Autocrine Growth Hormone: Effects on Growth Hormone Receptor ...

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Molecular Endocrinology 21(11):2832–2846 Copyright © 2007 by The Endocrine Society doi: 10.1210/me.2007-0092

Autocrine Growth Hormone: Effects on Growth Hormone Receptor Trafficking and Signaling Monique J. van den Eijnden and Ger J. Strous Department of Cell Biology, Institute of Biomembranes, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands GH and GH receptor are expressed in many extrapituitary tissues, permitting autocrine/paracrine activity. Autocrine GH has regulatory functions in embryonic development and cellular differentiation and proliferation and is reported to be involved in the development and metastasis of tumor cells. To understand the principles of transport and signaling of autocrine GH and GH receptor, we used a model system to express both proteins in the same cell. Our experiments show that GH binds the GH receptor immediately after synthesis in the endo-

plasmic reticulum and facilitates maturation of GH receptor. The hormone-receptor complexes arrive at the cell surface where exogenously added GH is unable to bind these receptors. Autocrine GH activates the GH receptors, but signal transduction occurs only after exiting the endoplasmic reticulum. This model study explains why autocrine GHproducing cells may be insensitive for GH (antagonist) treatment and clarifies autocrine signaling events. (Molecular Endocrinology 21: 2832–2846, 2007)

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routes include the JAK-signal transducer and activator of transcription (STAT) pathways, the MAPK pathway, the phosphatidylinositol-3 kinase pathway, and possibly a nuclear factor-␬B route (14–16). Negative regulation of GHR signaling occurs through phosphatases and members of the suppressors of cytokine signaling (SOCS) family (15, 17). After synthesis and dimerization in the endoplasmic reticulum (ER), the GHR is complex glycosylated in the Golgi apparatus and arrives at the cell surface. There, either the receptor binds GH (if available) or its extracellular domain is proteolytically cleaved by the metalloprotease TNF␣converting enzyme (18). Eventually the GHR, or its C-terminal remnant, is recruited into clathrin-coated pits (19) to be internalized via ubiquitin system-dependent endocytosis (20, 21). After internalization, the GHR ends up in the endosomal-lysosomal system, where it is degraded (22). Traditionally, the GHR is ubiquitously expressed, and GH is secreted by the pituitary gland (4). However, in the last decades, several sites of extrapituitary GH production became known. Among them are neuronal cells (23, 24) and several types of immune cells (25– 27). A number of embryonic cell types produce GH before the ontogenic differentiation of the pituitary somatotrophs (7, 28). Lung cells (29) and certain tumor cells were also found to synthesize both GH and GHR (30, 31). Autocrine GH is involved in breast development during puberty (32). These autocrine systems behave differently from the normal endocrine GH system. Autocrine GH may assist in oncogenic transformation and metastasis of (tumor) cells (33, 34). Some GH-producing tumor cells are sensitive to GH antagonist (GHA) (35) treatment, but others are not (36, 37). Some autocrine GH-producing cells do not respond to exogenous GH (34, 38). In addition, autocrine GH syn-

H AND ITS RECEPTOR, GH receptor (GHR), regulate longitudinal growth (1) and cellular differentiation, proliferation, and metabolism (2, 3). Incorrect GH or GHR functioning results in dwarfism, gigantism, and acromegaly (4) but has also been implicated in the development of cancers (5). Besides this, GH has immunomodulatory functions (6) and roles in early embryonic development (7). GH is a protein of 191 amino acids. GHR is a single transmembrane protein of 620 amino acids and a member of the superfamily of cytokine receptors (8). Other members include the prolactin, erythropoietin, thrombopoietin, and leptin receptors, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and several IL receptors (9). GH binds the N-terminal part of the extracellular domains of two dimerized receptors (10). This results in a conformational change of the extracellular domains and a rotation of the intracellular domains of the receptors (11, 12). Through this rotation, associated Janus tyrosine kinase 2 (JAK2) molecules are brought in close proximity. They cross-phosphorylate themselves, phosphorylate the receptors, and start the signal transduction cascades (13). Signal transduction First Published Online July 31, 2007 Abbreviations: BFA, Brefeldin A; btGH, biotinylated GH; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; EV, empty vector; GHA, GH antagonist; GHR, GH receptor; GHR-T, tail of the GHR; JAK2, Janus kinase 2; NEM, Nethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; PY, phosphotyrosine; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription; TfR, transferrin receptor. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

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thesis is differently regulated when compared with GH secretion from pituitary cells (39–42). Little is known about the cell biology and signaling capacity of the autocrine GH system. Because in vivo, the endocrine, paracrine, and autocrine actions of GH are acting simultaneously in varying degrees on different tissues, we created a model cell system in which we could exclusively study the effects of autocrine GH. In this report, we investigated how GH and GHR behave when they are expressed in the same cell with regard to receptor formation, localization, and signal transduction. RESULTS Autocrine GH Binds GHR in the ER To investigate whether GH and GHR interact when expressed in the same cell, we have used a model

system (ts20 cells) in which we coexpressed both GHR and GH at a 1:1 ratio on the DNA level. Lysates from ts20 cells, transfected with cDNA encoding GHR and GH or, as a control, GHR and empty vector (EV), were immunoblotted with an anti-GHR antibody (Fig. 1A, upper panel). Two forms of the GHR can be identified: a high-mannose glycosylated precursor form, residing in the ER and running at 110 kDa (p in Fig. 1A), and a 130-kDa mature form (m in Fig. 1A) that is mainly at the plasma membrane (11, 43). Upon GH expression, the amount of precursor form was reduced by approximately 60% (calculated from four independent experiments). The amount of mature GHR remained almost the same. The reason for this will be explained in Fig. 3. To investigate whether autocrine GH interacts with the GHR, transfected cells were lysed in the presence or absence of an excess of biotinylated GH (btGH). The btGH binds all the GHRs that are not complexed to the autocrine GH before lysis. In the

Fig. 1. Autocrine GH Binds GHR in the ER Transiently transfected ts20 cells expressing GHR and EV or GHR and GH were lysed in the presence (⫹) or absence (⫺) of an excess of btGH as indicated. A, Lysates were immunostained with anti-GHR-C (upper panel) or anti-GH antibodies (lower panel). The arrow represents unconjugated GH, the arrowhead btGH. B, Immunoblotting of btGH-GHR complexes, isolated through streptavidin pull-down, with anti-GHR-C. C, (Co)immunoprecipitations and reincubations of the lysates with streptavidin beads (SV) were immunoblotted with anti-GHR-C. IP, Immunoprecipitation; m, mature GHR; p, precursor GHR; WB, immunoblotting. Relative molecular mass standards are shown on the left. Data are representative of three independent experiments.

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Fig. 2. Different Ratio of Precursor-Mature When GHR Is Coexpressed with GH Ts20 cells were transiently transfected with cDNA encoding GHR and EV, GHR and GH, or GHR and Myc-tagged TfR. Lysates were immunoblotted with anti-GHR-B (A), anti-Myc polyclonal antibody (B), and anti-GH antibody (C), and equal loading is controlled by actin detection (D). Relative molecular mass standards are shown on the left. Data are representative of two experiments.

lower panel of Fig. 1A, GH was detected in the lysates running at 22 kDa. The btGH is also visible, running somewhat higher, due to conjugated biotin (arrowhead). Next, btGH was isolated via a pull-down procedure with steptavidin beads. The GHRs that were able to bind to btGH can be seen in Fig. 1B. In the GHR/EV-overexpressing cells, a large amount of GHR bound the btGH. In the GHR/GH transfections, virtually no GHRs could be pulled down, indicating that most of the GHRs have bound autocrine GH before lysis. To visualize the interactions between autocrine GH and GHR, coimmunoprecipitations were performed in the lysates of Fig. 1, A and B, which were partly incubated with streptavidin beads. When GH was immunoprecipitated from cell lysates of double-transfected cells (GHR and GH), GHRs could be detected by

Fig. 3. Folding and Maturation of GHR in the Presence of GH Ts20 cells, overexpressing GHR and EV or GHR and GH, were pulse-labeled for 10 min and chased for the indicated times. A, Lysates were immunoprecipitated with anti-GHR-T and subjected to reducing SDS-PAGE. p represents the 110kDa precursor form and m the 130-kDa mature form. B, ⫹, Cells were preincubated with 20 ␮M MG132 for 1 h and subsequently treated with 20 ␮M MG132 during pulse and chase periods; ⫺, samples were mock treated. Samples were immunoprecipitated with anti-GHR-T and subjected to reducing SDS-PAGE. C, Ts20 cells were transiently transfected with cDNA encoding GHR(C83S), GHR(C108A-C122A), or GHR(D152H) in combination with either EV or GH, and 48 h after transfection, cells were pulse-labeled for 10 min and chased for the indicated times. Lysates were immunoprecipitated with anti-GHR-T and subjected to reducing SDS-PAGE. Relative molecular mass standards are indicated. Data are representative of three (A and B) and two (C) independent experiments.

Western blotting, indicating that GHR is present in a complex with GH (Fig. 1C, right panel, lane 4). The precursor form was coimmunoprecipitated as well, indicating that in the ER, the receptors could bind GH. The other lanes represent control experiments; a second incubation with streptavidin beads did not result in a GHR signal (Fig. 1C, right panel, lane 3). In contrast, in the GHR/EV situation, more GHRs could be pulled down after a second round of incubation with streptavidin beads (Fig. 1C, left panel, lane 3). GHR signal can be observed after GH immunoprecipitation as well (Fig. 1C, left panel, lane 4). These are the btGH molecules connected to GHRs that were not removed by


the first incubation with streptavidin beads. No GH was present before lysis in these samples. Immunoprecipitation with GH after lysis without btGH did not result in a GHR signal (Fig. 1C, left panel, lane 5). These experiments show that when GH and GHR are expressed in the same cell, nearly all GHRs have bound GH before lysis. Thus, it is likely that in the ER, the (dimerized) receptors have bound a GH molecule directly after synthesis. The Amount of Precursor GHR Is Reduced upon GH Coexpression Because GH is able to bind the GHR in the ER, we investigated a possible role for GH in folding or maturation of the GHR. On Western blot, a smaller amount of precursor form of the GHR was observed when it was cotransfected with GH (Fig. 1). To test whether this effect was specifically due to the presence of GH, double transfections with cDNA encoding GHR and EV, GHR and GH, or GHR and myc-tagged transferrin receptor (TfR) were compared (Fig. 2A). When GHR and TfR were coexpressed, the amount of precursor form was equal to the GHR/EV transfection, whereas the amount of mature GHR remained almost the same. Thus, the 60% reduction of precursor form in the GHR/GH transfection was due to the presence of GH in the ER. In Fig. 2, B and C, correct transfection and expression of GH and TfR is controlled through detection with anti-Myc tag or anti-GH antibodies. In Fig. 2D, equal loading was controlled by actin detection of the lysates. GH Assists in Maturation of the GHR To further understand the role of GH in folding and maturation of the GHR, pulse-chase experiments were performed using an antibody against the tail of the GHR (anti-GHR-T). The double band at 110 kDa represents the ER precursor form of the GHR, being correctly folded within 2 min and capable of binding ligand (Fig. 3A) (43). After 30 min of chase, the complex-glycosylated, 130-kDa mature form of the GHR becomes visible. These GHR molecules were in or passed the Golgi apparatus. In the left panel, the precursor forms did not disappear after long chase times but tended only to become slightly smaller. This is due to mannose trimming and indicates the action of the ER-associated degradation system (ERAD) (43, 44). When GH and GHR were cotransfected (Fig. 3A, right panel), the pulse-chase pattern is different; the precursor forms disappeared after longer chase times. Because the mature form appeared with the same kinetics as in the control situation, the data indicate that the rate of ER-toGolgi transport did not change. In the presence of GH, the ER form of the GHR is either subjected to ERAD or allowed to mature and continue to the cell surface. To investigate this, pulsechase analysis was performed in the presence or ab-

sence of MG132, a proteasome inhibitor and inhibitor of ERAD (Fig. 3B). In the GHR/EV cotransfection, both GHR species were increased at 480 min of chase due to a block in endocytosis and inhibition of ERAD. For the GHR/GH double transfection, the 110-kDa bands still disappeared in the presence of MG132. They were apparently not subjected to ERAD but were allowed to mature. When more GHRs become mature, more 130kDa form is expected. This is not observed in Figs. 1 and 2. In the presence of MG132, an accumulation of 130-kDa GHR can be seen, which suggests an increased rate of internalization in the GHR/GH situation. To investigate the nature of GH action and answer the question whether a (stoichiometric) binding between GH and GHR is required, or whether a conformational change is required, we performed pulsechase analysis with mutated GHRs (Fig. 3C). Mutant C83S has one side of a disulfide bond changed into a serine and does not bind GH (43). In the left panel, virtually no differences can be observed on the 110kDa form when either GH or EV was cotransfected with the mutant GHR. The precursor form remains visible after long chase times. Besides this mutant, three other mutant GHRs, which could not bind GH, were tested. Most of them did not show the reduction in precursor form after coexpression with GH (results not shown). Thus, GH binding is required for the disappearance of the ER form. To control this experiment we used the mutated GHR(C108A-C122A). This mutant lacks a disulfide bridge, but can still bind GH and perform signal transduction (43). For GHR(C108AC122A) cotransfected with GH, the precursor form disappeared, like the wild-type GHR (Fig. 3C, middle panel). Another mutant, D152H, can bind GH but does not allow the conformational change necessary for activation of the GHR (11). In the right panel, after cotransfection with GH, the precursor form disappeared after 300 min chase time. In conclusion, GH assists in maturation of the GHRs. Apparently, a direct GH-GHR interaction, but no activation of the GHR, is necessary for this effect. GHRs at the Cell Surface of Autocrine GHProducing Cells Cannot Bind or Be Activated by Exogenous GH In Fig. 1, it was observed that the GHRs at the cell surface of autocrine GH-producing cells have bound GH. The data do not exclude the possibility that GHRs at the plasma membrane have bound GH that was first secreted into the medium. To investigate this, a GHR-expressing ts20 cell line was transiently transfected with cDNA encoding GH or enhanced green fluorescent protein (EGFP), a red-shifted variant of wild-type GFP (Fig. 4A). The cells were washed to remove the secreted GH in the medium, an excess of Cy3-labeled GH was added, and the cells were incubated for 2 h at 30 C. All newly synthesized GHRs that arrive at the cell surface and

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did not bind GH in the cell will bind the Cy3-GH. GH-transfected cells were stained with an anti-GH antibody (Fig. 4A, upper left panel, green signal) and imaged with confocal immunofluorescence microscopy. The ER is strongly stained in the transfected cells. GH-expressing cells did not take up the Cy3-GH present in the medium, as can be clearly observed when the green signal is omitted (Fig. 4A, upper right panel, red signal). In contrast, the untransfected cells or control EGFP-transfected cells (Fig. 4A, lower panels) did internalize the Cy3-GH. This indicates that GHRs at the cell surface of autocrine GH-producing cells are unable to bind exogenous GH. To further characterize this finding, ts20 cells were transiently transfected with cDNA encoding GHR and EV or GHR and GH. These cells were washed and subsequently incubated for 2 h with an excess of btGH. The newly synthesized GHRs that arrive at the cell surface and do not bind autocrine GH will then bind the btGH. Western blots of lysates were stained with an anti-GHR antibody to control the transfections (Fig. 4B, left panel). The middle panel shows the btGHGHR complexes isolated with streptavidin beads. In the control situation, newly formed GHRs have bound btGH. In the GHR/GH double transfections, no GHR signal can be seen, again indicating that all GHRs were occupied with GH when they arrived at the cell surface. Phosphorylated tyrosines can be labeled with an anti-phosphotyrosine (anti-PY) antibody. This is an indicator of activated GHRs. Only the GHRs pulled down from the GHR/EV-overexpressing cells have PY staining (Fig. 4B, right panel). When all the GHRs are occupied with GH upon arrival at the cell surface, no signal transduction can occur originating from exogenous GH (culture medium or serum). GHR, JAK2, and STAT5 Phosphorylation in Autocrine GH-Producing Cells Because virtually all GHRs are occupied with GH before arrival at the cell surface, and GH from the medium cannot activate them anymore, we asked the question whether the GHRs in these cells are activated by the autocrine GH. Double transfections with cDNA encoding GHR and EV, GHR and GH, and a ts20 cell line stably expressing GHR were used. The cells were incubated for 15 min in the presence or absence of

GH. GHR, JAK2, and STAT5 were immunoprecipitated and immunostained for GHR, JAK2 or STAT5 (Fig. 5, left panels), and PY (Fig. 5, right panels) to visualize activation of these molecules. Even though the amount of GHRs in the stable cell line is lower than the amount of GHRs after transient transfection, the PY signals for GHR, JAK2, and STAT5 are much stronger in the stable cell line than in the transiently transfected cells. This is probably because the transient transfection efficiency is 20–50%. For cells overexpressing GHR and GH, the PY signals are consequently low but just above the background signals. The intensity of the PY signal does not change when exogenous GH is added. This confirms the results of Fig. 4. The upper form of STAT5 (Fig. 5C, left panel) is the phosphorylated form. This allows calculation of the amounts of phosphorylated STAT5, which are indicated as percentages in Fig. 5C, left panel. The amount of phosphorylated STAT5 has increased 1.5 times (average of four independent experiments) in the GHR/GH situation compared with the background levels (lanes 1 and 4). In conclusion, although it is minimally, GHRs have been activated by autocrine GH. STAT5 Target Genes Transcribed in Autocrine GH-Producing Cells To investigate downstream signaling events in the autocrine situation, luciferase assays with a STAT5 reporter construct were performed (Fig. 6). As a negative control, a luciferase reporter without the STAT5-responsive element was used. In the control transfection (GHR⫹EV), addition of GH to the medium resulted in a strong signal, three times above background. In the GHR/GH transfection, there was also a strong STAT5 reporter signal, with correct controls. This indicates that despite the low transfection efficiency and the relatively low amounts of phosphorylated signaling molecules on Western blots, STAT5 target genes are transcribed in the autocrine situation. Autocrine Activation of the GHR Originates Only from the Mature GHR According to the previous experiments, GH can bind the GHR in the ER and remains constantly connected to the receptor. To discriminate whether the JAKSTAT signal originates from the precursor or mature

Fig. 4. GHR, Coexpressed with Autocrine GH, Is Invisible for Exogenous GH A, Ts20 cells, stably expressing GHR, were grown on coverslips and transiently transfected with cDNA encoding GH (upper panels) or EGFP (lower panels), and 48 h after transfection, cells were washed and incubated with 25⫻ excess Cy3-labeled GH (red signals). After 2 h, the cells were washed, fixed, and labeled with an anti-GH antibody, followed by a Cy5-labeled antirabbit secondary antibody (upper panel, green signal). Cells were visualized by confocal immunofluorescence microscopy. The right panels represent the same images as the left panels but without the green signals. B, Ts20 cells were transiently transfected with cDNA encoding GHR and EV or GHR and GH. Cells were washed and incubated for 2 h with 10⫻ excess btGH. Lysates are shown in the left panel. btGH-GHR complexes, isolated with streptavidin beads, are shown in the middle and right panels. Samples were immunoblotted with anti-GHR-C or anti-PY, as indicated. WB, Immunoblotting. Relative molecular mass standards are shown on the left. Data are representative of three independent experiments.


Fig. 5. GHR Signaling in the Presence of Autocrine GH Ts20 cells were stably transfected with GHR [ts20(GHR)], transiently transfected with cDNA encoding GHR and EV or GHR and GH, or not transfected (⫺), as indicated (DNA). Cells were used 48 h after transfection and incubated in the presence (⫹) or absence (⫺) of 180 ng/ml GH for 15 min (GH medium). A, Immunoblotting of anti-GHR-T immunoprecipitates with anti-GHR-C antibody (left panel) or anti-PY antibody (right panel); B, immunoblotting of anti-JAK2 immunoprecipitates with anti-JAK2 antibody (left panel) or anti-PY antibody (right panel); C, immunoblotting of anti-STAT5 immunoprecipitates with anti-STAT5 antibody (left panel) or anti-PY antibody (right panel). Percentages of phosphorylated STAT5 (upper band) with regard to total STAT5 are indicated. IP, Immunoprecipitation; WB, immunoblotting. Relative molecular mass standards are shown on the left. Data are representative of at least three independent experiments.

GHR, we reexamined the PY signal on the GHR species. Cells overexpressing GHR and EV or GHR and GH were not treated. They were immunoprecipitated with an anti-GHR-T antibody. When the PY signal of the GHR/GH transfection was detected (Fig. 7A, right panel), only the mature species was phosphorylated and not the ER form. To accumulate the precursor form of the GHR, brefeldin A (BFA) was used. This drug destabilizes the

Golgi complex. BFA treatment indeed gave accumulation of the 110-kDa form of the GHR in both the control and the GHR/GH transfection (Fig. 7B, upper panel). Another control transfection, GHR and EV, is shown in the right panel of Fig. 7B. GH was added to these cells, resulting in a PY signal at 130 kDa, which is shown in the lower right panel of Fig. 7B. No PY signal is visible in the BFA-treated control transfection (Fig. 7B, lower panel, first three lanes), and a faint PY


Fig. 6. STAT5 Target Genes Are Transcribed in Autocrine Situation Ts20 cells were transiently transfected with cDNA encoding GHR and EV or GHR and GH and cotransfected with a luciferase reporter construct (STAT5) or a luciferase reporter without the STAT5-responsive element (negative control) and a Renilla construct as a positive control for transfection efficiency. Cells were used 24 h after transfection, serum starved for 2 h, and incubated overnight with (⫹) or without (⫺) 500 ng/ml GH (GH medium). Next, cells were harvested, and a dual-luciferase assay was performed. Luciferase activities are indicated in luminescence units (RLU ⫻ 106). Firefly luciferase activity is indicated in dark gray, Renilla luciferase in light gray columns. Neg., Negative. The values represent the mean ⫾ SD of a triplicate transfection. The data shown are representative of three independent experiments.

signal is visible for the first two time points of the GHR/GH situation. This is, however, a signal at 130 kDa. Even after accumulation of the ER form of the GHR, no phosphorylation of the precursor form can be seen. In conclusion, activation of the GHR can occur only when the receptor is in or past the Golgi apparatus.

DISCUSSION When GH and its receptor are expressed in the same cell, our model system shows the following events (see Fig. 8, right panel). GH and GHR bind each other in the ER, where GH assists the receptor in maturation. GH and GHR both arrive at the cell surface, where only fully occupied GHRs reside. Therefore, exogenous GH cannot activate the receptors anymore. Internalization (and degradation) of GHR-GH complexes occurs at a higher rate than that of unoccupied GHRs, rendering the GHR short lived. Autocrine GH induces the JAK/ STAT signaling routes, but signaling does not occur when the GHR is still in the ER. In contrast, in the endocrine situation (Fig. 8, left panel), maturation of the GHR is relatively slow, and only extracellular GH

binds and activates the GHRs at the plasma membrane. Although our model system is based on relatively high expression levels of GH and GHR, it is valid for situations wherein the molar ratio of expression levels (GH/GHR) exceeds 0.5 in any given cell. In nature, the cellular levels of autocrine GH and GHR vary widely between individuals (27). The relative amounts of GH and GHR determine the fraction of GHRs occupied before arriving at the cell surface. Treatment of autocrine GH-producing tumors with GHA (pegvisomant) is suggested (5, 45) and has been tested. Not all tumors were growth inhibited with pegvisomant (36, 37). This can be explained by our model system. When all GHRs are occupied with GH upon arrival at the cell surface, GHA cannot bind any more. It is therefore advisable to examine the amount of free GHRs at the cell surface before GHA is administered. The same explanation holds for autocrine GH-producing cells that are insensitive for exogenous GH (34, 38). Studies in an MCF-7 mammary carcinoma cell line stably transfected with human GH, by the group of Peter Lobie, confirm that these cells are not sensitive for exogenous GH (33, 34). Other studies performed by the same group in the same model system show inhibition of proliferation


Fig. 7. Signaling Does Not Occur at the ER Ts20 cells were transiently transfected with cDNA encoding GHR and EV or GHR and GH and used 48 h after transfection. A, Immunoblot of untreated anti-GHR-T immunoprecipitates with anti-GHR-C antibody (left panel) or anti-PY antibody (right panel). B, Cells were treated with 25 ␮g/ml BFA for indicated times or with 180 ng/ml GH for 15 min (positive control), and immunoblotting of anti-GHR-T immunoprecipitates was done with anti-GHR-C antibody (upper panel) or anti-PY antibody (lower panel). IP, Immunoprecipitation; Pos., positive; WB, immunoblotting. Relative molecular mass standards are shown on the left. The data shown are representative of two independent experiments.


and signal transduction and abrogation of protection against apoptosis when GHA is used (46, 47). This is a discrepancy that cannot be explained with our model system, because GHA would not bind GHRs that have already bound GH inside the cell. When cells produce both GH and GHR, the GH molecules that bind the receptors are diverted from their normal fate, storage in secretory vesicles and release upon a GHRH signal. In autocrine GH-producing cells, the GH molecules that did not bind a receptor will probably be stored in secretory vesicles. It is unclear how the release is controlled. In our studies, we detected secreted GH in the medium, with no significant GH storage in granules. Probably, the ts20 cells are unable to form specialized GH granules. From our experiments, we conclude that GH and GHR interact in the ER. In the pulse-chase assay, we found that more GHRs become mature when GH is present. A remaining question is whether GH serves as a ligand chaperone. Some ligands are known to act in this way, assisting folding of the protein in the ER (48). The GHR is fully folded and GH-binding capable in 2 min (43). In the experiments described in this report, the first chase time is after a 10-min pulse labeling period. This means that a role for GH as a ligand chaperone in folding of the GHR could not be observed. Besides this, because the GHR is folding relatively quickly, the role of GH in this process may be marginal. However, more GHRs are allowed to mature when GH is coexpressed. GH does not increase the speed of maturation, because mature receptors do not appear before 30 min chase time in both the GHR/EV and GHR/GH situation. Most likely, GH assists the GHR in the ER, allowing all molecules to pass through the quality control system. Dimerization may be a prerequisite for ER exit, and GH may assist in this process. Although in our experiments, exogenously added GH or GHA have no effect, the autocrine GH-producing cells do have activated signal transduction routes. The phosphorylation signals on Western blot were weak and perhaps the result of a balance between activating factors, such as the JAK and STAT molecules and down-regulating factors, like phosphatases and the SOCS proteins. Raccurt et al. (49) report that SOCS gene expression is elevated in breast carcinoma, which may be a response to autocrine/paracrine GH. Apparently, activation of the GHR can occur only when the receptor is in or past the Golgi apparatus. Perhaps the GHR needs to be modified in the Golgi system to be capable of signaling or the signal transduction molecules cannot reach the GHR when it is in the ER. JAK2 may be absent from precursor GHR, which is different from the situation in the erythropoietin receptor (50). Others have looked at the signal transduction routes in cells expressing autocrine GH and found JAK2 and STAT1, -3, and -5 to be activated (51, 52). Several antiapoptotic pathways also appeared to be stimulated (16, 45). Some genes are expressed only in the

autocrine situation (5). The difference in signaling mediated through autocrine or exogenous GH may be caused by the fact that the GHRs are already activated within the cell. Alternative signal transduction pathways might become activated through signaling from the Golgi system. In the autocrine situation, GH is constantly present in high local concentrations. In the endocrine situation, GH levels vary because of pulsatile secretion (4). This invokes differences in transient or sustained activation of signal transduction molecules, which may result in alternative signal transduction routes (5). Why would a cell make its own GH? In embryonic development, when the pituitary has not been developed, GH might be necessary (7). When cells make their own GH, they can also react more strongly on developmental (differentiation) decisions. In the immune system, the endocrine levels of GH might be too low to induce effects. And for cells of the mammary tissue, which change drastically at puberty, pregnancy, and lactation (53), local high levels of GH might be required (32). For cancer cells, signaling routes resulting in growth and protection against apoptosis might be instrumental. When a cell is signaling in an autocrine manner and all GHRs at the surface are occupied, the cancer cell can overrule inhibiting signals from the medium. It is interesting to note that the more aggressive a tumor cell is, the more GH it produces (31, 54). In addition, GH induces GHR expression. Not only autocrine GH has effects on survival, growth, differentiation, and proliferation, but prolactin and the IGFs also have strong roles. The prolactin and IGF pathways are closely connected to GHR signaling, and prolactin also interacts with the GHR (5, 6, 45). Wennbo et al. (55) suggest that the oncogenic effects of GH are conducted only through interaction with the prolactin receptor. Others describe direct roles of GH on the GHR in human mammary carcinoma cell behavior (47). In this study, the effects on the prolactin or IGF systems were not measured. The results we obtained in our signaling experiments are most likely caused only by autocrine GH activating the GHR because of their high expression levels. The complexity of autocrine effects of GH and its regulators is beyond the scope of this model study. The amounts of autocrine GH that are produced and the number of GHRs can be up- or down-regulated in a single cell at certain time points (25, 32, 56). It is unclear whether GHRH, ghrelin, or somatostatin have any influence (42, 56). Pit-1, the regulator of pituitary GH transcription, was found to have no influence in autocrine GH transcription (39, 40), even though its presence has been reported in mammary (carcinoma) cells (57). Roles for progesterone, estrogens, and vitamin D have been suggested (36, 41, 58). Our research data explain why certain autocrine GH-producing cells are not sensitive to exogenous GH or GHA. They also show that GHRs are activated in an autocrine situation, but only when the GHR has exited the



ER. Unclear is how a cell regulates its own GH transcription. It is important to understand this regulation to be able to interfere with autocrine GH production as a possible therapeutic approach against tumor formation.

MATERIALS AND METHODS Materials and Antibodies Anti-GHR rabbit antisera generated against amino acid residues 271–320 (anti-GHR-T), 327–493 (anti-GHR-B), and 493–620 (anti-GHR-C) were raised as previously described (20, 59). Antiserum against human GH was raised in rabbits. Antibody Mab5, recognizing the extracellular domain of the GHR was obtained from AGEN Inc. (Parsippany, NJ). Monoclonal anti-actin antibody (clone C4) was obtained from MP Biomedicals Inc. (Irvine, CA). Monoclonal antibody 4G10 (PY), recognizing PY residues and polyclonal anti-JAK2 antibody were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal antibody Stat5 (C17) was obtained from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA). Human GH was kindly provided by Eli Lilly & Co. Research Labs (Indianapolis, IN). The btGH was created according to the manufacturer’s instructions and tested for GHR affinity, which is equal to non-btGH (Pierce, Rockford, IL). MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) was from Calbiochem (San Diego, CA). BFA was from ICN Biomedicals Inc. (Aurora, OH). Myc-tagged human TfR cDNA in pCB7 was prepared as previously described (60). A pEGFP-N1 vector was obtained from Clontech (BD Biosciences, Palo Alto, CA). Human GH was cloned in a pcDNA3 vector, and the EV control construct was a pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA). GHR Mutant and Cell Lines Mutations of the GHR extracellular domain were created by QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described before (11, 43). Chinese hamster ts20 cells, bearing a thermolabile ubiquitin-activating (E1) enzyme, were used for all experiments (61). A clonal cell line stably expressing wtGHR [ts20(GHR)] was obtained with the calcium phosphate transfection method. Transient transfections and a clonal cell line stably expressing GH were created with FuGENE6 (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s description. Cells were cultured at permissive temperature (30 C) in MEM␣ (GIBCO BRL, Carlsbad, CA) as described (12, 47). Pulse-Chase Assay Subconfluent ts20 cells, grown in 6-cm dishes, were used 48 h after transfection for pulse-chase analysis as described (62). Briefly, the cells were washed in PBS and preincubated in starvation medium lacking methionine and cysteine for 15

min at 30 C. Cells were pulse labeled for 10 min with 125 ␮Ci/ml Redivue PRO-MIX L-[35S] in vitro cell labeling mix (Amersham Life Sciences, Arlington Heights, IL) and chased with excess cold methionine/cysteine and 1 mM cycloheximide in MEM␣ (with HEPES) at 30 C for the indicated times. Incubations were stopped by transferring the cells to ice, aspirating the medium, and adding ice-cold PBS containing 0.5 mM MgCl2, 1 mM CaCl2, and 20 mM N-ethylmaleimide (NEM) (Sigma-Aldrich, St. Louis, MO). The cells were lysed in ice-cold lysis buffer containing 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 20 mM NEM in PBS. Cell lysates were centrifuged to pellet the nuclei, and postnuclear supernatants were used for immunoprecipitation with anti-GHR-T antibody in 1% Triton X-100, 1% SDS, 0.5% sodium deoxycholate, 1% BSA, 1 mM EDTA, 1 mM PMSF, 50 mM NaF, 1 mM Na3VO4, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 20 mM NEM. Immune complexes were isolated with protein A-conjugated agarose beads (Repligen Co., Waltham, MA) and subjected to SDS-PAGE. Gels were stained in a Coomassie Brilliant Blue solution, destained in 10% acetic acid and 10% methanol, dried, and visualized using a Amersham Biosciences (Piscataway, NJ) phosphorimager. Microscopy Ts20 cells, stably expressing GHR, were grown on coverslips and transiently transfected with cDNA encoding GH or EGFP. Cells were treated for 16 h with 10 mM butyrate to increase the GHR expression and used 48 h after transfection. Cy3labeled GH was prepared with a FluoroLink Cy3 label kit (Amersham Life Sciences, Piscataway, NJ) according to the supplier’s instructions. Cells were washed, serum-free medium was added, and the cells were incubated for 2 h at 30 C with 10 ␮g/ml Cy3-labeled GH (25⫻ excess related to secreted GH per milliliter medium from GH-transfected cells). Next, cells were washed with PBS and fixed for 60 min in 4% paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.4) at room temperature. Cells were permeabilized with 2% saponin, 10% BSA in PBS and subsequently incubated with primary and fluorescently labeled secondary antibodies, as indicated in figure legends, for 60 min each. Unbound antibodies were removed by washing with permeabilization buffer. Coverslips were embedded in the antifading agent, Mowiol (Calbiochem). Confocal laser scanning microscopy was performed with a Zeiss CSLM 510 Meta live-cell imaging station. Luciferase Assay The ts20 cells were grown in 12-well plates and transiently transfected with cDNA encoding GHR and EV or GHR and GH (400 ng/well) and cotransfected with a luciferase reporter construct (STAT-5 reporter pSPI_Luc) or a luciferase reporter without the STAT5-responsive element (pTK_luc) as a negative control (100 ng/well) and a Renilla construct (pRL_SV40) as a positive control for transfection efficiency and viability (2 ng/well). The luciferase constructs were a kind gift from Dr. Amilcar Flores-Morales (Karolinska Institute, Stockholm, Sweden). Cells were used 24 h after transfection, serum

Fig. 8. Model for Endocrine vs. Autocrine GH Actions on the GHR Schematic representation of the endocrine (left side) vs. the autocrine situation (right side). In the endocrine situation, exogenous GH binds the GHR at the plasma membrane. The complex is internalized via clathrin-coated pits and degraded in the endosomal/lysosomal system. In the autocrine situation, GH binds GHR after synthesis in the ER, facilitating its maturation. Signal transduction can start from the Golgi apparatus and may continue until internalization and degradation of the GHR. Exogenous GH cannot bind the GHRs at the cell surface. The GH-GHR complex is eventually internalized and degraded faster than GHR without GH. PM, Plasma membrane.


starved for 2 h, and incubated overnight with (⫹) or without (⫺) 500 ng/ml GH in starvation medium. Next, cells were lysed and a dual-luciferase reporter assay system was used according to the manufacturer’s instructions (Promega, Madison, WI). Luciferase activities were measured in a Lumat LB9507 luminometer (Berthold, Nashua, NH). The values represent the mean ⫾ SD of a triplicate transfection. The data shown are representative of three independent experiments. Coimmunoprecipitations Cells were grown in 6-cm dishes and used 48 h after transfection. Dishes were put on ice and washed three times with ice-cold PBS. The cells were lysed in ice-cold buffer containing 0.5% Triton X-100, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 1 mM PMSF in PBS and, when indicated, supplemented with 20 ␮g/ml btGH (10⫻ excess related to GH concentration in lysates). Cell lysates were centrifuged to pellet the nuclei, and postnuclear supernatants were incubated as indicated with Immunopure immobilized streptavidin (Pierce), anti-GHR-T, or anti-GH antibodies. Immune complexes were isolated with protein A-conjugated agarose beads (Repligen). The immunoprecipitates or btGH-GHR complexes were washed twice with PBS. The lysates, immunoprecipitates, and btGH-GHR complexes were subjected to reducing SDS-PAGE. Lysis, Immunoprecipitations, and Pull-Downs Cells were grown in 6-cm dishes and used 48 h after transfection. When indicated, cells were washed, serum-free medium (MEM␣) was added, and the cells were incubated for 2 h at 30 C with 4 ␮g/ml btGH (10⫻ excess related to secreted GH per milliliter medium from GH-transfected cells). Dishes were put on ice and washed three times with ice-cold PBS. The cells were lysed in ice-cold lysis buffer containing 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 1 mM PMSF in PBS. Cell lysates were centrifuged to pellet the nuclei, and postnuclear supernatants were used for immunoprecipitation or btGH pull-down. GHR molecules were immunoprecipitated with anti-GHR-T, JAK2, or STAT5 antibodies in 1% Triton X-100, 1% SDS, 0.5% sodium deoxycholate, 1% BSA, 1 mM EDTA, 1 mM PMSF, 50 mM NaF, 1 mM Na3VO4, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin. Immune complexes and btGH-GHR complexes were isolated as described above. Lysates, immunoprecipitates, or btGH-GHR complexes were subjected to reducing SDS-PAGE. GH Incubation (for Tyrosine Phosphorylation) The ts20 cells stably transfected with cDNA encoding GHR or transiently transfected with cDNA encoding GHR and EV were grown in 6-cm dishes and used 48 h after transfection. Cells were incubated for 15 min in the presence of 180 ng/ml GH or mock treated at 30 C. After incubation, cells were put on ice and washed three times with ice-cold PBS, supplemented with 0.4 mM Na3VO4. Cells were lysed in the same ice-cold lysis buffer as described above. Immunoblotting After SDS-PAGE, proteins were transferred to Immobilon-FL polyvinylidene difluoride membrane (Millipore). Blots were immunostained with the indicated antibodies followed by Alexa Fluor 680 (Molecular Probes, Portland, OR) or Alexa800 IRDye (Rockland, Gilbertsville, PA) conjugated goat antimouse or goat antirabbit antibodies or IRDye 800-conjugated protein A (Rockland). When needed, blots were reprobed after stripping twice for 15 minutes with stripping

buffer (25 mM glycine, pH 2.0, and 1% SDS in H2O). Detection and quantification were performed with an Odyssey System (LI-COR Biosciences, Lincoln, NE).

Acknowledgments We thank Rene´ Scriwanek and Marc van Peski for assistance with the figures, Daniele Tauriello and Joyce Putters for assistance with the luciferase assay, and Peter van Kerkhof, Peter van der Sluijs, Ineke Braakman, Madelon Maurice, and the rest of the Strous group for stimulating discussions.

Received February 19, 2007. Accepted July 24, 2007. Address all correspondence and requests for reprints to: Ger J. Strous, Department of Cell Biology, University Medical Center Utrecht, Heidelberglaan 100, Room G02.525, 3584 CX Utrecht, The Netherlands. E-mail: [email protected]. The investigations were in part supported by the Research Council for Earth and Life Sciences (ALW) with financial aid from the Netherlands Organization for Scientific Research (NWO-814.02.011) and by the Network of Excellence “Rubicon” (LSHG-CT-2005-018683). Disclosure Statement: The authors have nothing to disclose.

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Course Title: 2nd Annual International Adrenal Cancer Symposium: Clinical and Basic Science Course Date: March 13-16, 2008 Location: Biomedical Science Research Building University of Michigan Medical School Ann Arbor, MI 48109 Websites: http://www.med.umich.edu/intmed/endocrinology/acs.htm http://cme.med.umich.edu/events/ Contact: Registrar Office of Continuing Medical Education University of Michigan Medical School G1200 Towsley Center 1500 E. Medical Center Drive, SPC 5201 Ann Arbor, MI 48109-5201 Phone: 734-763-1400; Fax: 734-936-1641