P)-STAT3 levels were elevated after 5 h of incubation in the absence of GH after the initial GH treatment (Fig. 5, C and D, dashed line, 0 time point). On the top of this elevated basal Tyr(P)-STAT3 levels, there was no significant induction of Tyr(P)-STAT3 observed in cells exposed to the second GH treatment (Fig. 5, C and D). However, leukemia inhibitory factor, another JAK activator, was still able to fully stimulate phosphorylation of STAT1 and STAT3 after removal of GH and incubation in GH-free, serum-free medium (Fig. 5, A and C, rows 1). Therefore, similar to the MEK/ERK pathway, but unlike STAT5, the first GH treatment led to desensitization of the ability of a second GH treatment to induce Tyr(P)-STAT1 and Tyr(P)-STAT3.

Insulin Restored a Second GH-induced ERK1/2 Phosphorylation—In adult male rats, insulin normally peaks in the middle of GH interpulse interval (28). Insulin is known to maintain GHR levels in vivo, which may be related to the growth-promoting effects of insulin (29–33). But the possible effects of insulin on repeated GH-induced signaling have yet to be studied. Therefore, we examined whether insulin affected GH-induced signaling pathways after a second exposure to GH. To mimic the in vivo GH and insulin secretory pattern, H4IIE cells were exposed to GH for 1 h, incubated in the absence of GH for 3 h, and then treated with insulin for 2 h followed by a second GH treatment (the time between two GH treatments was held at 5 h).

As shown in Fig. 4, in the absence of GH for 5 h after the first GH treatment, a second GH-induced P-ERK1/2 was barely observed compared with the first GH treatment (Fig. 6, A, row 1, lanes 2 versus 4, and B). However, with insulin pretreatment for 2 h, the second GH treatment significantly induced P-ERK1/2, reaching ~70% that induced by the first GH treatment (Fig. 6, A, row 1, compare lanes 2 and 4 to lane 7, and B). This induction was much greater than the sum of P-ERK1/2 induced by the second GH for 10 min without insulin treatment (Fig. 6A, row 1, lane 4) plus the P-ERK1/2 induced by insulin treatment for 2 h (Fig. 6A, row 1, lane 6).

Because the GH interpulse interval in male rats is about 3 h, we then asked whether insulin had similar effects when given in the middle of a 3-h GH interpulse interval. Similarly, insulin pretreatment for 1 h also significantly restored the ability of a second GH treatment to induce P-ERK1/2 (Fig. 6, C and D). Therefore, we chose 3-h GH interpulse interval and 1-h insulin treatment in the following investigation of the mechanisms by which insulin restored GH-induced P-ERK1/2.

Because insulin also activates ERK1/2, it could not be excluded that the second GH exposure potentiates insulin activation via ERK1/2. The other major signaling pathway activated by insulin is the PI3K/Akt pathway; therefore, the effects of the second GH treatment on insulin-induced phosphorylation of Akt was also investigated. GH-induced P-Akt in H4IIE cells was weak (Figs. 6, A and C, rows 3, lanes 2). Insulin-induced P-Akt remained highly elevated after insulin treatment for 2 or 1 h (Figs. 6, A and C, rows 3, lanes 6), and the second addition of GH did not affect insulin-induced P-Akt levels (Figs. 6, A and C, rows 3, lanes 7). Our previous work (34) also suggests that the time course of ERK1/2 activation by insulin and by GH in H4IIE cells are different. GH induced P-ERK1/2 with a maximal induction by 10 min and returned toward basal levels after 20 min. In contrast, insulin induced P-ERK1/2 peaks by 5 min, which quickly decreased after 5 min, reaching a secondary plateau level and returning toward basal levels between 60 and 120 min. In the present study, after the first GH exposure, incubation in serum-free medium plus insulin treatment, and then a second exposure to GH, P-ERK1/2 peaked at 10 min and then returned toward basal levels after 20 min (data not shown). Therefore, the resensitization of P-ERK1/2 after the second GH addition occurred over the normal GH time course not the insulin time course (quicker and more prolonged). Our data strongly suggest that insulin re-sensitizes GH-induced ERK1/2 signaling.


Figure 6
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  		FIGURE 6.
Insulin restored a second GH-induced ERK1/2 phosphorylation. H4IIE cells were treated with GH1 or vehicle for the indicated times, washed, and incubated in GH-free, serum-free medium for 5 or 3 h. They were then treated without or with 10 nM insulin (Ins) for 2 h or 1 h and followed by GH2. Western blot analysis was performed with antibody for P-ERK1/2 and P-Akt followed by stripping and reprobing with antibody for total ERK1/2 and total Akt as loading controls. A and C, representative Western blots. B and D, densitometric analysis of autoradiographs from three or more separate experiments were performed to quantify P-ERK1/2 levels. The data are expressed as the mean ± S.E. The P-ERK1/2 levels in untreated samples were arbitrarily set to 1. * or $, p < 0.05; ##, p < 0.01; NS, not significantly different.

 
Insulin Did Not Affect a Second GH-induced Tyrosine Phosphorylation of STAT5, STAT1, and STAT3—Whether insulin affected the ability of a second GH treatment to activate STAT protein phosphorylation was then determined. The first GH treatment resulted in a 20–25-fold induction of Tyr(P)-STAT5 (Fig. 7, A, row 1, lanes 1 versus 2, and B). Consistent with our previous findings (25), after 3 h of GH-free, serum-free incubation, the ability of a second GH treatment to induce Tyr(P)-STAT5 recovered by about 60–70% of that induced by the initial GH treatment (Fig. 7, A, row 1, lanes 2 versus 4, and B). Important for this work, the Tyr(P)-STAT5 induced by a second GH treatment was not significantly altered by insulin treatment (Fig. 7A, row 1, lanes 4 versus 7, and B).

As described in Fig. 5, a previous GH treatment also resulted in a desensitization of Tyr(P)-STAT1 and Tyr(P)-STAT3 (Fig. 7A, rows 2 and 3, lanes 2 versus 4). In contrast with ERK1/2, however, insulin did not restore the ability of a second GH treatment to induce Tyr(P)-STAT1 or Tyr(P)-STAT3 (Fig. 7A, rows 2 and 3, lanes 4 versus 7). Therefore, the effects of insulin on a second GH-induced signaling were selective to the ERK1/2 pathway, but not to STATs including STAT5, STAT1, or STAT3.


Figure 7
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  		FIGURE 7.
Insulin did not affect a second GH-induced tyrosine phosphorylation of STAT1, STAT3, or STAT5. Western blot analysis was performed as described in the legend for Fig. 6, except for the use of antibodies for Tyr(P)(PY)-STAT1, Tyr(P)-STAT3, Tyr(P)-STAT5, total STAT1, and total STAT3. A, representative Western blots of at least 3 separate experiments with insulin pretreatment for 60 min. B, densitometric analysis of autoradiographs from at least three separate experiments were performed to quantify Tyr(P)-STAT5 levels. The data are expressed as mean ± S.E. The Tyr(P)-STAT5 levels in untreated samples were arbitrarily set to 1. NS, not significantly different.

 
Insulin Did Not Alter GHR Levels or a Second GH-induced Activation of JAK2 and Raf-1—As previously reported (25), GH treatment for 10 min resulted in a reduction of GHR levels by ~50% (Fig. 8, A, lanes 1 versus 2, and B). After 3 h in GH-free, serum-free medium, the GHR levels recovered to 60–70% that in cells untreated with GH (Fig. 8, A, lanes 1 versus 3, and B (25)), and a second GH treatment further reduced GHR levels by about 35% (Fig. 8, A, lanes 3 versus 4, and B (25)). Insulin treatment for 1 h did not alter the GHR protein abundance (Fig. 8, A, lanes 3 and 5 versus 6, and B) nor alter the further reduction of GHR by the second GH exposure (Fig. 8, A, lanes 4 versus 7, and B).

As described in Fig. 1, Tyr(P)-JAK2 induced by a second GH treatment peaked at 5 min (Fig. 8, C, row 1, lanes 1–3, and D). After 3 h in GH-free, serum-free medium, Tyr(P)-JAK2 induced by the second GH treatment at 5 and 10 min was between 60 and 75% that induced by the first GH (Fig. 8, C, row 1, lanes 2 versus 5 and lanes 3 versus 6, and D), correlating with the recovery of GHR levels. There was no significant effects of insulin on a second GH-induced Tyr(P)-JAK2 (Fig. 8, C, row 1, lanes 7–9 and D). It was also found that insulin pretreatment did not affect the second GH-induced Raf-1 kinase activity (data not shown), determined by an in vitro kinase assay. Therefore, the resensitization of GH-induced ERK1/2 phosphorylation by insulin was not due to a change of GHR levels or activation of JAK2 and Raf-1.


Figure 8
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  		FIGURE 8.
Effects of insulin on GHR levels and a second GH-induced Tyr(P)-JAK2. Western blot analysis was performed as described in the legend for Fig. 6 except for the use of antibodies for GHR, Tyr(P)-JAK2, and total JAK2. A and C, representative Western blots. B and D, densitometric analysis of autoradiographs from at least three separate experiments were performed to quantify GHR and Tyr(P) (PY)-JAK2 levels. The data are expressed as the mean ± S.E. The GHR and Tyr(P)-JAK2 levels in untreated samples were arbitrarily set to 1. Ins, insulin. NS, not significantly different.

 


Figure 9
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  		FIGURE 9.
Insulin restored the ability of a second GH treatment to induce MEK1/2 phosphorylation. Western blot analysis was performed as described in the legend for Fig. 6 except for the use of antibodies for Ser(P) (PS)-MEK1/2 and total MEK1/2. A, representative Western blots. B, densitometric analysis of autoradiographs from at least three separate experiments was performed to quantify Ser(P)-MEK1/2 levels. The data are expressed as the mean ± S.E. The Ser(P)-MEK1/2 levels in untreated samples were arbitrarily set to 1. Ins, insulin. NS, not significantly different; # or $, p < 0.05.

 
Insulin Restored the Ability of a Second GH Treatment to Induce MEK1/2 Phosphorylation—We next examined whether a second GH after insulin treatment was able to induce Ser(P)-MEK1/2. When H4IIE cells were treated with GH for 1 h followed by 3 h of GH-free incubation, the second GH-induced Ser(P)-MEK1/2 was greatly diminished compared with the first GH (Fig. 9A, row 1, lanes 2 versus 4, and B (25)), as was basal Ser(P)-MEK1/2 (Fig. 9, A, row 1, lanes 3 versus 1, and B). After insulin treatment for 1 h, cellular Ser(P)-MEK1/2 approached control levels (Fig. 9, A, row 1, lanes 6 versus 1, and B), and the second GH after insulin treatment significantly induced Ser(P)-MEK1/2 (Fig. 9, A, row 1, lanes 6 versus 7, and B), suggesting that insulin reversed GH-induced desensitization of Ser(P)-MEK1/2 and resulted in a resensitization of the MEK/ERK pathway in response to a GH treatment.

GH and Insulin Synergized in Promoting Cell Proliferation—It was then examined whether the combined action of insulin and a second GH exposure on the ERK1/2 pathway led to any effects in a biological response to GH. GH is a known hepatic mitogen, and liver size is increased compared with other organs and body size in response to GH (35–37). When cells were treated with one GH treatment (G1), two repeated GH treatments (G1/G2), or one GH treatment followed by insulin (G1/I), the cell proliferation rate was similar to control cells (30% of the cells were BrdUrd-positive), suggesting a lack of effects of these treatments on cell proliferation (Fig. 10). However, two GH treatments in the presence of insulin (G1/I/G2) significantly increased cell proliferation (40% of the cells were BrdUrd-positive) compared with control cells. As a positive control, 70% of the cells were BrdUrd-positive when grown in complete medium. Therefore, a GH treatment was capable of increasing liver cell proliferation when and only when cells had been pretreated with insulin.


Figure 10
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  		FIGURE 10.
GH and insulin synergized in promoting cell proliferation. H4IIE cells were untreated (CTL) or treated with the following. G1, the first GH treatment for 1 h; G1/G2, the first GH treatment for 1 h, incubation in serum-free and GH-free medium for 3 h followed by the second GH treatment; G1/I, the first GH treatment for 1 h, incubation in serum-free, and GH-free medium for 2 h followed by insulin treatment; G1/I/G2, the first GH treatment for 1 h, incubation in serum-free, GH-free medium for 2 h, then insulin treatment for 1 h followed by the second GH treatment; CM, complete medium. Cell proliferation was examined by the BrdUrd incorporation assay. The data are expressed as the mean ± S.E. *, p < 0.05; ***; p < 0.001.

 
Insulin Increased the Amount of MEK Bound to KSR and Restored a Second GH-induced Tyrosine Phosphorylation of KSR—Because insulin restored the ability of a second GH treatment to activate MEK/ERK without affecting GHR levels and activation of JAK2 and Raf-1, we next investigated the underlying mechanisms. KSR, a scaffolding protein for the Ras/Raf-1/MEK/ERK pathway, brings Raf-1 and MEK closer to each other and facilitates the activation of MEK by Raf-1 (38–41). The interaction between Raf-1 or MEK and KSR was examined by co-immunoprecipitation assay. Compared with cells untreated with insulin, the amount of KSR immunoprecipitated with KSR antibody consistently decreased after 1 h of insulin treatment in the absence or presence of a second GH (Fig. 11A, row 1, lanes 1–5 versus lanes 6 and 7). However, when whole cell lysates were directly subjected to Western blot analysis, the total cellular KSR abundance was not changed (Fig. 11A, row 5), and there was also no change in total STAT1 protein, which was used as a loading control (Fig. 11A, row 6). This suggests that pretreatment with insulin, possibly by inducing a conformational change or by some other mechanism, modestly decreases the ability of KSR to be immunoprecipitated.


Figure 11
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  		FIGURE 11.
Insulin increased the amount of MEK bound to KSR and restored a second GH-induced tyrosine phosphorylation of KSR. H4IIE cells were treated as described in the legend for Fig. 6. Whole cell lysates were immunoprecipitated (IP) with anti-KSR antibody and then subjected to Western blot (WB) analysis by antibodies for KSR, Raf-1, MEK1, and tyrosine phosphorylation (PY)(rows 1–4, respectively), or whole cell lysates were directly subjected to Western blot analysis with antibodies for KSR and STAT1 (rows 5 and 6, respectively). A, representative Western blots of at least three separate experiments. HC, heavy chain of the anti-KSR antibody used for immunoprecipitation. B–D, densitometric analysis of autoradiographs from three separate experiments was performed to quantify the ratio of KSR-associated Raf-1 to KSR, the ratio of KSR-associated MEK to Raf-1, and the ratio of Tyr(P)-KSR to KSR. The data are expressedasthe mean ± S.E. The levels in untreated samples were arbitrarily set to 1. Ins, insulin. * or #, p < 0.05; ### or $$$, p < 0.001; NS, not significantly different.

 
The amount of Raf-1 or MEK bound to KSR was not significantly affected by the first GH treatment (Fig. 11A, rows 2 and 3, lanes 2 versus 1), whereas the amount of Raf-1 bound to KSR significantly increased after the first GH treatment and incubation in serum-free, GH-free medium (SFM) for 3 or 2 h, respectively (Fig. 11A, row 2, lanes 3 and 5). After insulin pretreatment for 1 h without or with the second GH treatment, the amount of MEK co-immunoprecipitated with KSR actually increased when normalized to the reduced amount of immunoprecipitated KSR (Fig. 11, A, row 3, lanes 6 and 7, and C). For this normalization, the amount of Raf-1 or MEK bound to KSR was divided by the levels of KSR immunoprecipitated on the same blot. Although insulin significantly increased KSR-bound MEK, it did not affect the ratio of KSR-bound Raf-1 versus KSR (Fig. 11B). The second GH treatment did not alter the amount of Raf-1 bound to KSR, and this was true when the cells were pretreated with insulin or not (Fig. 11B). The second GH after insulin pretreatment did not alter the KSR-bound MEK that was increased by insulin pretreatment (Fig. 11C). Taken together, this suggests that, after insulin pretreatment, the second GH treatment induced activation of MEK and ERK without further increasing the ability of Raf-1 or MEK to associate with KSR.

Studies suggest a potential role of KSR phosphorylation in its function (42, 43). To ask whether GH or insulin treatment could regulate KSR function, the effects of GH or insulin on the phosphorylation status of KSR were examined. The levels of Tyr(P)-KSR were also normalized to the levels of immunoprecipitated KSR. The first GH treatment induced tyrosine phosphorylation (Tyr(P)) of KSR by 8-fold, whereas a second GH treatment induced only a 4-fold increase of Tyr(P)-KSR (Fig. 11, A, row 4, lanes 2 and 4, respectively, and D). After incubation in GH-free, serum-free medium for 2 or 3 h, Tyr(P)-KSR levels returned to basal values. After insulin treatment for 1 h, Tyr(P)-KSR levels were elevated ~4-fold, an effect of insulin. However, a second exposure to GH after insulin treatment induced a large, 14-fold increase of Tyr(P)-KSR (Fig. 11D). This large induction of Tyr(P)-KSR above the induction by insulin suggests that the insulin pretreatment restored the ability of a second GH to robustly induce Tyr(P)-KSR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We previously reported that, after an initial GH treatment, ERK1/2 phosphorylation becomes desensitized to a second GH treatment, possibly due to post-receptor mechanisms (25). In human IM-9 lymphocytes, two continuous GH pulses without an interpulse interval resulted in a desensitized tyrosine phosphorylation of JAK2 (44). However, in the present study of rat H4IIE hepatoma cells, where there was a GH-free interval between GH treatments, a second GH was able to activate JAK2. The extent of JAK2 phosphorylation correlated well with the recovery of GHR levels and GH-induced STAT5 phosphorylation. This indicates that the absence of GH between two GH applications is required for the recovery of JAK2/STAT5 phosphorylation. In agreement with this, in male rats, even a nominal GH pulse during the usual GH-devoid interpulse interval of the masculine GH secretory profile results in a complete repression of STAT5-dependent male-specific expression of CYP2C11, CYP3A2, and CYP2A2 (45).

The recovery of JAK2 activation after the GH-free period led to a similar recovery of activation of STAT5, Ras, and Raf-1 but not MEK/ERK. This suggests that GH-induced homologous desensitization of the MEK/ERK pathway is due to a defect in signal transduction from Raf-1 to MEK1/2. Several recent studies have focused on the mechanisms underlying homologous desensitization of intracellular signaling. Treatment with insulin or growth factors results in homologous desensitization of the Ras/Raf-1/MEK/ERK pathway, and this desensitization can occur at receptor or post-receptor levels. Ras desensitization induced by epidermal growth factor or platelet-derived growth factor is due to the loss of their functional cell surface receptors, whereas insulin-induced desensitization of Ras results from the dissociation of SOS from Grb2 (46). Our data indicate that GH-induced desensitization of the ERK1/2 signaling pathway is not caused by the change of GH receptor levels or Ras desensitization but by a defective signal transduction from Raf-1 to MEK1/2, thus suggesting a novel post-receptor mechanism of homologous desensitization.

Although STAT5 is the predominant STAT utilized by GH to regulate gene transcription, STAT1 and STAT3 are also involved in GH-induced gene expression (19, 47–50). We previously reported that GH activates all three STATs in H4IIE cells, STAT1, STAT3, and STAT5 (27). In the present study, previous GH treatment desensitized the ability of a second GH to induce phosphorylation of STAT1 and STAT3 but not STAT5, which is consistent with in vivo findings (19). After a GH pulse, activation of STAT5 in rat liver is fully restored when a second GH pulse is given 4 h later. The low steady-state level of tyrosine-phosphorylated STAT1 and STAT3 in male rat liver may be due to desensitized STAT1 and STAT3 (19).

The mechanisms underlying the differential desensitization of STAT1, STAT3, and STAT5 are unknown but may involve an activation of negative regulators specific for STAT1 and STAT3 proteins or their phosphotyrosine-binding sites on the GHR·JAK2 complex. The phosphotyrosine-binding sites on GHR·JAK2 complex used by STAT5 are different from those of STAT1 and STAT3. Binding to phosphorylated tyrosines in the cytoplasmic domain of GHR is required for GH-induced STAT5 activation (51, 52) but not for GH to activate STAT1 and STAT3 (53–57). This is further supported by a C-terminal GHR mutation recently identified in a patient with severe short stature and biochemical features of GH insensitivity. This GHR mutant results in impaired STAT5 but normal STAT3 signaling (58). Furthermore, JAK2 but not GHR, contains STAT3 association motifs and a STAT1-like association motif (59, 60), suggesting that JAK2 not only phosphorylates STAT1 and STAT3 but also provides binding sites for their association with the GHR·JAK2 complex.

Growth hormone-activated STAT signaling is also negatively regulated by a number of proteins, including the suppressors of cytokines signaling (SOCS) (SOCS1–7 and cytokine-inducible SH2 protein (CIS)) and protein-tyrosine phosphatases. Of the eight known members of the SOCS family, GH induces expression of SOCS-1, -2, and -3 and CIS in rat liver (61–63). SOCS/CIS proteins bind to GHR or JAK2 via their phosphotyrosine-binding SH2 domains and inhibit the kinase activity of JAK2 or compete with STAT5 for receptor docking sites (63, 64). In addition, the SH2 domain-containing phosphatases 1 and 2 have been implicated in dephosphorylation of tyrosines on GHR or JAK2 (65–69). However, negative regulators specific for STAT1 or STAT3, but not for STAT5, have not been reported. Therefore, the putative role of SOCS and phosphatases in STAT1/STAT3 desensitization, but not STAT5 desensitization, can only be hypothesized.

Insulin plays an important role in regulating GH signaling and action, which may partially explain the insulin growth-promoting effect. In vivo, insulin appears to be necessary for normal liver GH responsiveness, in part by maintaining liver GHR levels (29–32). It was reported nearly 20 years ago that insulin peaks in the middle of a GH interpulse interval in male rats (28), but the possible effects of insulin on repeated GH pulse-induced signaling have not been studied. In the present study we found that insulin selectively restored the ability of a second GH treatment to induce MEK/ERK phosphorylation. Liver ERK1/2 can be activated by a single GH injection (70), but it is not known whether it can be activated by multiple GH pulses or whether the steady-state levels of phosphorylated ERK1/2 is high in intact male rat liver. The current study suggests that multiple GH pulses may be able to reactivate ERK1/2 in vivo after a peak of insulin secretion.

The synergistic effects of GH and insulin in promoting cell proliferation suggest that repeated GH pulse-induced ERK1/2 activation in the presence of insulin may be necessary for liver growth. GH transgenic mice demonstrate significantly increased growth of internal organs, but the liver in particular is enlarged compared with other organs (35, 37), and there are life-long high levels of hepatocellular replication in this model (36). GH also plays a critical role in liver regeneration after hepatectomy (71). It is not clear which pathway is responsible for the action of GH in hepatocyte proliferation, but the ERK1/2 pathway is a potential candidate in the growth response to many growth factors including GH, given that activation of the ERK1/2 pathway is frequently associated with cell proliferation. Furthermore, the decreased growth in children with type 1 diabetes has been substantially improved by daily multiple-dose insulin treatment compared with single-dose insulin treatment (72–74). It is possible that improvements in growth are related to, in part, the effects of insulin on GH-induced ERK1/2 activation.

Although insulin restored a second GH-induced MEK/ERK phosphorylation, it did not affect GHR levels and GH-induced phosphorylation of JAK2 and STAT5. Tyrosine phosphorylation of STAT1 and STAT3 remained insensitive to a second GH treatment in H4IIE cells no matter whether insulin was present or not. This is consistent with the in vivo findings that liver tyrosine-phosphorylated STAT1 and STAT3 levels in intact male rats are as low as in female rats (19). The low levels of tyrosine-phosphorylated STAT1 and STAT3 in male liver may be due to the desensitization of STAT1 and STAT3 after a previous exposure to GH and an inability to become resensitized during the low GH concentration interpeak interval. This suggests that STAT1 and STAT3 are not primary players in the sex-specific patterns of liver gene expression and body growth because these two STATs, unlike STAT5, may not be reactivable by the masculine GH secretory pattern. In addition, STAT3 plays an important role in the acute phase response, and liver-specific STAT3 knock-out mice exhibited an impaired acute phase response in liver (75). GH can regulate the expression of acute phase proteins (76–78), but the desensitization of STAT3 to repeated GH pulses suggests that STAT3 may not be the major pathway utilized by GH to regulate acute phase protein expression.

In H4IIE cells, after the initial GH treatment plus a GH-free incubation, a second GH treatment was able to activate JAK2, Ras, and Raf-1 but not MEK/ERK. Insulin restored the ability of a second GH treatment to activate MEK/ERK but did not alter the activation of JAK2 and Raf-1, suggesting that insulin restores the disrupted signal transduction from Raf-1 to MEK1/2 caused by the previous GH treatment. KSR plays an integral role in scaffolding of Ras/Raf-1/MEK/ERK pathway proteins. After growth factor stimulation, KSR co-localizes MEK1/2 into close proximity with Raf-1 and allows Raf-1 to phosphorylate MEK1/2 (38–41). In the KSR knock-out mouse, Ras and Raf-1 activation is normal, but MEK1/2 and ERK1/2 activation is defective, indicating KSR is required for proper activation of MEK1/2 and ERK1/2 in the Ras/Raf/MEK/ERK pathway (79).

Similar to the disruption of the MEK/ERK pathway in the KSR knock-out mouse, a second GH treatment in the present study normally activated Ras and Raf-1 but not MEK1/2 and ERK1/2. The desensitization of MEK1/2 in response to the second GH treatment was associated with increased Raf-1/KSR interaction but reduced tyrosine phosphorylation of KSR. Insulin pretreatment increased the amount of MEK bound to KSR. More importantly, insulin also restored the ability of a second GH treatment to activate MEK, and this was correlated with a recovery of tyrosine phosphorylation of KSR. The function of tyrosine phosphorylation of KSR is unknown; however, the present findings lead us to hypothesize that it may cause a conformational change of KSR protein and thereby position the activator Raf-1 in close proximity to its substrate MEK so that activation of MEK by Raf-1 could proceed (Fig. 12).


Figure 12
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  		FIGURE 12.
Proposed model for the interaction of Raf-1, MEK, and KSR after repeated GH treatments in the absence or presence of insulin. A, in untreated cells, Raf-1 and MEK binds to KSR. KSR is not tyrosine-phosphorylated (PY), and Raf-1 and MEK are inactive. When cells are exposed to the first GH treatment, KSR is tyrosine-phosphorylated, and Raf-1 is activated. We hypothesize that due to a conformational change caused by its tyrosine phosphorylation, KSR properly orients MEK with respect to Raf-1, and MEK is activated by Raf-1. B, after the first GH treatment and incubation in SFM, the amount of Raf-1 bound to KSR increases, but the amount of MEK bound to KSR remains unchanged. The second GH treatment activates Raf-1 but only partially induces Tyr(P)-KSR, as indicated by the smaller size of Tyr(P). The partial tyrosine phosphorylation of KSR fails to lead to a conformational change. Consequently, KSR does not bring Raf-1 and MEK together, and MEK is not activated by Raf-1. C, in the presence of insulin, the amount of MEK bound to KSR increases, and the second GH treatment is able to induce Tyr(P)-KSR. Tyrosine-phosphorylated KSR changes its conformation and brings Raf-1 and MEK together, and MEK is activated by Raf-1. Compared with the first GH treatment (A), the association of Raf-1/KSR and MEK/KSR increases after insulin pretreatment (C). This coincides with resensitization to GH; that is, the ability of a second GH exposure after insulin pretreatment to induce MEK and ERK phosphorylation is returned to a level similar to that observed after the first GH treatment.

 
In summary, GH-induced desensitization of MEK/ERK phosphorylation was not due to a deficiency in activation of JAK2, Ras, or Raf-1 but resulted from a defective signal transduction from Raf-1 to MEK1/2, possibly due to the reduced tyrosine phosphorylation of KSR. Insulin restored the ability of a second GH treatment to induce MEK/ERK phosphorylation without affecting GHR levels and phosphorylation/activation of JAK2 and Raf-1 possibly by increasing the amount of MEK bound to KSR and also restoring GH-induced tyrosine phosphorylation of KSR. Previous GH treatment also induced a desensitization of STAT1 and STAT3 phosphorylation. However, resensitization by insulin was selective for the MEK/ERK pathway, since insulin failed to reverse the impaired phosphorylation of STAT1 and STAT3.

Therefore, adequate insulin secretion may be necessary for multiple GH pulses to reactivate the MEK/ERK pathway. Activation of ERK1/2 may mediate GH-induced cell proliferation, differentiation, and gene expression (9, 11, 70, 80, 81). ERK1/2 may also play an important role in mediating the growth-promoting effects of GH (82–85). However, when insulin secretion is deficient, such as in type 1 diabetes or with malnutrition, all of these important GH actions mediated by ERK1/2 may be lost due to the desensitization induced by prior GH exposure.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK DK40456 and DK62071 (to J. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pathology, Division of Molecular and Cellular Pathology, Volker Hall, G019, 1670 University Blvd., University of Alabama at Birmingham, Birmingham, AL 35294-0019. Tel.: 205-934-4921; Fax: 205-975-1126; E-mail: messina{at}path.uab.edu.

2 The abbreviations used are: GHR, growth hormone (GH) receptor; ERK1/2, extracellular signal-regulated kinases 1 and 2; JAK2, Janus-activating kinase 2; KSR, kinase suppressor of Ras; MEK1/2, mitogen-activated protein kinase/ERK kinases 1 and 2; STAT, signal transducer and activator of transcription; SOCS, suppressors of cytokine signaling; P-, phosphorylated; BrdUrd, bromodeoxyuridine; SFM, serum-free, GH-free medium; SOS, Son of Sevenless. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Stuart J. Frank, and Dr. Crystal Johnson, and Vanessa Williams for helpful and insightful discussions and suggestions in the preparation of this manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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