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Endocrinology Vol. 138, No. 8 3207-3215
Copyright © 1997 by The Endocrine Society

ARTICLES

Growth Hormone-Induced Reorganization of the Actin Cytoskeleton Is Not Required for STAT5 (Signal Transducer and Activator of Transcription-5)-Mediated Transcription1

Eyleen L. K. Goh, Tony J. Pircher, Timothy J. J. Wood, Gunnar Norstedt, Ralph Graichen and Peter E. Lobie

Institute of Molecular and Cell Biology and Defense Medical Research Institute (E.L.K.G., R.G., P.E.L.), National University of Singapore, Singapore 119260, Republic of Singapore; Karolinska Institute (T.J.P., T.J.J.W., G.N.), Department of Medical Nutrition, NOVUM, 14186 Huddinge, Sweden

Address all correspondence and requests for reprints to: Peter E. Lobie, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, 119260 Singapore, Republic of Singapore. E-mail: mcbpel{at}leonis.nus.sg


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the effect of GH on the organization of the actin cytoskeleton within the cell. Human GH (hGH) treatment (50 nM) of Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR1–638) resulted in a reorganization of actin filaments in the cell that was not observed upon GH treatment of the untransfected parental CHO cell line. hGH initially induced depolymerization of actin stress fibers similar in magnitude to that induced by treatment of the cells with 100 nM human insulin-like growth factor I. This loss of stress fibers was observed as early as 30 sec after addition of hGH to the medium, and maximal depolymerization of stress fibers was observed between 1–4 min after addition of hGH. This was followed by a slow, but submaximal, repolymerization of the stress fibers and the formation of localized focal filamentous actin containing complexes. Similar cytoskeletal changes were observed after hGH treatment in Swiss 3T3 fibroblasts and BRL cells stably transfected with rat GH receptor complementary DNA (BRL-GHR1–638). Pretreatment of CHO-GHR1–638 cells with wortmannin (a phosphatidylinositol 3-kinase inhibitor) and verapamil (a calcium channel antagonist) both inhibited the hGH-induced actin reorganization. The integrity of the actin cytoskeleton was not required for GH-induced STAT5 (signal transducer and activator of transcription-5)-mediated transcription, as treatment of cells with cytochalasins B and D did not alter the fold stimulation of the STAT5-mediated transcriptional response to GH. We conclude that GH induces a rapid reorganization of the actin cytoskeleton by a process requiring phosphatidylinositol 3-kinase activation and calcium influx, but this cytoskeletal reorganization is not required for the STAT5-mediated transcriptional response to GH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS THE major regulator of postnatal body growth (1). It has diverse and pleiotropic actions on the growth, differentiation, and metabolism of cells, engaging a wide array of intracellular proteins in its signaling cascade (2). These diverse actions include the stimulation of chemotaxis and migration of monocytic cells (3). Such changes in cell motility, shape, and degree of attachment to the substratum require rearrangement of the cytoskeleton (4). Actin filament reorganization is an important event during this process, and such reorganization of the actin cytoskeleton has been observed upon cellular stimulation with other growth factors, such as insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) (5, 6). The reorganization of actin filaments usually involves an initial depolymerization of actin stress fibers followed by the formation of membrane ruffles containing filamentous actin (5, 6).

The depolymerization of stress fibers is thought to result at least in part from messengers released by the activity of phosphatidylinositol 3-kinase (PI 3-kinase) (6, 7). GH has recently been reported to promote the association of the p85 subunit of PI-3 kinase with both insulin receptor substrate (IRS)-1 and IRS-2 (8, 9, 10) and to increase the PI-3 kinase activity associated with IRS-1 (8). Although no effect of GH on the actin microfilament network has been reported, the fact that GH is able to increase the migration of certain cell types (3) and has the capacity to activate the signal transduction pathways required for such migration (2, 6, 7, 8, 9, 10) is strongly suggestive that it affects actin filament organization.

We, therefore, used well characterized cell lines (11, 12) to investigate the effect of GH on the actin microfilament network. We show that GH causes an initial depolymerization of the actin stress fibers followed by the formation of focal filamentous actin-containing complexes. We also partly delineated the mechanism of the GH-induced reorganization of the actin microfilaments and defined the role of the microfilament network in GH-stimulated STAT5 (signal transducer and activator of transcription-5)-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). Human insulin-like growth factor I (hIGF-I) was a gift from Pharmacia-Upjohn (Stockholm, Sweden). Phalloidin-TRITC, cytochalasin B, cytochalasin D, verapamil, wortmannin, and cell culture reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell lines
Chinese hamster ovary (CHO) and CHO cells stably transfected with rat GH receptor complementary DNA (cDNA) (11) were maintained in Ham’s F-12 medium supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin as previously described. Swiss 3T3 fibroblasts and BRL cells stably transfected with rat GH receptor cDNA were maintained in DMEM supplemented as above and as previously described (12).

Stimulation of cells by hGH and hIGF-I
CHO, CHO-GHR1–638, Swiss 3T3 fibroblasts, and BRL-GHR1–638 cells were grown on coverslips in medium containing 10% FCS for 48 h before changing to serum-free medium (serum deprivation) for 12–15 h. Serum-deprived cells were treated with 50 nM hGH or 100 nM hIGF-I for the indicated time periods. For pharmacological inhibition, serum-deprived cells were preincubated with 50 nM wortmannin or 10 nM verapamil for 20 min before treatment with 50 nM hGH or 100 nM hIGF-I.

Fluorescence microscopy
At the end of the respective treatment periods, cells were fixed in ice-cold 4% paraformaldehyde, washed with PBS, permeabilized for 10 min with 0.1% Triton X-100, and incubated with phalloidin-TRITC (0.2 mg/ml) (5). Excess phalloidin-TRITC was removed by extensive washing in PBS. Labeled cells were visualized with a Carl Zeiss Axioplan microscope (New York, NY) equipped with a Bio-Rad MRC600 confocal optics system (Richmond, CA). Images were converted to the tagged-information-file format and processed with the Adobe Photoshop program (Adobe Systems, Inc.).

Quantitation of actin cytoskeletal changes
Cytoskeletal changes were quantitated blindly by counting at least 150 cells in random fields at each time point from each experiment as previously described (6). Experiments were repeated at least three times. Individual cells displaying parallel actin stress fibers extending across the nucleus and actin filaments concentrating as focal complexes were scored as positive for stress fibers and positive for focal filamentous actin-containing complexes, respectively. The percentages of the total counted cells displaying each phenotype were used as the perimeter for the plotting of the graphs presented.

Quantitation of total cellular filamentous actin
CHO-GHR1–638 cells were cultured to 50% confluence in six-well plates before serum deprivation for 12 h. Cells were treated with vehicle or 50 nM hGH. Filamentous actin content was measured as described previously (13). Briefly, the treated cells were washed once in ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 30 min at 4 C. The cells were permeabilized with 0.2% Triton X-100 for 30 min, washed in PBS, and incubated with TRITC-phalloidin (0.2 mg/ml) for 1 h. The cells were washed three times in PBS, and the bound TRITC-phalloidin was extracted with methanol for 1 h. The fluorescence intensity was measured with an excitation wavelength of 465 nm and an emission wavelength of 535 nm. The results are expressed as a relative index calculated from the ratio of the fluorescence intensity of stimulated cells to that of unstimulated cells.

Transient transfection and reporter assay
BRL-GHR1–638 cells were cultured to confluence in six-well plates. Transient transfection was performed in serum-free DMEM with DOTAP according to the manufacturer’s instructions. One microgram of reporter plasmid (SPI-GLE1-LUC) and 1 µg cytomegalovirus-chloramphenicol acetyl transferase (CMV-CAT) were transfected per well. Cells were incubated with DOTAP/DNA (DOTAP, Boehringer Mannheim, Mannheim, Germany) for 12 h before the medium was changed to serum-free DMEM containing 50 nM hGH or vehicle. Cytochalasin B (10 µM) or cytochalasin D (100 µM) was added 1 h before stimulation with hGH. After an additional 24 h, cells were washed in PBS and scraped into lysis buffer. The protein content of the samples were normalized, and CAT and luciferase assays were performed as previously described (14). Results were normalized to the level of CAT expression to control for transfection efficiency and were calculated as the fold stimulation of unstimulated (nonhormone-treated) cells.

Statistics
All data are expressed as the 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 induces stress fiber breakdown in CHO cells stably transfected with GH receptor cDNA
We first wished to determine whether GH could stimulate the reorganization of the actin cytoskeleton. To do this we resorted to the use of CHO cells stably transfected with rat GH receptor cDNA (CHO-GHR1–638 cells) (11). Filamentous actin was visualized by the use of TRITC-labeled phalloidin. CHO-GHR1–638 cells that had been serum deprived for at least 12 h displayed a fibroblast-type morphology, with bundles of parallel stress fibers traversing the cell (Figs. 1aGo and 2aGo). For control purposes we also treated other cells with 100 nM hIGF-I (Fig. 1Go), which has previously been reported to induce stress fiber breakdown and the formation of membrane ruffles in various cell types (15). CHO-GHR1–638 cells were, therefore, grown on glass coverslips to 50% confluence and stimulated for 1, 2, 4, 10, 15, 30, and 60 min with either 50 nM hGH (Fig. 2Go) or 100 nM hIGF-I (Fig. 1Go). Cytoskeletal changes were quantitated blindly. Both hGH and IGF-I induced a rapid depolymerization of the stress fibers within the cell. This depolymerization was observed as rapidly as 30 sec after the addition of either hormone. Maximum depolymerization was obtained between 1–4 min (Figs. 1Go- 3). After the initial depolymerization event there was a gradual repolymerization of actin into stress fibers 30 min after hormone stimulation. Beginning as early as 5 min after exposure to the hormones there was also the formation of localized filamentous actin-containing complexes, which often appeared at the leading edge of the cell ( Figs. 1–3GoGoGo). Other investigators have described these complexes as membrane ruffles rich in filamentous actin (6). hIGF-I (100 nM) was more potent in the promotion of these complexes than was 50 nM hGH despite the fact that the level of stress fiber breakdown was similar in magnitude with the two hormones. To determine whether the hGH-induced changes in the actin cytoskeleton were mediated through the transfected rat GH receptor, we also examined the response of the actin cytoskeleton to hGH and hIGF-I in untransfected parental CHO cells (Fig. 4Go). hGH failed to stimulate any reorganization of the actin cytoskeleton in CHO cells despite the fact that hIGF-I stimulated the depolymerization of stress fibers in CHO cells to the same extent as observed in CHO-GHR1–638 cells. hGH, but not hIGF-I, also failed to promote the formation of focal filamentous actin-containing complexes in CHO cells (Fig. 4Go). Therefore, the hGH-induced cytoskeletal reorganization observed in CHO-GHR1–638 cells was mediated through the transfected rat GH receptor.



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  		Figure 1. Representative photomicrographs of IGF-I-induced reorganization of the actin cytoskeleton in CHO-GHR1–638 cells. CHO-GHR1–638 cells were grown on glass coverslips, serum deprived for 12 h, and stimulated for the indicated periods (A, 0 min; B, 1 min; C, 2 min; D, 4 min; E, 10 min; F, 15 min; G, 30 min; H, 60 min) with 100 nM hIGF-I. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Specimens were viewed and photographed as described in Materials and Methods. Magnification bar, 10 µm.

 


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  		Figure 2. Representative photomicrographs of hGH-induced reorganization of the actin cytoskeleton in CHO-GHR1–638 cells. CHO-GHR1–638 cells were grown on glass coverslips, serum deprived for 12 h, and stimulated for the indicated periods (A, 0 min; B, 1 min; C, 2 min; D, 4 min; E, 10 min; F, 15 min; G, 30 min; H, 60 min) with 50 nM hGH. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Specimens were viewed and photographed as described in Materials and Methods. Magnification bar, 10 µm.

 


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  		Figure 3. Quantitation of the hIGF-I- and hGH-induced reorganization of the actin cytoskeleton in CHO-GHR1–638 cells. CHO-GHR1–638 cells were grown on glass coverslips, serum deprived for 12 h, and stimulated for the indicated periods with either 100 nM hIGF-I or 50 nM hGH. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Specimens were viewed and quantitated as described in Materials and Methods. Results represent the mean ± SD of 3 separate experiments, with at least 150 cells at each time point counted in each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. a, Effects of hIGF-I and hGH on the polymerization state of actin stress fibers in CHO-GHR1–638 cells. b, Effects of hIGF-I and hGH on the formation of focal filamentous actin-containing complexes in CHO-GHR1–638 cells.

 


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  		Figure 4. Quantitation of the hIGF-I- and hGH-induced reorganization of the actin cytoskeleton in untransfected parental CHO cells. CHO cells were grown on glass coverslips, serum deprived for 12 h, and stimulated for the indicated periods with either 100 nM hIGF-I or 50 nM hGH. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Specimens were viewed and quantitated as described in Materials and Methods. Results represent the mean ± SD of 3 separate experiments, with at least 150 cells at each point counted in each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. a, Effects of hIGF-I and hGH on the polymerization state of actin stress fibers in CHO cells. b, Effects of hIGF-I and hGH on the formation of focal filamentous actin-containing complexes in CHO cells.

 
We also quantitated the total amount of filamentous actin within CHO-GHR1–638 after hGH treatment cells by absorption emission spectroscopy. As shown in Fig. 5Go, hGH caused an initial decrease in the total amount of filamentous actin within the cell concordant with the previously demonstrated hGH-induced depolymerization of stress fibers. However, the total amount of filamentous actin within the cell rapidly returned to baseline levels at 10 min and then increased, presumably due to the formation of the focal filamentous actin-containing complexes and the repolymerization of stress fibers observed morphologically.



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  		Figure 5. Quantitation of the total cellular filamentous actin content in CHO-GHR1–638 cells. CHO-GHR1–638 cells were cultured to 50% confluence in six-well plates before serum deprivation for 12 h. Cells were treated with vehicle or 50 nM hGH. The filamentous actin content was measured as described in Materials and Methods. The results are expressed as a relative index calculated from the ratio of the fluorescence intensity of stimulated cells to that of unstimulated cells. Assays were performed in triplicate, and results are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Effects of hGH on actin cytoskeleton in other cell types
We also sought to determine whether other cell types responded to hGH with similar cytoskeletal changes. We, therefore, treated Swiss 3T3 fibroblasts with 50 nM hGH and 100 nM hIGF-I. Again, treatment with both hormones resulted in the initial fast depolymerization of stress fibers, followed by the slower formation of focal filamentous actin-containing complexes (Fig. 6Go). Such results were also observed in the BRL cell line stably transfected with rat GH receptor cDNA (BRL-GHR1–638; Fig. 7Go), although the peripheral filamentous actin-containing complexes were more extensive and semicircular in appearance, reminiscent of filamentous actin-containing membrane ruffles (6). Thus, hGH is able to induce the reorganization of the actin cytoskeleton in several different cell types, suggesting a generalized cell response to GH stimulation.



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  		Figure 6. Representative photomicrographs of hGH and IGF-1 induced reorganization of the actin cytoskeleton in Swiss 3T3 fibroblasts. Swiss 3T3 cells were grown on glass coverslips, serum deprived for 12 h, and stimulated for the indicated periods with either 100 nM hIGF-I or 50 nM hGH. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Specimens were viewed and photographed as described in Materials and Methods. Magnification bar, 10 µM.

 


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  		Figure 7. Representative photomicrographs of hGH and IGF-1 induced reorganization of the actin cytoskeleton in BRL cells stably transfected with GH receptor cDNA (BRL-GHR1–638). BRL-GHR1–638 cells were grown on glass coverslips, serum deprived for 12 h, and stimulated for the indicated times periods with either 100 nM hIGF-I or 50 nM hGH. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Specimens were viewed and photographed as described in Materials and Methods. Magnification bar, 10 µM.

 
Mechanism of hGH-induced cytoskeletal reorganization
We next sought to define the mechanism by which hGH stimulated the reorganization of the actin cytoskeleton. We, therefore, resorted to pharmacological inhibition of pathways known to be involved in the reorganization of the actin cytoskeleton by other agents. PI-3 kinase has been demonstrated to be required for PDGF- and insulin-induced depolymerization of actin stress fibers (6, 7). To determine whether PI-3 kinase is required for the hGH-induced actin reorganization, CHO-GHR1–638 cells were pretreated with 50 nM wortmannin for 20 min before stimulation with 50 nM hGH or 100 nM hIGF-I. Such treatment of cells with 50 nM wortmannin did not alter the level of [125I]hGH binding at the cell surface (data not shown) (16). As shown in Figs. 8Go and 9Go, wortmannin was able to completely prevent both hGH- and hIGF-I-induced depolymerization of stress fibers and hGH- and hIGF-I-induced formation of the focal filamentous actin-containing complexes. GH has also been previously reported to cause a rise in the intracellular free calcium concentration (17, 18). This GH-induced rise in free calcium is prevented by the calcium channel antagonist verapamil (18). CHO-GHR1–638 cells were pretreated with 10 nM verapamil for 20 min before stimulation with 50 nM hGH or 100 nM IGF-I. Such treatment of cells with 10 nM verapamil did not alter the level of GH receptor at the cell surface (data not shown). As shown in Figs. 8Go and 9Go, verapamil was able to prevent hGH- and hIGF-I-induced depolymerization of stress fibers and hGH- and hIGF-I-induced formation of the focal filamentous actin-containing complexes. Therefore, GH-induced reorganization of the actin cytoskeleton requires PI-3 kinase and a rise in the intracellular free calcium concentration.



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  		Figure 8. Quantitation of the effects of wortmannin and verapamil on the hIGF-I-induced reorganization of the actin cytoskeleton in CHO-GHR1–638 cells. CHO-GHR1–638 cells were grown on glass coverslips, serum deprived for 12 h, and treated with either wortmannin or verapamil 20 min before stimulation for the indicated periods with 100 nM hIGF-I. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Specimens were viewed and quantitated as described in Materials and Methods. Results represent the mean ± SD of 3 separate experiments, with at least 150 cells at each point counted in each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. a, Effects of wortmannin and verapamil on the hIGF-I-induced depolymerization of actin stress fibers in CHO-GHR1–638 cells. b, Effects of wortmannin and verapamil on the hIGF-I-induced formation of focal filamentous actin-containing complexes in CHO-GHR1–638 cells.

 


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  		Figure 9. Quantitation of the effects of wortmannin and verapamil on the hGH-induced reorganization of the actin cytoskeleton in CHO-GHR1–638 cells. CHO-GHR1–638 cells were grown on glass coverslips, serum deprived for 12 h, and treated with either wortmannin or verapamil 20 min before stimulation for the indicated periods with 50 nM hGH. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Specimens were viewed and quantitated as described in Materials and Methods. Results represent the mean ± SD of 3 separate experiments, with at least 150 cells at each time point counted in each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. a, Effects of wortmannin and verapamil on the hGH-induced depolymerization of actin stress fibers in CHO-GHR1–638 cells. b, Effects of wortmannin and verapamil on the hGH-induced formation of focal filamentous actin-containing complexes in CHO-GHR1–638 cells.

 
Role of actin cytoskeleton in STAT5-mediated transcription
To determine whether the actin cytoskeleton was involved in hGH-induced transcriptional activation, we used a reporter assay requiring STAT5 for function (14, 19). BRL-GHR1–638 cells were treated with 10 µm cytochalasin B or 100 µM cytochalasin D before stimulation of the cells with hGH. We have previously demonstrated that these concentrations of cytochalasins B and D induce gross morphological changes in the CHO-GHR1–638 cell, including bulging of the nucleus and complete disruption of the microfilament system (20). Neither cytochalasin B nor D affected the fold stimulation of luciferase production from SPI-GLE1-LUC induced by hGH (Fig. 10Go). It is, therefore, apparent that an intact microfilament system is not required for GH stimulation of STAT5-mediated transcription.



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  		Figure 10. Effect of microfilament disruption with cytochalasins B and D on the hGH-induced STAT5-mediated transcriptional activation through SPI-GLE1. BRL-GHR1–638 cells were grown to confluence and transiently transfected with SPI-GLE1-LUC and CMV-CAT as described in Materials and Methods. Cells were treated with 50 nM hGH and processed for luciferase activity as described. Cytochalasins B and D were used at 10 and 100 µM, respectively. Vehicle was used as the control. Results represent the mean ± SD of triplicate estimations. The results presented are representative of three experiments. These results were also observed in CHO-GHR1–638 cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated here that GH induces a rapid reorganization of the actin filament system in a variety of cell types. To our knowledge, this is the first demonstration that GH causes a reorganization of the actin cytoskeleton, although GH has previously been reported to up-regulate the expression level of several cytoskeletal proteins in adipocytes (21, 22) and alter the morphology of mammary epithelial cells grown in long term culture (23). GH has also been reported to act as a chemotactic factor for certain cell types (3). We have further partly defined the mechanism of this filamentous actin reorganization and defined its functional significance. A variety of other growth factors, including insulin (5, 6), platelet-derived growth factor, and IGF-I (5, 15), have also been reported to stimulate stress fiber breakdown and the formation of membrane ruffles. The effect of GH on the actin cytoskeleton in this study cannot be mediated by autocrine/intracrine IGF-I because CHO-GHR1–638 cells have been demonstrated not to express IGF-I (11).

We have investigated the mechanism by which GH promotes stress fiber breakdown and the formation of membrane ruffles. Stress fiber breakdown and membrane ruffling induced by insulin and IGF-I both require PI-3 kinase activity (6, 24). GH has been reported to promote the association of the p85 PI-3 kinase subunit with IRS-1 (8, 9), and GH stimulation increases the PI-3 kinase activity associated with IRS-1 (8). GH has also been reported to promote the association of PI-3 kinase with IRS-2 (10). In both cases the association of PI-3 kinase with the IRS molecule is dependent upon tyrosine phosphorylation in the cell mediated by JAK1 or -2 (9, 10). Pretreatment of CHO-GHR1–638 cells with wortmannin inhibited the initial stress fiber breakdown induced by GH and also the formation of the focal filamentous actin-rich complexes. This demonstrates that this biological effect of GH requires the p110 subunit of PI-3 kinase. It has also been reported previously that expression of the constitutively active p110 is sufficient to promote both stress fiber breakdown and membrane ruffling (6). Other growth factors that activate PI-3 kinase, such as EGF and PDGF, promote stress fiber breakdown in cells with a high stress fiber content (5). Other studies have demonstrated that PI-3 kinase is required for activation of the Rac protein (25), which is involved in membrane ruffling (26). We also investigated the requirement for the GH-induced increase in intracellular free calcium by the use of the calcium channel antagonist verapamil. Verapamil has been reported to completely prevent the GH-induced rise in intracellular free calcium in CHO-GHR1–638 cells (18). Pretreatment of the cells with verapamil inhibited the GH-induced stress fiber breakdown and the formation of filamentous actin-containing complexes. This is presumably because actin-severing proteins, such as gelsolin, require transient increases in the intracellular calcium concentration to mediate the depolymerization of actin filaments (27). In any case, one of the functions of the GH-induced rise in the intracellular free calcium concentration is to participate in reorganization of the actin cytoskeleton.

We also investigated the requirement of an intact microfilament system for GH-stimulated transcription mediated by STAT5. Complete disruption of the microfilaments with cytochalasin B or D failed to prevent the GH-induced fold stimulation of luciferase activity generated via the STAT5-responsive element of the SPI gene. This is interesting because the STAT factors have been proposed to translocate from the cytoplasm to the nucleus upon phosphorylation, where they mediate transactivation (28). Thus, the microfilament network appears not to be involved in such a translocation of STAT factors. Other molecules have been previously demonstrated to undergo nuclear translocation independent of the microfilament system, including GH (20), the adenovirus E1A protein, and the 72- and 90-kDa heat shock proteins (29). One possibility is that the JAK (30) and STAT molecules (31) resident in the nucleus may mediate 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 (31). Support for our observation that an intact microfilament network is not required for STAT5-mediated transcription is an old report demonstrating that PRL induction of ß-casein messenger RNA in mammary gland explants is not affected by treatment of the explants with cytochalasin B (32). ß-Casein is now known to be a STAT5-regulated gene (33). Activation of some signaling molecules has been reported to be affected by cytochalasin B [protein kinase C (34) and p125 FAK (35)], but these molecules are apparently not involved in STAT-mediated transcriptional activation. In this regard it is interesting that cytochalasin B treatment of BRL-GHR1–638 cells prevents GH induction of the non-STAT-regulated LPL messenger RNA (Pircher, T. J., T. J. J. Wood, G. Norstedt, and P. E. Lobie, unpublished observations).

There exists the possibility of a direct association between the GH receptor and the actin cytoskeleton, for example, the EGF receptor is an actin-binding protein (36). However, we could not locate an actin binding sequence in the GH receptor as exists for the EGF receptor. One difference between these two receptors is that the EGF receptor has intrinsic tyrosine kinase activity, whereas the GH receptor is associated with the nonreceptor kinase JAK2 (2). Therefore, such possible direct physical associations between the GH receptor and actin may be mediated by an associated molecule. It is possible as well that the GH receptor undergoes a ligand-dependent translocation to the cytoskeleton, as does the EGF receptor (36).

In conclusion, we have demonstrated that GH induces the breakdown of stress fibers, with the subsequent formation of focal filamentous actin-containing complexes and that this reorganization of the cytoskeleton requires both PI-3 kinase activity and an increase in intracellular free calcium. The reorganization of the cellular actin cytoskeleton induced by GH is likely to be pivotal in mediating many of its pleiotropic actions, including, among others, cell migration (such as into wounds) and chemotaxis.


   Footnotes
 
1 This work was supported by an Institute of Molecular and Cell Biology project grant (Singapore; to P.E.L.). Back

Received February 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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