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Endocrinology Vol. 139, No. 10 4364-4372
Copyright © 1998 by The Endocrine Society

ARTICLES

Growth Hormone Promotion of Tubulin Polymerization Stabilizes the Microtubule Network and Protects Against Colchicine-Induced Apoptosis1

Eyleen L. K. Goh, Tony J. Pircher and Peter E. Lobie

Institute of Molecular and Cell Biology and Defence Medical Research Institute (E.L.K.G., P.E.L.), National University of Singapore, Singapore 117609, Republic of Singapore; and Karolinska Institutet (T.J.P.), Institution för Medicinsk Näringslära, NOVUM, Huddinge 14186, Sweden

Address all correspondence and requests for reprints to: Peter E. Lobie, Institute of Molecular and Cell Biology, An Institute Affiliated to National University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore. E-mail: mcbpel{at}mcbsgs1.imcb.nus.edu.sg


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the effect of GH on microtubular physiology in Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR1–638). We show here that after 30 min of human GH (hGH) treatment of CHO-GHR1–638 cells, there was a significant increase in the level of polymerization of all four tubulin isoforms ({alpha}-, ß-, {gamma}-, and tyrosinated {alpha}-tubulin) compared with the serum-deprived state. However, this transient increase in the levels of polymerized tubulin after hGH treatment was particularly pronounced for ß- and tyr {alpha}-tubulin. For {alpha}- and {gamma}-tubulin, the hGH-induced increase in polymerization state lasted to approximately 3 h and then declined by 7 h, whereas for ß- and tyr {alpha}-tubulin there was a decrease in the polymerization state at 1–2 h after hGH treatment compared with the level at 30 min (but still greater than the serum-deprived state) followed by a second but lesser wave of increased polymerization lasting to 7 h. The changes in the polymerization state of the tubulins were not accompanied by comparative changes in the level of total cellular tubulin. The proline rich box 1 region of the GH receptor was required for hGH to stimulate tubulin polymerization indicative that this event is JAK dependent. Increased tubulin polymerization still occurred in response to hGH in a receptor truncation lacking the carboxyl terminal half of the intracellular domain of the GH receptor indicative that hGH induced changes in intracellular calcium concentration is not required for tubulin polymerization. Prior treatment of CHO-GHR1–638 cells with hGH retarded colchicine induced microtubule depolymerization and also prevented colchicine induced apoptotic cell death. The integrity of the microtubule network was not required for GH-induced STAT5 mediated transcription as treatment of cells with colchicine, vincristine, or vinblastine did not alter the fold stimulation of the STAT5 mediated transcriptional response to GH. Thus one consequence of cellular treatment with GH is alteration in microtubule physiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH HAS DIVERSE and pleiotropic actions on cellular metabolism, growth, and differentiation (1, 2). GH-induced changes in cell motility, shape, and substratum attachment (3) require the rearrangement of the cellular cytoskeleton (4). The cellular cytoskeletal network is composed of three main components termed the microfilament, intermediate filament, and microtubule networks, respectively. We have recently investigated the effect of GH on the actin microfilament in Chinese hamster ovary (CHO) cells stably expressing the rat GH receptor (CHO-GHR1–638) and demonstrated that GH causes an initial depolymerization of the actin stress fibers followed by the formation of focal filamentous actin-containing complexes (4). In these cells, GH stimulation also results in the tyrosine phosphorylation of molecules associated with the actin cytoskeleton such as focal adhesion kinase, paxillin, and tensin (5). GH has also been shown to up-regulate the expression of the cytoskeletal protein vinculin, which is involved in linking the actin cytoskeleton to extracellular matrix in 3T3 preadipose cells (6, 7).

On the other hand, the effect of GH on the microtubule network has not been extensively documented. Tubulin expression is slightly increased as part of adipogenic differentiation in the GH-induced differentiation of 3T3 preadipocytes (8). The effect of GH on other parameters of microtubule function has not been investigated. Interestingly, some parameters of microtubule physiology such as total cellular tubulin and the level of polymerized tubulin are altered in cells stimulated with insulin or PRL (9, 10, 11, 12); two hormones that exhibit overlapping signal transduction pathways and cellular effects with GH (13, 14). Changes in the microtubule network in response to platelet-derived growth factor and epidermal growth factor have also been documented (10). To investigate the effect of GH on the microtubule network and their role in GH signal transduction, we have examined several aspects of microtubule physiology in CHO-GHR1–638 cells upon GH stimulation. We have found that GH causes specific changes in polymerized tubulin levels, an effect that is dependent on the proline-rich box1 domain of the GH receptor required for JAK association (15). Prior treatment of cells with GH also retards the colchicine-induced depolymerization of microtubules and consequently diminishes the colchicine-induced apoptotic cell death. In contrast, the microtubule network is not required for GH-stimulated STAT5 mediated transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human GH (hGH) was a generous gift of Novo-Nordisk (Singapore) and Pharmacia-Upjohn (Stockholm, Sweden). Colchicine, sodium orthovanadate, phenylmethylsulfonyl fluoride (PMSF), and monoclonal antibodies against {alpha}-, ß-, {gamma}- and tyrosinated {alpha}-tubulin and common chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Secondary anti-IgG antibodies conjugated to FITC and the enhanced chemiluminescence (ECL) kit were from Amersham Life Science.

Cell lines
CHO and CHO cells stably transfected with rat GH receptor complementary DNAs (cDNAs) (16) [CHO-GHR1–638 (17), CHO-GHR1–294 (17), CHO-GHR1–638{Delta}297–311 (15), CHO-GHR1–638P300,301,303,305A (15), and CHO-GHR1–454 (17)] were maintained in Ham’s F-12 medium supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin as previously described (4). CHO-GHR1–638 cells do not synthesize nor secrete IFG-1 either in the serum-deprived state or upon GH stimulation (4, 17)

Treatment of cells with hGH
CHO and CHO-GHR1–638 (17), CHO-GHR1–294 (17), CHO-GHR1–638{Delta}297–311 (15), CHO-GHR1–638P300,301,303,305A (15) and CHO-GHR1–454 (17) were grown in medium containing 10% FCS for 36–48 h before changing to serum-free medium (serum deprivation) for 12–15 h. Serum-deprived cells were treated with 50 nM hGH for the indicated time periods. For colchicine treatment, serum deprived cells were preincubated with 50 nM hGH for 2 h before treatment with 1 µM colchicine. For staurosporine treatment, serum deprived cells were preincubated with either 50 nM or 500 nM staurosporine for 1 h before hGH treatment for 30 min. Polymerized fractions were then extracted as below.

Extraction of soluble and polymerized tubulin fractions
CHO cells were grown to about 80% confluence, incubated for 12–15 h in serum-free medium, washed twice in serum-free medium, and incubated with 50 nM hGH for the indicated time periods. Cells were rinsed twice with prewarmed (25 C) PBS, scraped in prewarmed microtubule stabilizing buffer (0.1 M piperazine-N, N' bis-2-ethanesulfonic acid (Pipes), pH 6.75, 1 mM EGTA, 1 mM MgSO4, 1 mM sodium orthovanadate, 1 µg/µl protease inhibitor cocktail of aprotinin, pepstatin, and leupeptin, and 0.1 mM PMSF). After vortexing, the cell suspension was centrifuged at 14,000 x g for 10 min at room temperature. The pellet was resuspended in TBST buffer (25 mM Tris, pH 7.4, 0.4 M NaCl, 0.1% Triton X-100, 1 mM sodium orthovanadate, 1 µg/µl protease inhibitor cocktail and 0.1 mM PMSF) and kept on ice for 60 min to depolymerize microtubules. The suspension was then centrifuged at 14,000 x g for 10 min at 4 C. The supernatant was collected and designated the "polymerized tubulin fraction."

SDS-PAGE and Western blot analysis
Proteins in the tubulin fractions were assayed using a Bio-Rad (Hercules, CA) protein assay kit with BSA as standards. 2 x SDS-PAGE sample buffer (50 mM Tris (pH 6.8), 2% SDS, 2% ß-mercaptoethanol and bromophenol blue) was added to 8 µg protein of each sample and the samples were boiled for 5 min. Samples were subjected to discontinuous SDS-PAGE gels with a 10% resolving gel and transferred to nitrocellulose membranes (Hybond C-extra, Amersham Life Science, Chicago, IL) using standard electroblotting procedures. Membranes were blocked with 2% BSA overnight at 4 C, immunolabeled with either anti-{alpha}, ß, {gamma}, or tyrosinated {alpha}-tubulin for 1 h at room temperature. Immunolabeling was detected by the ECL kit according to the manufacturer’s instructions.

Quantitation of changes in polymerized ß-tubulins
The intensity of the bands on Western blots are quantitated using a Bio-Rad GS-700 imaging densitometer and analyzed with the MultiAnalyst (version 1.0.1) program (Bio-Rad). In brief, x-ray films of appropriate exposure time with band intensity within a linear optical density range were used. Bands were marked, and their total integrated volume were quantitated against the film background. Corrected integrated volumes (in arbitrary units) were plotted against time/treatment.

Confocal laser scanning microscopy
At the end of the respective treatment period, cells were rinsed with ice-cold PBS, fixed in ice-cold 4% paraformaldehyde, permeabilized for 10 min with 0.1% Triton X-100, blocked in 2% BSA, and incubated with a monoclonal antibody against ß-tubulin followed by antimouse IgG conjugated with FITC at room temperature. Labeled cells were visualized with a Carl Zeiss Axioplan microscope (Jena, Germany) equipped with epifluorescence optics and a Bio-Rad MRC600 confocal laser system. Images were converted to the tagged-information-file format and processed with the Adobe Photoshop program (Adobe Systems, Inc., Seattle, WA).

Oligonucleosomal laddering assay
CHO-GHR1–638 cells were grown to 80% confluence, serum-deprived, pretreated for 2 h with various concentrations of hGH, and then treated with 1 µM colchicine as before. Cells were extracted in extraction buffer (50 mM Tris, pH 7.5, 20 mM EDTA, and 1% Nonidet P-40). SDS was then added to 1%. The mixture was incubated at 37 C for 2 h with RNase A added to 1 µg/ml followed by incubation at 42 C for 3 h with proteinase K added to 0.5 µg/ml. The mixture was then extracted once with phenol-chloroform (1:1) and the DNA precipitated with one volume of 5 M ammonium acetate and 2.5 volume of absolute ethanol. Precipitated DNA was analyzed by electrophoresis on a 2% agarose gel. The enrichment of oligonucleosomes was also measured by the Cell Death Detection ELISAPLUS kit (Boehringer Mannheim GmbH, Mannheim, Germany). CHO-GHR1–638 cells were grown in 96-well plates, serum-deprived, pretreated for 2 h with different concentrations of hGH, and then treated with 1 µM colchicine as before and processed according to the working procedure for ELISA as per the manufacturer’s instruction protocol. The ELISA plates were then read at 410 nm with a reference wavelength at 495 nm.

Transient transfection and reporter assay
CHO-GHR1–638 cells were cultured to 50% confluence in six-well plates. Transient transfection was performed in serum-free DMEM with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) according to the manufacturer’s instructions (Boehringer Mannheim GmbH, Mannheim, Germany). A total of 1.4 µg of reporter plasmid (SPI-GLE1-LUC) and 0.05 µg of cytomegalovirus-ß-galactosidase were transfected per well. Cells were incubated with DOTAP/DNA for 12 h before the media was changed to serum-free DMEM containing 50 nM hGH or vehicle. Colchicine, vinblastine, or vincristine (each at 10 µM) were added 1 h before stimulation with hGH. After a further 24 h, cells were washed in PBS and scraped into lysis buffer. The protein content of the samples were normalized and ß-galactosidase and luciferase assays were performed as previously described (18). Results were normalized to the level of ß-galactosidase activity to control for transfection efficiency and calculated as the fold stimulation of unstimulated (nonhormone-treated) cells.

Statistical analysis and presentation of data
All experiments were performed at least three times, usually five to seven times each. In the case of Western blot analysis, representative data from one experiment are presented. Numerical data are expressed as mean ±SD. Data were analyzed using the two tailed t test or ANOVA. Results were considered significant at the 5% level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hGH alters the polymerization state of tubulin in CHO-GHR1–638 cells
To determine if GH induces alterations in the cellular microtubule network, we examined the effect of hGH in CHO cells stably transfected with rat GH receptor cDNA (CHO-GHR1–638) (16). We first wished to determine if GH would affect the total cellular expression levels of the different tubulin molecules. We therefore treated cells with 50 nM hGH for 0, 5, 10, 15, and 30 min and also for 1, 2, 3, 5, and 7 h. When assessed by quantitative Western immunoblot analysis, the total amount of {alpha}-, ß- and {gamma}-tubulin did not change significantly over time in different experiments except for a slight transient increase at 60 min. Interestingly though, there was a gradual increase in the levels of tyrosinated (tyr) {alpha}-tubulin over the 7 h period of measurement (Fig. 1AGo). Because the total level of cellular {alpha}-, ß- and {gamma}- tubulin was not altered significantly, we could now proceed to study the effect of GH on the level of tubulin polymerization. We therefore selectively extracted the polymerized tubulin fraction after varying times of hGH treatment and subjected the extracts to quantitative Western blot analysis for {alpha}-, ß-, {gamma}-, and tyrosinated {alpha}-tubulin. The polymerized levels of all four tubulin isoforms were increased at 30 min of hGH treatment compared with the serum-deprived state. However, this transient increase in the levels of polymerized tubulin after hGH treatment was particularly pronounced for ß- and tyr {alpha}-tubulin. For {alpha}- and {gamma}-tubulin, the hGH-induced increase in polymerization state lasted to approximately 3 h and then declined by 7 h, whereas for ß- and tyr {alpha}-tubulin there was a decrease in the polymerization state at 1–2 h after hGH treatment compared with the level at 30 min (but still greater than the serum-deprived state) followed by a second but lesser wave of increased polymerization lasting to 7 h. To emphasize that the changes in polymerized tubulin are not related to potential changes in total tubulin we compared the changes in total vs. polymerized ß-tubulin by densitometric analysis. The increase in total cellular ß-tubulin from time 0 to 30 min (1.11x) was significantly lower than the increase in polymerized ß-tubulin (11.5x) comparing the same time points (Fig. 1BGo). Thus, the increase in polymerized ß-tubulin could not be accounted for by an increase in total ß-tubulin. Human GH treatment, therefore, results in a transient increase specifically in the level of polymerized tubulin. We have also demonstrated that hGH stimulation of NIH3T3 fibroblasts results in an increase in the level of polymerized tubulin (data not shown). To determine if the changes in polymerized tubulin levels induced by hGH translate into gross changes in microtubule architecture, we examined the microtubule network of {alpha}- or ß-tubulin by confocal laser scanning microscopy in serum-deprived CHO-GHR1–638 cells and CHO-GHR1–638 cells treated for the above time points with 50 nM hGH. CHO-GHR1–638 cells possessed a well-developed microtubule cytoskeleton in the serum- deprived state. Gross changes in the microtubule architecture were not evident upon hGH treatment, although some thickening of microtubule was observed (data not shown).



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  		Figure 1. A, Western immunoblot analysis of total and polymerized tubulin in CHO-GHR1–638 cells stimulated with 50 nM hGH. Serum-deprived CHO-GHR1–638 cells were stimulated with 50 nM hGH for the indicated time periods, polymerized (lower panel) and total (upper panel) tubulin fractions were prepared as described in Materials and Methods, subjected to SDS-PAGE, Western blotted and probed with monoclonal antibodies against either {alpha}-, ß-, {gamma}- or tyrosinated (tyr) {alpha}-tubulin. The blots are representative of at least three separate experiments. B, Quantitative analysis by densitometry of the level of polymerized ß-tubulin after stimulation of CHO-GHR1–638 cells by 50 nM hGH. The graph is representative of at least three experiments. The arbitrary units were obtained as the values relative to the unstimulated cells, which has the unit of 1.

 
The GH receptor regions required for the increase in polymerized ß-tubulin
We then proceeded to identify region(s) of the GH receptor required for the hGH-induced increase in polymerized ß- tubulin. We used well characterized CHO cell clones as described previously (15, 16, 17). These clones stably express the following recombinant constructs: the wild-type receptor (CHO-GHR1–638), a receptor truncation containing only 5 of 349 amino acids in the intracellular domain (CHO-GHR1–294), a receptor truncation containing 184 of 349 amino acids in the intracellular domain (CHO-GHR1–454), a receptor mutation in which the proline rich box 1 region has been deleted (CHO-GHR1–638{Delta}297–311) and a receptor mutation in which the individual proline residues of box 1 have been converted to alanine (CHO-GHR1–638P300,301,303,305A) (Fig. 2AGo). Each cell line was stimulated with 50 nM hGH and the level of polymerized ß-tubulin was estimated as before and compared with the unstimulated control for each. Only those CHO cells expressing the wild-type receptor (CHO-GHR1–638) and the receptor truncation containing 184 of 349 amino acids in the intracellular domain (CHO-GHR1–454) displayed an hGH-dependent increase in polymerized ß-tubulin after 30 min of hGH stimulation. The increase in the level of polymerized ß-tubulin after hGH stimulation in both cell types was of comparable magnitude [approximately 10-fold (Fig. 2Go, B and C)]. The transfection of the GH receptor cDNA in parental CHO cells does not alter the basal level of tubulin polymerization as may be interpreted in Fig. 2Go. The microtubule system is dynamic, and the differences in the basal level of polymerized tubulin between the cell clones presumably relate to the state of the individual cell clones at the time of the experiment (e.g., state of confluence, time in culture). We have performed repeat experiments where the basal level of tubulin polymerization in CHO-GHR1–638 cells was actually higher than that observed in the other cell clones and upon stimulation with hGH, an increase in the level of polymerized tubulin was still observed. Therefore, regardless of the basal level of tubulin polymerization, GH stimulated an increase in polymerized tubulin only in CHO cells expressing the full-length receptor or the receptor mutation GHR1–454. Thus, the normalization of the level of the polymerized tubulin upon GH stimulation to the basal level of polymerized tubulin for each cell line is valid because we are investigating the changes in polymerized tubulin upon GH stimulation. The ability of GHR1–454 to mediate tubulin polymerization is indicative that the hGH stimulated increase in intracellular free calcium concentration is not required for this effect of GH (2). No changes in the level of polymerized ß-tubulin after hGH stimulation was observed in the cell lines expressing the other GH receptor deletions or mutations; nor in the parental wild-type CHO cell line. These remaining constructs are either lacking the proline rich Box 1 or possessing a defective Box 1 region. Because Box 1 is the postulated site of association of the JAK kinases to the GH receptor, these results suggest that the hGH-stimulated increase in polymerized ß-tubulin level is dependent on JAK signal transduction.



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  		Figure 2. A, An illustration of the various GH receptor mutations and truncations used to analyze the regions of the GH receptor required for hGH stimulation of tubulin polymerization. B, One experiment demonstrating a Western immunoblot analysis of polymerized ß- tubulin in parental wild-type CHO cells, CHO-GHR1–638 cells, CHO-GHR1–294 cells, CHO-GHR1–638{Delta}297–311 cells, CHO-GHR1–638 P300,301,303,305A cells, and CHO-GHR1–454 cells. Each cell line was unstimulated or stimulated with 50 nM hGH for 30 min. The polymerized tubulin fractions were prepared as described in Materials and Methods, subjected to SDS-PAGE, Western blotted, and probed with a monoclonal antibody against ß-tubulin. The blot is representative of at least three independent experiments where hGH only stimulated tubulin polymerization in CHO-GHR1–638 and CHO-GHR1–454 cells regardless of the basal level of tubulin polymerization (see results). C, Quantitative analysis by densitometry of the level of polymerized ß-tubulin after stimulation of CHO-GHR1–638 cells, CHO-GHR1–294 cells, CHO-GHR1–638{Delta}297–311 cells, CHO-GHR1–638 P300,301,303,305A cells, and CHO-GHR1–454 cells by 50 nM hGH. The graph is representative of at least three experiments. The values of unstimulated for each pair of experiments are arbitrarily set to 1, and the values for the stimulated in each pair are then plotted as fold increase over the unstimulated value, resulting in the bar chart.

 
Staurosporine inhibits the GH-induced increase in polymerized ß-tubulin
To further demonstrate that the GH-stimulated increase in polymerized ß-tubulin is tyrosine kinase dependent, we use staurosporine to inhibit tyrosine kinase activity. Staurosporine has previously been demonstrated to inhibit tyrosine phosphorylation stimulated by GH (19). The pretreatment of CHO-GHR1–638 cells with 50 nM or 500 nM staurosporine prevents the GH-stimulated increase in polymerized ß-tubulin (Fig. 3Go). Thus, tyrosine kinase activity is required for the GH stimulation of polymerization of tubulin in accord with the previous results demonstrating the requirement of Box1 region of the GH receptor for hGH stimulation of tubulin polymerization.



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  		Figure 3. The effect of staurosporine on the hGH-induced increase in polymerized ß-tubulin. Serum-deprived CHO-GHR1–638 cells were pretreated for 1 h with either 50 nM or 500 nM staurosporine before stimulation with 50 nM hGH for 30 min. Polymerized tubulin fractions were prepared as described in Materials and Methods, subjected to SDS-PAGE, Western blotted, and probed with monoclonal antibodies against ß-tubulin. The blots are representative of at least three separate experiments.

 
hGH reduces colchicine-induced microtubule depolymerization in CHO-GHR1–638 cells
Colchicine is a microtubule disrupting agent that binds tightly to tubulin monomers and prevents their polymerization into microtubule networks (20). Because the microtubule network is not static, prevention of polymerization of tubulin will effectively result in the depolymerization or disassembly of the microtubule network. We have shown above that hGH stimulation of cells promotes tubulin polymerization. Thus, it was reasonable to postulate that hGH should therefore prevent or retard the colchicine induced depolymerization of the microtubule network. We therefore examined the integrity of the microtubule network cells (by confocal laser scanning microscopy) of CHO-GHR1–638 cells either in the serum-deprived state or treated for 2 h with 50 nM hGH before addition of 0.1 µM colchicine for 0, 1, or 2 h. Colchicine treatment of CHO-GHR1–638 cells in the serum-deprived state resulted in almost complete depolymerization of the microtubule network within 2 h. In contrast, most hGH pretreated CHO-GHR1–638 cells treated with colchicine (Fig. 4Go, lower panels) still displayed reasonably intact microtubule networks. Human GH treatment of wild-type CHO cells not expressing the GH receptor did not offer protection against the colchicine-induced depolymerization of the microtubule network (data not shown).



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  		Figure 4. The effect of colchicine on the microtubule network of GH treated or untreated CHO-GHR1–638 cells. CHO-GHR1–638 cells were grown on coverslips, serum deprived for 15 h, either unstimulated (upper panels) or stimulated with 50 nM hGH for 2 h (lower panels) before 1 µM colchicine treatment for the indicated time periods. Cells were then fix with 4% paraformaldehyde and processed for confocal laser scanning microscopy using a monoclonal antibody against ß-tubulin (see Materials and Methods). Bar, 10 µm. The photomicrographs presented are representatives of at least three independently preformed experiments.

 
hGH reduces colchicine-induced cell death in CHO- GHR1–638 cells
Another consequence of colchicine treatment is apoptotic cell death (21, 22, 23). Proteins that stabilize the microtubule network such as bcl-2 are also antiapoptotic (24). We therefore wished to examine if hGH could prevent colchicine-induced apoptosis since hGH was able to prevent colchicine-induced depolymerization of the microtubule network in CHO-GHR1–638 cells. Genomic DNA prepared from serum-deprived and colchicine treated CHO-GHR1–638 cells displayed an oligonucleosomal laddering pattern that is characteristic of apoptotic cell death (Fig. 5AGo). Human GH reduces the intensity of the oligonucleosomal laddering in colchicine-treated cells in a dose-dependent manner with the maximal effect achieved at 50 nM. We also quantitated the apoptotic process in colchicine treated CHO-GHR1–638 cells by measuring oligonucleosomal formation by ELISA (Fig. 5BGo). Again it was evident that pretreatment with hGH exerted an antiapoptotic effect in CHO-GHR1–638 cells treated with colchicine. The antiapoptotic effects of hGH were not observed in the untransfected parental CHO cell line (data not shown).



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  		Figure 5. A, Oligonucleosomal laddering analysis of the dosage dependent antiapoptotic effect of hGH on colchicine-treated CHO-GHR1–638 cells. CHO-GHR1–638 cells were grown for 48 h, serum-deprived for 15 h, pretreated with the indicated hGH doses, and then treated with 1 µM colchicine. DNA samples were prepared and analyzed by agarose gel electrophoresis as described in Materials and Methods. The gel photograph presented is representative of at least three independently preformed experiments. B, The enrichment of oligonucleosomes was also measured by the Cell Death Detection ELISAPLUS kit (Boehringer Mannheim GmbH, Mannheim, Germany). CHO-GHR1–638 cells were grown in 96-well plates, serum-deprived, pretreated with hGH, and treated with colchicine as previously described, ELISA was performed and analyzed as described in Materials and Methods. Data presented are representative of at least three independently preformed experiments.

 
Role of microtubule network in STAT5 mediated transcription
We also examined the role of the microtubule network in hGH-induced STAT5 mediated transcription using a reporter assay requiring STAT5 for function. CHO-GHR1–638 cells were treated with either 10 µM colchicine, 10 µM vinblastine, or 10 µM vincristine before stimulation of the cells with hGH. These concentrations of colchicine, vinblastine, and vincristine result in complete disruption of the microtubule network in CHO-GHR1–638 cells (25). Thus, the microtubule network is depolymerized before cellular stimulation with hGH. Neither colchicine, vinblastine, nor vincristine affected the fold stimulation of luciferase production from SPI-GLE1-LUC induced by hGH (Fig. 6Go). It is therefore apparent that an intact microtubule network is not required for GH-stimulated STAT5-mediated transcription.



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  		Figure 6. Effect of microtubule disruption with colchicine, vinblastine, and vincristine on the hGH-induced STAT5-mediated transcriptional activation through SPI-GLE1. CHO-GHR1–638 cells were grown to confluence and transiently transfected with SPI-GLE1-LUC and cytomegalovirus-CAT as described in Materials and Methods. Cells were treated with 50 nM hGH and processed for luciferase activity as described. Colchicine, vincristine, and vinblastine were each used at 10 µM final concentration. Vehicle was used as control. Results represent the mean ±SD of triplicate estimations. Results presented are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrate here that GH stimulation of cells induces large and transient changes in the polymerization state of tubulin that results in stabilization of the microtubule network. To investigate the effect of GH on the microtubule network, we have used CHO cells stably transfected with GH receptor cDNA (16). CHO cells do not possess endogenous functional GH receptors and therefore require the expression of a transfected receptor cDNA to elicit a functional response to GH. The use of these cells also allows us to examine the receptor regions (15, 16, 17) involved in the GH stimulated increase in polymerized tubulin. We have also demonstrated that GH stimulation of another cell line expressing the endogeneous functional GH receptor (NIH3T3) results in increased tubulin polymerization (Goh, E. L. K., and P. E. Lobie, unpublished observations). Thus, the changes in tubulin polymerization level induced by GH are not due to some receptor cDNA transfection artifact. hGH-treated CHO-GHR1–638 cells showed an increase in the level of polymerized ß-tubulin, which could not be accounted for by a corresponding increase in total ß-tubulin. Interestingly though, neither {alpha}- nor {gamma}- tubulin demonstrated parallel increases in their polymerization state. Tubulin can be posttranslationally modified in several ways (26). One such modification is the reversible removal of the C-terminal tyrosine of {alpha}-tubulin involving a tubulin carboxypeptidase and a tubulin specific tyrosine ligase (27). The C-terminal tyrosine of {alpha}-tubulin has a high turnover in various cells and tissues (27), and the cyclic detyrosination/tyrosination of {alpha}-tubulin may serve to differentiate subclasses of microtubules for specific activities (27, 28). From our data, it is apparent that GH stimulation of cells promotes the accumulation of tyrosinated {alpha}-tubulin in cells as well as the accumulation of tyrosinated {alpha}-tubulin in the polymerized state. In this regard, it is interesting that GH stimulation of the polymerization of both tyrosinated {alpha}-tubulin and ß-tubulin is in correspondence. Tubulin tyrosine ligase (TTL) forms a one-to-one complex with the {alpha}/ß-tubulin dimer, requiring a binding site on ß-tubulin for high specificity interaction (29, 30, 31). Thus, it appears that GH preferentially promotes the incorporation of the tyrosinated {alpha}-tubulin into polymerized microtubules. Because GH also promotes the accumulation of tyrosinated {alpha}-tubulin in the cell, it may be that GH causes the activation of TTL (or the deactivation of the carboxypeptidase). The activated TTL would then catalyze the tyrosination of the {alpha}-tubulin of the {alpha}/ß-tubulin dimer before its conversion to the polymerized state. Such a hypothesis provides an explanation for the lack of precise correspondence between the polymerized levels of {alpha}- and ß-tubulin

The changes in the microtubule cytoskeleton of the CHO cells expressing various mutations and truncations in the GH receptor indicate that there is a requirement for the proline-rich box 1 region (2, 15). These results indicate that the hGH stimulated changes in polymerized tubulin levels are dependent on JAK activity and therefore presumably requires tyrosine phosphorylation of downstream effector molecules to modify the rates of polymerization. This requirement for tyrosine kinase activity is further exemplified by the prevention of the GH-stimulated increase in polymerized ß- tubulin by prior treatment of the cells with staurosporine. The concentrations of staurosporine used here have previously been demonstrated to inhibit JAK2 tyrosine phosphorylation stimulated by GH (19). On the other hand, cells with the GH receptor lacking the carboxyl terminal of the intracellular domain behave almost exactly like those with the full-length GH receptor. This domain is involved in GH-mediated increase in intracellular free calcium concentration (2). The lack of requirement of GH-induced increases in intracellular free calcium concentration for tubulin polymerization is concordant with the fact that Ca2+-calmodulin is actually involved in the depolymerization of microtubules (32). In contrast, we have previously shown that the GH-stimulated increases in intracellular free calcium is required for the hGH-mediated effect on actin microfilaments (4). Thus, the effects of GH on the microfilament and microtubule networks require, at least in part, distinct signal transduction pathways. It will be of interest to determine which GH activated signal transduction molecules are involved to produce the increase in polymerized tubulin. Interestingly, the related hormone PRL has been demonstrated to increase the level of polymerized tubulin in pituitary cells presumably related to secretion of peptide hormones (12). Other hormones and growth factors also regulate the level of tubulin polymerization (9, 10, 11, 12).

The {alpha}/ß tubulin heterodimer is the effective microtubule-forming subunit (33). Colchicine binds to tubulin monomers and prevents their polymerization (20). Our assessment by confocal laser scanning microscopy revealed almost complete disruption of the microtubule network after 2 h of colchicine treatment of CHO-GHR1–638 cells in serum free media. However, upon pretreatment of CHO-GHR1–638 cells with hGH, the disruption of the microtubular network is significantly reduced. Because the uptake of colchicine is not affected by the presence of hGH (data not shown), then presumably the prior promotion of tubulin polymerization by GH is the primary event in the reduced rate of microtubule disruption.

Colchicine treatment of cells also leads to apoptosis (21, 22, 23) as revealed by the appearance of oligonucleosomal ladders in colchicine treated cells. hGH pretreatment of CHO-GHR-1–638 cells greatly reduced the intensity of nucleosomal laddering in a dosage-dependent manner. It has been postulated that microtubule disruption and cell death may be coupled (21, 34). The protective effect of hGH against colchicine-induced apoptosis could then, at least in part, be attributed to its ability to preserve the integrity of the microtubule network in colchicine-treated cells. Different cytokines and growth factors can suppress apoptosis, although they activate different intracellular signal transduction pathway (35). Several molecules such as the insulin-like growth factor 1 (IGF-1), nerve growth factor, interleukin-2 (IL-2), and platelet-derived growth factor have all been shown to possess antiapoptotic or survival effects (36, 37, 38, 39, 40). Nerve growth factor (41) and insulin (10) treatment have also been shown to affect the microtubule network. GH has also been shown previously to suppress apoptosis in preovulatory rat follicles (42). hGH treatment may preserve the integrity of the microtubule network by other more indirect pathways. The antiapoptotic gene, bcl-2, has been shown to be important in preserving the integrity of the microtubule network (24). IGF-1 has been shown to up-regulate the level of bcl-xL gene product, a member of the antiapoptotic Bcl-2 family (37). hGH and IGF-1 share many overlapping effects in both physiological and cellular terms. They both use IRS-1 and IRS-2 (43, 44), activate PI-3 kinase (36, 44), MAP kinase (36, Zhu, T., and P. E. Lobie, unpublished results) and stimulate the phosphorylation of adaptor molecules such as c-crk (45, Zhu, T., and P. E. Lobie, unpublished results). JAK2-mediated signal transduction is the major pathway whereby GH exerts its cellular effects. The kinase domain of JAK2 has been shown to mediate induction of bcl-2 and delays cell death in hematopoietic cells treated with granulocyte-macrophage colony-stimulating factor, IL-3, and IL-5 (46). It is therefore conceivable that the up-regulation of the Bcl-2 family proteins may be a possible mechanism whereby hGH exerts a protective effect on the microtubule network. In fact, in the IL-3-dependent murine pro-B cell line expressing a chimeric cDNA containing the hGHR extracellular domain and the intracellular domain of human G-CSF, hGH treatment induces bcl-2 expression while preventing induction of bax, p53, and c-myc (all mediators of apoptosis) (47). Interestingly, PRL also induces bcl-2 expression and attenuates bax expression in the rat Nb2 lymphoma cells (48). Unfortunately, the unavailability of suitable antibodies directed against hamster (CHO) Bcl-2 family proteins precluded the testing of the above hypothesis in this study.

The alteration of the microtubule network as a result of hGH treatment has important physiological implications. A significant fraction of activated mitogen-activated protein kinases (49) and phosphatidylinositol 3-kinase (50) is associated with polymerized microtubules, which may play an important role in their signal transduction processes. The above-mentioned kinases have all been shown to be involved in hGH signal transduction (17, 51, 52, 53). As far as hGH-mediated gene transcription is concerned, disruption of the actin microfilament by drugs such as cytochalasin B does not affect STAT5-mediated gene transcription but prevents GH induction of the non-STAT-regulated LPL messenger RNA (4). Likewise, disruption of the microtubule network by either colchicine, vinblastine, or vincristine did not affect GH stimulated STAT5-mediated gene transcription, although these agents do prevent the GH-induced increase in LPL messenger RNA in BRL-GHR1–638 cells (Pircher, T. J., and P. E. Lobie, unpublished). This is interesting because the STAT factors have been proposed to translocate from the cytoplasm to the nucleus upon phosphorylation where they mediate transactivation (54). Thus, the microtubule network appears not to be involved in such a translocation of STAT factors. One possibility is that the JAK (55) and STAT molecules (54) resident in the nucleus may mediate the transcriptional activation entirely in the nucleus without the need for cytoplasmic to nuclear translocation. Nuclear STAT1 has previously been reported to be phosphorylated within the nucleus (54). PRL signal transduction to ß-casein gene transcription involves STAT5 and ß-casein is also a GH-regulated gene (56). Previous reports have demonstrated that PRL stimulation of ß-casein gene transcription is inhibited by colchicine (57, 58). We found no evidence here that STAT5-mediated gene transcription was affected by cellular treatment with colchicine. It may be that in the previous studies (57, 58) the use of colchicine affected some other cellular process leading to transcription of the endogenous gene product. Here we have used a reporter assay that is simply dependent on the activation of STAT5, and therefore we can conclude that the activation of STAT5 per se is not affected by colchicine.

In conclusion, we have demonstrated that hGH treatment causes transient increases in the level of polymerized tubulin. These changes in polymerization state are JAK dependent, but independent of GH-mediated changes in intracellular calcium concentration. hGH also confers a protective effect on the microtubule network from disruption by colchicine with subsequent antiapoptotic effects. The effect of GH on the microtubule network may be pivotal for many of the pleiotropic effects of GH, including the promotion of cell survival.


   Footnotes
 
1 Supported by monies from the National Science and Technology Board of Singapore (to P.E.L.). Back

Received February 10, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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