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Endocrinology Vol. 141, No. 4 1571-1584
Copyright © 2000 by The Endocrine Society

ARTICLES

Autocrine Human Growth Hormone Enhancement of Human Mammary Carcinoma Cell Spreading Is Jak2 Dependent1

Karmal K. Kaulsay, Hichem C. Mertani, Kok-Onn Lee and Peter E. Lobie

Department of Medicine, National University of Singapore (K.K.K., K.O.L.), Singapore 119074; and Institute of Molecular and Cell Biology (H.C.M., P.E.L.), Singapore 117069, Republic of Singapore

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of autocrine production of human (h) GH in the attachment and spreading of mammary carcinoma cells in vitro. We used a previously described model system for the study of the autocrine/paracrine role of GH in which the hGH gene (MCF-hGH) or a translation-deficient hGH gene (MCF-MUT) was stably transfected into MCF-7 cells. No differences in attachment to a collagen matrix between MCF-hGH and MCF-MUT cells were observed in either serum-free medium (SFM) or medium containing exogenous hGH, 5% serum, or 10% serum. In contrast, MCF-hGH cells spread more rapidly on a collagen matrix than did MCF-MUT cells. Exogenous hGH and 10% serum interacted with autocrine production of hGH in an additive manner to increase cell spreading. MCF-hGH cells formed filipodia and stress fibers earlier than MCF-MUT cells during the process of cell spreading and possessed marked differences in morphology after spreading. MCF-MUT cells displayed uniform and symmetrical formation of stress fibers, whereas MCF-hGH cells displayed irregular and elongated stress fiber formation. The level of cytoplasmic phosphotyrosine was increased in MCF-hGH compared with MCF-MUT cells during spreading and displayed colocalization with Janus kinase 2 (JAK2). Basal JAK2 tyrosine phosphorylation was increased, and it increased further on spreading in MCF-hGH cells compared with MCF-MUT cells. Transient transfection of JAK2 complementary DNA resulted in interaction with autocrine hGH to increase the rate of cell spreading in MCF-hGH cells compared with MCF-MUT cells. Treatment with a selective JAK2 tyrosine kinase inhibitor (AG 490) reduced the rate of MCF-hGH cell spreading to the rate of MCF-MUT cell spreading. Thus, we conclude that autocrine production of hGH enhances the rate of mammary carcinoma cell spreading in a JAK2-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GH GENE is expressed in the normal and tumorous mammary gland of the cat and dog (1). In human mammary gland, human (h) GH messenger RNA (mRNA) identical to pituitary hGH mRNA is also expressed by nontumorous tissue and by benign and malignant tumoral tissue; immunoreactive hGH being restricted to epithelial cells (2). Accordingly, GH receptor (GHR) mRNA, and protein have been detected in the mammary gland epithelia of murine and rabbit (3, 4, 5), bovine (6), and human (7, 8) species. Both endocrine GH and autocrine-produced GH therefore possess the capacity to exert a direct effect on the development and differentiation of mammary epithelia in vitro (9) and in vivo (10). We recently generated a model system to study the role of autocrine-produced hGH in mammary carcinoma by stable transfection of either the hGH gene or a translation-deficient hGH gene into mammary carcinoma (MCF-7) cells (11). The autocrine hGH-producing cells display marked insulin-like growth factor I (IGF-I)-independent hyperproliferation in both serum-free and serum-containing conditions as well as a specific increase in STAT5 (signal transducer and activator of transcription-5)-mediated transcription (11). Thus, autocrine production of hGH by mammary carcinoma cells may direct mammary carcinoma cell behavior to impact on the final clinical prognosis.

Cell adhesion and spreading are critical to the pathological progression of cancer (12). We have previously demonstrated that cellular stimulation with hGH results in the reorganization of both the microfilament (13) and microtubule networks (14). Further, we demonstrated that cellular stimulation with hGH results in the Janus kinase 2 (JAK2)-dependent phosphorylation of p125FAK (15) and the formation of a multiprotein signaling complex centered around CrkII and p130Cas (16). Formation of a p130Cas-CrkII complex has been demonstrated to be sufficient for cell migration (17), and we demonstrated that CrkII-dependent actin cytoskeletal reorganization is due to enhanced phosphoinositide 3-kinase (PI-3 kinase) activity (Goh, E. L. K., T. Zhu, and P. E. Lobie, manuscript in preparation). Thus, autocrine hGH is likely to affect cytoskeletal architecture and lead to changes in the motile behavior of mammary carcinoma cells.

We therefore investigated the effect of the autocrine production of hGH in human mammary carcinoma cells on the rate of attachment to and spreading on a collagen substrate. We demonstrate that autocrine production of hGH in mammary carcinoma cells results in an enhanced rate of cell spreading that is dependent on the kinase activity of JAK2. Thus, autocrine production of hGH may affect the metastatic potential of mammary carcinoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant hGH was a gift from Novo-Nordisk (Singapore). SuperFect transfection reagent was obtained from QIAGEN (Hilden, Germany), and rat tail type 1 collagen was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Ca2+- and Mg2+-free PBS solution used for cell dissociation was obtained from Life Technologies, Inc. (Grand Island, NY), and AG 490 was purchased from Calbiochem (La Jolla, CA). All other tissue culture materials were obtained from HyClone Laboratories, Inc. (Logan, UT). Phalloidin-tetramethylrhodamine isothiocyanate (TRITC), Cy3-conjugated sheep antimouse IgG, fluorescein isothiocyanate-conjugated sheep antirabbit IgG, and protein G cell suspension were obtained from Sigma (St. Louis, MO). JAK2 polyclonal antiserum (directed against amino acids 758–776 of murine JAK2), used for Western immunoblotting, immunoprecipitation, and immunofluorescence, was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antiphosphotyrosine monoclonal Ab PY20 used for immunofluorescence and Western immunoblot was obtained from Transduction Laboratories (Lexington, KY). Peroxidase-conjugated antirabbit and antimouse IgGs were obtained from Pierce Chemical Co. (Rockford, IL). Enhanced chemiluminescence detection reagents were purchased from Amersham Pharmacia Biotech (Aylesbury, UK). The expression plasmids containing the JAK2 complementary DNA [cDNA; pRc/cytomegalovirus (CMV) mJAK2] and vector (pRc/CMV) were gifts from Dr. Stuart J. Frank (18).

The MCF-7 cell line was obtained from American Type Culture Collection (Manassas, VA) and stably transfected with an expression plasmid containing the wild-type hGH gene (pMT-hGH) under the control of the metallothionein-1a promoter (19) (designated MCF-hGH). For control purposes the ATG start site in pMT-hGH was disabled via a mutation to TTG generated by standard techniques (pMT-MUT), and MCF-7 cells stably transfected with this plasmid were designated MCF-MUT. MCF-MUT cells therefore transcribe the hGH gene but do not translate the mRNA into protein. A detailed description of the characterization of these cell lines has been previously published (11). Neither MCF-7 nor MCF-MUT cells produce detectable amounts of hGH protein, whereas MCF-hGH cells secrete approximately 100 pM hGH into 2 ml medium over a 24-h period under the culture conditions described here. MCF-7 and MCF-MUT cells behave identically to each other in terms of proliferation, transcriptional activation (11), and cell spreading (this study).

Cell culture
MCF-hGH and MCF-MUT cells (11) were cultured at 37 C in 5% CO2 in RPMI supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine.

Cell substrate attachment assay
Attachment, operationally defined as the number of single isolated cells or small cell aggregates resistant to shear forces, was measured by a modification of a method previously described (20). In brief, MCF-hGH and MCF-MUT cell lines were maintained in 10% FBS-supplemented RPMI before being serum deprived for 12 h. Cells were then washed in 1 x PBS, detached in Ca2+- and Mg2+-free PBS-based cell dissociation buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4), and collected by centrifugation at 300 x g for 10 min. Cells were resuspended in serum-free medium (SFM) and plated in a crosswise mixing movement on rat tail tendon collagen-coated dishes (5 µg/cm2, according to manufacturer’s instructions) to a final concentration of 1 x 104 cells in a total volume of 2 ml, and incubated for various time periods in the indicated serum conditions. Medium was then gently aspirated, the dishes were rinsed twice in fresh SFM to remove unattached cells, and the number of attached cells were counted under a microscope eyepiece grid. Data are expressed as the mean number of cells per grid field (±SD). Ten grid fields were counted for each cell line at each time interval.

Single cell spreading assays
Cell spreading in single cell preparations was measured by counting the number of cells that possessed a spread morphology (epithelioid, phase-dark with lamelloid extensions) in each microscope field (21). Spread cells were expressed as the proportion of total cells attached in each respective microscope field (±SD). Ten grid fields were counted for each time interval.

Confocal laser scanning microscopy
MCF-hGH and MCF-MUT cells were plated on collagen-coated glass coverslips, fixed in ice-cold 4% paraformaldehyde at the end of the respective time intervals, washed with 1 x PBS, permeabilized for 10 min with 0.1% Triton X-100, and incubated with phalloidin-TRITC (0.2 mg/ml), mouse antiphosphotyrosine PY20 (1:1000), or rabbit anti-JAK2 (1:800). Either Cy3-labeled sheep antimouse IgG (1:1000) or a combination of Cy3-labeled sheep antimouse IgG and fluorescein isothiocyanate-labeled antirabbit IgG (1:1000) second antibodies was used in the detection of phosphotyrosine and the colocalization of phosphotyrosine with JAK2, respectively. Controls included 1) substitution of PY20 with a species-specific monoclonal antibody directed against the rabbit GH receptor (Mab 7) at the same protein concentration, and 2) substitution of the JAK2 antisera with normal rabbit serum at the same concentration. Control experiments were consistently negative. Labeled cells were visualized with a Carl Zeiss Axioplan microscope (New York, NY) equipped with a Bio-Rad Laboratories, Inc., MRC600 confocal optics system (Richmond, CA). All labeling and acquisition parameters were maintained constant for comparison between cell lines. Images were converted to the tagged information file format and processed with the Adobe Photoshop program (Adobe Systems, Inc., Seattle, WA).

Immunoprecipitation of proteins from cell extracts
MCF-hGH and MCF-MUT cells were grown to confluence in 10% serum-supplemented medium, incubated for 12 h in serum-free medium, washed twice in SFM, detached, replated in collagen-coated dishes, and allowed to spread for the indicated time periods. Cells were lysed at 4 C in 1 ml lysis buffer [50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.2 mM sodium orthovanadate, 0.5% Nonidet P-40, and 0.1% phenylmethylsulfonylfluoride] for 30 min with regular vortices. Cell lysates were then centrifuged at 14,000 x g for 15 min, the resulting supernatants were collected, and protein concentration was determined by the Lowry method using BSA as a standard. Eight hundred micrograms of total protein were used for each immunoprecipitation. Immunoprecipitation was performed routinely by incubating cell lysates with 4 µg/ml of the respective antibody for 2 h at 4 C. Immunocomplexes were collected by incubation with 50 µl protein G cell suspension for 1 h at 4 C and subsequent centrifugation of lysates at 14,000 x g for 5 min. Immunoprecipitates were washed three times in ice-cold lysis buffer. The pellets were resuspended in 2 x SDS-sample buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 2% ß-mercaptoethanol, and bromophenol blue], boiled for 10 min, and centrifuged at 14,000 x g for 5 min. The supernatants were collected and subjected to 7% SDS-PAGE. Proteins were transferred to nitrocellulose membranes using a standard semidry electroblotting apparatus in Laemmli buffer containing 10% methanol.

Western blot analysis
Nitrocellulose membranes were blocked with 5% insulin-free BSA in PBS with 0.1% Tween 20 (PBST) for 2 h at 22 C. Blots were then immunolabeled for 1 h at 22 C with either mouse phosphotyrosine antiserum (1:1,000) or rabbit JAK2 antiserum (1:800). After six washes for 10 min each time in PBST, membranes were incubated in either goat antimouse (1:1,000) or goat antirabbit IgG (1:10,000) horseradish peroxidase-conjugated second antibodies, respectively, for 1 h at 22 C. Membranes were further washed six times for 10 min each time in PBST before immunolabeling was detected by ECL according to the manufacturer’s instructions.

Transient transfection of MCF-hGH and MCF-MUT cells with JAK2 cDNA and treatment of cells with AG 490
MCF-hGH and MCF-MUT cells were cultured to subconfluence in six-well plates. Transient transfection was performed in serum-free RPMI with SuperFect reagent according to the manufacturer’s instructions. Four micrograms of wild-type JAK2 plasmid (pRc/CMV mJAK2) and vector plasmid (pRc/CMV) were transfected per well. Cells were incubated with the SuperFect/DNA complex for 12 h in 10% serum-containing medium before medium was changed to growth medium for an additional 12 h and finally serum deprived for an additional 12 h in SFM alone or in SFM supplemented with 20 µM AG 490. Cells were then detached in cell detachment buffer and either replated on collagen-coated dishes and subjected to attachment and spreading assays in the presence or absence of AG 490 or scraped into lysis buffer, normalized for protein content, and analyzed by Western immunoblot.

Statistics
All experiments were repeated at least three to five times. All numerical data are expressed as the mean ± SD. Data were analyzed using the two-tailed t test or ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of autocrine production of hGH on cell morphology
Live photography was used to examine and compare the motile behavior of MCF-hGH and MCF-MUT cells (Fig. 1Go). Single cell suspensions or small clusters (three to six cells) of MCF-hGH and MCF-MUT cells were allowed to adhere to collagen-coated dishes. Both MCF-MUT (Fig. 1Go, A–I) and MCF-hGH cells (Fig. 1Go, J–R) initially attached as phase-bright rounded cells before spreading to form phase-dark epithelioid cells over a 4-h time course [as previously described for other cell types (21)]. MCF-hGH (Fig. 1Go, L–R) and MCF-MUT (Fig. 1Go, F–I) cells incubated in SFM displayed a characteristic flattening of the cell base upon contact with the substrate, followed by phase-darkening of the cell appearing first at the peripheral edge of the cell, then darkening of the entire cell area in a spread state. MCF-hGH cells demonstrated a highly polarized morphology, with locomotor activity initiated by formation of long protrusions as early as 5 min after plating (Fig. 1KGo). This was continued at 15 min by protrusion formation originating from the ventral margins (Fig. 1LGo) and subsequently at 2, 3, and 4 h (Fig. 1Go, P–R, respectively) of increasingly elongated and narrow processes, resulting in an increased surface area of cellular attachment to the substrate. MCF-MUT cells also demonstrated the formation of protrusions, but beginning at a later time point (15 min) compared with MCF-hGH cells (Fig. 1Go, C vs. K, respectively) and in fewer numbers compared with MCF-hGH cells (6.2 ± 1.8 vs. 2.1 ± 0.6 for MCF-hGH and MCF-MUT, respectively). In contrast to MCF-hGH cells, MCF-MUT cells demonstrated formation of shorter and thicker cellular processes, first observed at 45 min (Fig. 1EGo). Also in contrast to MCF-hGH, the development of cellular protrusions over the course of 4 h in MCF-MUT cells did not result in an elongation of the individual cell, but, rather, in a more rounded-cuboidal and symmetrically shaped cell (Fig. 1IGo). The remainder of the cell periphery remained relatively morphologically quiescent and was characterized by a concave, curving profile (Fig. 1Go, F and H). Thus, autocrine production of hGH affects the morphology of mammary carcinoma cells.



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  		Figure 1. Morphological differences between MCF-MUT and MCF-hGH cells allowed to spread on a collagen matrix as observed by phase contrast microscopy. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). Photomicrographs demonstrate the morphological characteristics of MCF-MUT (A–I) and MCF-hGH (J–R) cell spreading when cultured in SFM. Single cells or small cell aggregates (three to six cells) were allowed to adhere to collagen-coated dishes for the indicated time intervals. MCF-MUT cells displayed less prominent cellular processes (arrows; C) and rounded, refringent morphology (A–I) throughout the time course. In contrast, MCF-hGH cells displayed motile cell features, such as extensive elongation of bases with cellular protrusions (arrows; K–L, N, and P–R) extending from the active leading edges of the cell. Scale bar, 6 µm.

 
Effect of autocrine hGH production on MCF-hGH and MCF-MUT cell adhesion and spreading
We first quantified the rate of attachment and spreading of MCF-MUT and MCF-hGH cells plated on collagen substrata in SFM and in SFM supplemented with 100 nM exogenous hGH (Fig. 2Go, A and B). No significant reproducible differences in the rates of attachment were seen between MCF-MUT and MCF-hGH cells in either SFM or 100 nM hGH-supplemented SFM (Fig. 2AGo). MCF-hGH cells, however, spread at a faster rate than MCF-MUT cells in SFM (Fig. 2BGo). A greater percentage of MCF-hGH cells than MCF-MUT cells were observed to have spread in SFM (Fig. 2BGo; 29% vs. 2%, respectively) at the 15 min point. The percentage of cells spread in SFM was greater for MCF-hGH than for MCF-MUT cells for all indicated time intervals, although at 4 h the percentage of MCF-MUT cells spread approximated that of MCF-hGH cells. The addition of 100 nM exogenous hGH increased the rate of MCF-MUT cell spreading at all time points compared with the spreading rate in SFM (Fig. 2BGo), but not to the same extent as that of MCF-hGH cells for the corresponding time points in either serum-free or hGH supplemented spreading conditions. For example, at 15 min, 15% vs. 2% of MCF-MUT cells and 55% vs. 29% of MCF-hGH cells were spread in hGH-supplemented medium vs. SFM, respectively (Fig. 2BGo). MCF-hGH cells also spread at a faster rate after the addition of exogenous hGH (Fig. 2BGo). Similar autocrine hGH-enhanced cell spreading was observed for another set of independently generated MCF-7 clones stably transfected with either the expression plasmid encoding the wild-type hGH gene (MCF-hGH2) or the hGH expression plasmid containing the disabled start codon (MCF-MUT2) (data not shown).



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  		Figure 2. Quantitative analysis of the attachment and spreading of MCF-MUT and MCF-hGH cells plated on a collagen matrix cultured in either SFM or 100 nM exogenous hGH-supplemented SFM. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). Single cells were incubated in collagen-coated dishes in SFM alone or in SFM supplemented with 100 nM hGH. After the indicated time periods, dishes were washed to remove nonadherent cells as described in Materials and Methods. Cell attachment (cells/field; A) and cell spreading (percentage of the total number of attached cells; B) were measured and are presented as the mean ± SD of triplicate determinations (n = 10). Results presented are representative of at least three (usually five to seven) independent experiments. *, P < 0.01; **, P < 0.001 (MCF-MUT vs. MCF-hGH for the corresponding time intervals). #, P < 0.01; ##, P < 0.001 (MCF-MUT in SFM vs. MCF-MUT in 100 nM hGH-supplemented SFM for the corresponding time intervals). +, P < 0.01; ++, P < 0.001 (MCF-hGH in SFM vs. MCF-hGH in 100 nM hGH-supplemented medium for the corresponding time intervals).

 
We also examined the attachment and spreading characteristics of MCF-MUT and MCF-hGH cells in the presence of 5% (Fig. 3AGo) and 10% (nonprimate) FBS (Fig. 3BGo). Any GH present in nonprimate serum will not bind to either the primate PRL receptor or the primate GH receptor (22), thereby allowing study of the interaction between autocrine hGH and various heterologous factors in serum. Five percent serum did not alter the rate of attachment (data not shown) or spreading of either cell line compared with serum-free conditions (Fig. 3AGo). The attachment rate of MCF-MUT and MCF-hGH also remained unaffected by the addition of 10% FBS (data not shown). Ten percent FBS minimally enhanced spreading of both MCF-hGH and MCF-MUT cells compared with their rates of spreading for each time point in SFM (Fig. 3BGo). However, the fold differences in the rate of spreading between MCF-hGH and MCF-MUT were maintained in 10% FBS.



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  		Figure 3. Quantitative analysis of the rate of spreading of MCF-MUT and MCF-hGH cells plated on a collagen matrix cultured in 5% and 10% FBS. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). Single cells were incubated in collagen-coated dishes in SFM alone or in SFM supplemented with 5% (A) or 10% (B) serum. After the indicated time periods, dishes were washed to remove nonadherent cells as described in Materials and Methods. Cell spreading (percentage of the total number of attached cells; A and B) was measured and presented as the mean ± SD of triplicate determinations (n = 10). The results presented are representative of at least three (usually five to seven) independent experiments. *, P < 0.01; **, P < 0.001 (MCF-MUT vs. MCF-hGH for the corresponding time intervals). #, P < 0.01; ##, P < 0.001 (MCF-MUT in SFM vs. MCF-MUT in serum-supplemented medium for the corresponding time intervals). +, P < 0.01; ++, P < 0.001 (MCF-hGH in SFM vs. MCF-hGH in serum-supplemented medium for the corresponding time intervals).

 
Autocrine hGH-induced cell spreading is inhibited by a hGH receptor antagonist
To demonstrate that the increase in the rate of spreading of MCF-hGH compared with MCF-MUT cells was indeed due to autocrine hGH production, we used a competitive hGH receptor antagonist, hGH-G120R. hGH-G120R acts as a competitive antagonist to hGH due to deficient site 2 binding of the GH receptor, thereby preventing hGH receptor dimerization and consequent initiation of signal transduction (23). The percentage of cell spreading of MCF-hGH cells in SFM in the presence of 1000 nM hGH-G120R was reduced to that of MCF-MUT cells in either the presence or absence of hGH-G120R (Fig. 4AGo). In addition, enhancement of MCF-MUT cell spreading observed with exogenous hGH stimulation was suppressed with hGH-G120R treatment in the presence of 100 nM exogenous hGH to the same level as that observed under serum-free conditions for MCF-MUT (Fig. 4BGo). Thus, the enhanced cell spreading observed in MCF-hGH cells is completely abrogated with the use of a competitive hGH receptor antagonist.



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  		Figure 4. A, Inhibition of the hGH receptor by the nondimerizing receptor antagonist hGH-G120R prevents the enhancement of cell spreading by autocrine hGH. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). hGH-G120R was used at a concentration of 1000 nM. Cell spreading assays were performed as described in Materials and Methods. Results represent the mean ± SD of triplicate determinations. The results presented are representative of at least three (usually five to seven) independent experiments. a, Nonsignificant; c, P < 0.001 [differences between paired (bracketed) values]. ***, P < 0.001 (cell spreading at 30 min vs. that at the beginning of the experiment). B, Inhibition of hGH receptor-specific exogenous hGH enhancement of MCF-MUT cell spreading by hGH-G120R. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). Single cells were incubated in collagen-coated dishes in SFM alone or in SFM supplemented with 100 nM hGH (B). After the indicated time periods, dishes were washed to remove nonadherent cells as described in Materials and Methods. Cell spreading (percentage of the total number of attached cells; B) was measured and presented as the mean ± SD of triplicate determinations (n = 10). The results presented are representative of at least three (usually five to seven) independent experiments. c, P < 0.001 [differences between paired (bracketed) values]. ***, P < 0.001 (cell spreading at 30 min vs. that at the beginning of the experiment).

 
Cell spreading and actin stress fiber formation in MCF-hGH and MCF-MUT cells
GH has been previously demonstrated to stimulate the reorganization of the actin cytoskeleton (13). The morphological differences observed between MCF-hGH and MCF-MUT (Fig. 1Go, A—I and J–R) could, therefore, be expected to result from differences in actin cytoskeletal organization. We visualized cellular filamentous actin using TRITC-labeled phalloidin. MCF-hGH cells displayed rapid formation of filamentous actin-containing complexes accumulated most prominently within filipodia (green arrows) extending from marginal edges of the cell beginning at 5 min (Fig. 5KGo) and continuing to 15 min (Fig. 5LGo). Stress fibers first appeared at 30 min (Fig. 5MGo) oriented in a parallel manner to the long axis of the cell [as previously reported in spreading cells (24)] and continued to elongate as spreading progressed over 4 h to give the cells a pronounced elongated morphology (Fig. 5RGo). MCF-hGH cells also displayed numerous microspikes (blue arrows) oriented around the ventral margins perpendicular to the long axis of the cell at 30 min (Fig. 5MGo) and, to a lesser extent, from the leading edges of the cell at 45 min (Fig. 5NGo). Filamentous, actin-rich mature filipodia were obvious by 2, 3, and 4 h (Fig. 5Go, P–R, respectively) in MCF-hGH cells. MCF-MUT cells contained only small patches of filamentous actin distributed in the cytoplasm and at the cell membrane (Fig. 5BGo) or short linear deposits of filamentous actin in the cell margins (Fig. 5CGo) and demonstrated a rounded, symmetrical, and characteristically epithelial morphology as spreading occurred over 4 h (Fig. 5IGo). In addition, MCF-MUT cells demonstrated leading cellular processes (yellow arrows) at 2 h (Fig. 5GGo) at the cell margins from which filipodia developed at 3 h (Fig. 5HGo). MCF-MUT cells also demonstrated microspike activity, but it was distributed symmetrically around the cell periphery (Fig. 5FGo), in contrast to the distribution pattern observed in MCF-hGH cells. Thus, autocrine hGH-dependent changes in mammary carcinoma cell morphology are accompanied by differences in filamentous actin organization.



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  		Figure 5. Confocal laser scanning microscopic analysis of actin filament organization in MCF-MUT and MCF-hGH cells during cell spreading on a collagen matrix. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). MCF-MUT (A–I) and MCF-hGH (J–R) cells were allowed to spread on collagen-coated glass coverslips in SFM, fixed at the indicated time periods, and permeabilized. Filamentous actin within the cell was visualized with TRITC-labeled phalloidin. Control MCF-MUT cells displayed short bundles of actin filaments symmetrically distributed around the peripheral ring of the cell (C). In contrast, MCF-hGH cells possessed elongated actin fibers distributed in a polarized manner throughout the cell (M). Five minutes after plating, MCF-hGH cells displayed extensions of immature filipodia (green arrows) from the cell periphery that made contact with the substrate (K). At 30 min (M), MCF-hGH cells had flattened lengthwise, with maturing filipodia becoming evident at 45 min and 1 h (N and O) and fully developed, actin-rich filipodia observed at 2–4 h (P–R, respectively). Microspikes (blue arrows) were evident in MCF-hGH cells arising from the ventral edges perpendicular to the long axis of the cell at 30 min (M) and at 45 min from the leading edges of the cell (N). Scale bar, 25 µm.

 
Distribution of phosphotyrosine in MCF-hGH and MCF-MUT cells during cell spreading
Several studies have demonstrated the importance of tyrosine phosphorylation in cell spreading to extracellular matrix adhesion (25, 26). The distribution of phosphotyrosine in MCF-hGH and MCF-MUT cells plated on collagen-coated substrata in SFM for different time intervals was therefore examined by confocal laser scanning microscopy (Fig. 6Go, A–I and J–R). Phosphotyrosine-containing proteins were detected by use of the monoclonal antibody PY20. Both MCF-hGH and MCF-MUT cells demonstrated initial phosphotyrosine staining localized to the cell periphery (Fig. 6Go, J–K and A and B), with a perinuclear localization of the protein becoming evident at 15 min in both MCF-hGH (Fig. 6LGo) and MCF-MUT (Fig. 6CGo) cells. An elevated level of cytoplasmic phosphotyrosine was noted in MCF-hGH cells at each of the nine time intervals (Fig. 6Go, J–R), which increased in intensity with advancing time, in contrast to MCF-MUT cells, which consistently exhibited low levels of phosphotyrosine throughout the 4-h time course (Fig. 6Go, A-I). This difference was maintained as both cell lines achieved maximal spreading over the 4-h time course. Cytoplasmic phosphotyrosine aggregates were evident in MCF-hGH cells at 45 min, 1 h, 2 h, and 3 h (Fig. 6Go, N–Q, respectively), which disappeared by 4 h as MCF-hGH cells exhibited a more uniform distribution of phosphotyrosine in the same distinct perinuclear localization (Fig. 6RGo). In contrast to MCF-hGH cells, phosphotyrosine aggregates were not present in MCF-MUT cells at any time interval, as localization generally occurred in a diffuse pattern throughout the cell with some perinuclear localization evident at 2, 3, and 4 h (Fig. 6Go, G–I).



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  		Figure 6. Confocal laser scanning microscopic analysis of the distribution of phosphotyrosine within MCF-MUT and MCF-hGH during cell spreading on a collagen substrate. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). MCF-MUT (A–I) and MCF-hGH (J–R) cells were allowed to spread on collagen-coated glass coverslips in SFM and fixed at the indicated time periods. Phosphotyrosine was visualized with a monoclonal antiphosphotyrosine antibody (PY20) and detected with a Cy3-labeled second antibody. MCF-MUT cells demonstrated low levels of cytoplasmic phosphotyrosine (A–I), in sharp contrast to MCF-hGH cells (J–R). A prominent, granular distribution of phosphotyrosine was visible at 1, 2, and 3 h (O–Q) in a cytoplasmic localization in MCF-hGH cells changing to a uniform perinuclear localization instead by 4 h (R). Note the increase in total intracellular (mainly cytoplasmic) phosphotyrosine over advancing time in MCF-hGH cells (K–R) vs. control MCF-MUT cells (B–I), which demonstrated minimal differences in cytoplasmic phosphotyrosine levels over the course of 4 h. Scale bar, 25 µm.

 
Autocrine hGH enhances JAK2 tyrosine phosphorylation during cell spreading on a collagen matrix
GH-dependent tyrosine phosphorylation within the cell is mediated by the receptor-associated JAK2 kinase (27, 28). To determine whether JAK2 is differentially tyrosine phosphorylated in MCF-hGH and MCF-MUT cells upon attachment to a collagen substrate, we immunoprecipitated JAK2 from cell extracts prepared from MCF-hGH and MCF-MUT cells plated for the various indicated time intervals on collagen-coated substrata. SDS-PAGE and subsequent Western blotting for phosphotyrosine revealed a time-dependent increase in the phosphotyrosine content of JAK2 in MCF-hGH cells, but not MCF-MUT cells (Fig. 7AGo). JAK2 protein was identified as a single band at 130 kDa. In contrast, MCF-MUT cells demonstrated markedly lower levels and minimal kinetic changes in JAK2 tyrosine phosphorylation throughout the 4-h time course (Fig. 7AGo). Expression of JAK2 has previously been demonstrated to be equivalent between MCF-MUT and MCF-hGH (11). Equal amounts of JAK2 protein were immunoprecipitated at all time points, as indicated by the loading control (Fig. 7BGo). Thus, autocrine production of hGH in mammary carcinoma cells increases the phosphotyrosine content of JAK2 upon attachment to a collagen matrix.



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  		Figure 7. JAK2 tyrosine phosphorylation in MCF-MUT and MCF-hGH cells during cell spreading on a collagen matrix. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). MCF-MUT and MCF-hGH cells were serum deprived and replated onto collagen-coated dishes for the times indicated. Cell lysates were prepared and equalized for protein content, and JAK2 was immunoprecipitated with rabbit polyclonal anti-JAK2 as described in Materials and Methods. Immunoprecipitates were subjected to SDS-PAGE and Western blot analysis for phosphotyrosine using PY20 monoclonal antibody. The loading control for JAK2 is shown in B. Phosphorylated-JAK2 and JAK2 were detected as bands migrating at 130 kDa. The data are representative of at least three separate experiments.

 
Colocalization of JAK2 with phosphotyrosine during cell spreading
To determine whether JAK2 was appropriately spatially distributed to mediate tyrosine phosphorylation of proteins during cell spreading, we examined the colocalization of JAK2 with phosphotyrosine by confocal laser scanning microscopy. Cells were plated in SFM on a collagen substrate, fixed at the indicated time intervals, permeabilized, and subjected to dual labeling with a monoclonal antiphosphotyrosine antibody (PY20) and a polyclonal anti-JAK2 antibody. MCF-hGH cells (Fig. 8Go, J–R) demonstrated sharp colocalization of JAK2 with phosphotyrosine in a perinuclear distribution initially upon attachment (Fig. 8LGo). In contrast, MCF-MUT cells demonstrated a diffuse colocalization of JAK2 with phosphotyrosine (Fig. 8Go, A–I). A marked granular colocalization of PY20 with JAK2 in a cytoplasmic distribution was obvious in MCF-hGH cells at 30 min, 45 min, 1 h, 2 h, and 3 h (Fig. 8Go, M–Q), but was absent in MCF-MUT cells not only for the corresponding time points, but also throughout the 4-h time course (Fig. 8Go, A–I).



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  		Figure 8. Colocalization of JAK2 with phosphotyrosine in MCF-MUT and MCF-hGH cells during spreading on a collagen matrix. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). MCF-MUT and MCF-hGH cells were allowed to spread on collagen-coated glass coverslips in SFM, fixed at the indicated time points, and permeabilized. MCF-MUT (A–I) and MCF-hGH (J–R) cells were dual labeled with a mouse monoclonal antibody to phosphotyrosine (PY20) and a rabbit polyclonal antibody to JAK2. Detection was achieved using a Cy3-labeled antimouse IgG for phosphotyrosine (red) and a fluorescein isothiocyanate-labeled antirabbit IgG for JAK2 (green). Colocalization of JAK2 and phosphotyrosine is indicated in yellow. MCF-MUT cells displayed diffuse colocalization of JAK2 with PY20 throughout the time course (A–I). In contrast, immunoreactivity within MCF-hGH cells for JAK2 colocalization with PY20 was observed as large, punctate structures distributed in a cytoplasmic manner (N–Q). A distinct perinuclear colocalization of JAK2 with phosphotyrosine was evident by 30 min in MCF-hGH cells (M). Scale bar, 25 µm.

 
Autocrine hGH-induced cell spreading is inhibited by the JAK2 inhibitor, AG 490
To determine whether the increased rate of cell spreading in MCF-hGH compared with MCF-MUT cells was indeed due to autocrine hGH-mediated hyperphosphorylation of JAK2, we used the JAK2-specific inhibitor, AG 490 (29, 30). MCF-hGH and MCF-MUT cells were serum deprived and treated with 20 µM tryphostin AG 490 for 12 h before detachment, replated, and subjected to spreading assays on collagen substrata. Spreading of MCF-hGH cells cultured in 20 µM AG 490 was suppressed at 30 min, 1 h, 2 h, 3 h, and 4 h to the level of cell spreading observed with MCF-MUT cells cultured in either the presence or absence of AG 490 (Fig. 9Go). A similar effect on MCF-hGH cell spreading, but to a lesser extent, was observed when cells were treated with 10 µM AG 490 (data not shown). Thus, the enhancement in the rate of cell spreading stimulated by autocrine hGH is abrogated by the use of a JAK2 tyrosine kinase-specific inhibitor.



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  		Figure 9. Inhibition by the JAK2 protein tyrosine kinase (PTK) inhibitor AG 490 of the rate of MCF-hGH cell spreading on a collagen matrix. AG 490 was used at the indicated concentration for 12 h in SFM. Cell spreading assays were performed as described in Materials and Methods. Results represent the mean ± SD of triplicate determinations. The results presented are representative of at least three (usually five to seven) independent experiments. *, P < 0.01; **, P < 0.001 (MCF-MUT vs. MCF-hGH for the corresponding time intervals). +, P < 0.01; ++, P < 0.001 (MCF-hGH in SFM vs. MCF-hGH in 20 µM AG 490-supplemented medium for the corresponding time intervals).

 
Overexpression of the wild-type JAK2 protein enhances cell spreading in MCF-hGH, but not MCF-MUT, cells
To verify the role of JAK2 in the enhanced cell spreading observed in MCF-hGH cells, we transiently transfected MCF-hGH and MCF-MUT cells with an expression plasmid encoding for JAK2 containing residues 1–1129 (pRc/CMV mJAK2) and, for control purposes, with the empty vector (pRc/CMV) (18).

MCF-hGH cells demonstrated constitutive activation of the overexpressed JAK2 tyrosine kinase activity, as demonstrated by an increased level of JAK2 tyrosine phosphorylation upon transient transfection of JAK2 cDNA. The tyrosine phosphorylation was, however, commensurate with the JAK2 protein level compared with endogenous levels of JAK2 tyrosine phosphorylation (Fig. 10AGo) in vector and untransfected control cells (Fig. 10AGo). In contrast, overexpression of JAK2 in MCF-MUT cells did not result in constitutive activation of the overexpressed kinase, as a similar level of JAK2 tyrosine phosphorylation was observed between the transfected and untransfected control cells (Fig. 10AGo). Overexpression of JAK2 in both cell lines was confirmed by Western immunoblot analysis (Fig. 10BGo). JAK2 migrated as a 130-kDa protein, with increased levels of the protein observed in cell lysates of both MCF-hGH and MCF-MUT cells, after JAK2 cDNA transfection compared with the levels of endogenous JAK2 in both cell lines (Fig. 10AGo). Transient transfection of JAK2 cDNA significantly increased the rate of spreading in MCF-hGH, but not MCF-MUT, cells. Treatment with 20 µM AG 490 of MCF-hGH cells transiently transfected with JAK2 cDNA (Fig. 11Go) prevented the increased rate of cell spreading observed upon transient transfection of JAK2 cDNA in SFM alone (Fig. 10CGo). In addition, MCF-hGH cell spreading in the presence of AG 490 was suppressed close to the level observed with either transfected or untransfected MCF-MUT cells allowed to spread in the presence or absence of AG 490 at 30 min and 1 h (Fig. 11Go). Thus, it is apparent that the increase in the rate of cell spreading stimulated by autocrine production of hGH is JAK2 dependent.



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  		Figure 10. A and B, Overexpression of JAK2 in MCF-MUT and MCF-hGH cells upon transient transfection of JAK2 cDNA. MCF-MUT and MCF-hGH cells were grown to subconfluence and transiently transfected for 12 h with either the pRc/CMV mJAK2 expression plasmid or the vector alone (pRc/CMV), as described in Materials and Methods. Cell extracts were prepared and subjected to SDS-PAGE. A, Western blot analysis for tyrosine phosphorylation of JAK2 was performed using PY20 as described in Materials and Methods. B, Control blot for overexpression of transiently transfected JAK2 in MCF-MUT and MCF-hGH cells using rabbit polyclonal JAK2 antiserum as described in Materials and Methods. Phosphorylated JAK2 and JAK2 were detected as proteins migrating at 130 kDa. The data are representative of at least three separate experiments. C, Effect of overexpression of JAK2 on the rate of MCF-MUT and MCF-hGH cell spreading on a collagen matrix. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). MCF-MUT and MCF-hGH cells were grown to subconfluence and transiently transfected for 12 h with pRc/CMV mJAK2 or pRc/CMV vector alone as described in Materials and Methods. Cell spreading (percentage of the total number of attached cells) was measured as described in Materials and Methods, and values presented are the mean ± SD of triplicate determinations (n = 10). The results presented are representative of at least three (usually five to seven) independent experiments. a, Nonsignificant; b, P < 0.01; c, P < 0.001 [differences between paired (bracketed) values]. ***, P < 0.001 (differences between cell spreading at 30 min, 1 h, 2 h, 3 h, and 4 h vs. the beginning of the experiment).

 


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  		Figure 11. Inhibition by AG 490 of the rate of cell spreading on a collagen substrate of MCF-hGH cells transiently transfected with pRc/CMV mJAK2. MCF-MUT and MCF-hGH cells were grown to subconfluence and transiently transfected with either the pRc/CMV mJAK2 expression plasmid or the pRc/CMV vector control for 12 h, as described in Materials and Methods. Cells were treated with 20 µM AG 490 in SFM or were left untreated in SFM for an additional 12 h before being detached and subjected to cell spreading assays as described in Materials and Methods. Cell spreading (percentage of the total number of attached cells) was measured, and values presented are the mean ± SD of triplicate determinations (n = 10). The results presented are representative of at least three (usually five to seven) independent experiments. a, Nonsignificant; c, P < 0.001 [differences between paired (bracketed) values]. ***, P < 0.001 (differences between cell spreading at 30 min and 1 h vs. the beginning of the experiment).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated here that autocrine-produced hGH dramatically increases the rate of human mammary carcinoma cell spreading on a collagen substrate. This is concordant with our previous demonstration of hGH-stimulated reorganization of the actin cytoskeleton (13) and promotion of tubulin polymerization (14). GH has also previously been demonstrated to promote cell spreading (but not attachment) of fetal islet cells in vitro (31) and to promote the motile behavior of phagocytes characterized by the formation of branched lamellipodia and membrane ruffling (32). We further demonstrate here that autocrine-produced hGH is a more potent stimulator of mammary carcinoma cell spreading than exogenously administered hGH. This is particularly evident in molar terms, as autocrine production of hGH from MCF-hGH results in an extracellular concentration of approximately 100 pM hGH (11) in comparison to 100 nM exogenously added hGH. Such increased molar potency of autocrine hGH was also observed for mammary carcinoma cell proliferation, and some possible mechanisms are discussed (11). The effect of autocrine-produced hGH on human mammary carcinoma cell spreading is mediated via the hGH receptor, as we are able to inhibit the effect of autocrine hGH on cell spreading with the hGH receptor-specific antagonist B2036 (33) (Kaulsay, K. K., T. Zhu, K. O. Lee, and P. E. Lobie, manuscript in preparation). Also, the effect of hGH on cell morphology and spreading in this system is independent of IGF-I, as these cells have been demonstrated not to express IGF-I in the presence or absence of autocrine hGH (11).

We demonstrate that the hGH-dependent increase in the rate of cell spreading is dependent on the kinase activity of JAK2. To our knowledge, this is the first demonstration of the involvement of the Janus kinases in cell motility. JAK2 has been proposed to mediate GH-dependent tyrosine phosphorylation within the cell (34) and tyrosine phosphorylation has been demonstrated to be an important regulator of cell spreading in various cell types (25, 27, 35). We observed here that autocrine production of hGH allowed for an increase in JAK2 tyrosine phosphorylation and an increase in the level of cytoplasmic phosphotyrosine during the course of cell spreading. Thus, autocrine hGH must be interacting with signals from the extracellular matrix to increase tyrosine phosphorylation of JAK2. It is interesting that no change in JAK2 phosphorylation was observed during the course of cell spreading in the absence of hGH, indicating that cell spreading or integrin engagement by itself does not result in or require the activation of JAK2. The mechanism by which the extracellular matrix and autocrine hGH interact to produce an increase in the tyrosine phosphorylation of JAK2 requires delineation. In this regard it is interesting that the JAK2-associated protein, SH2-Bß, recently described by Carter-Su and colleagues (36), has been reported to be a potent cytoplasmic activator of JAK2 (37) and also a regulator of actin cytoskeletal dynamics in response to GH (38). Utilization of SH2-Bß upon integrin engagement would provide a mechanism for the increase in JAK2 tyrosine phosphorylation observed during cell spreading in MCF-hGH cells.

One possible mechanism for the interaction between autocrine hGH and the extracellular matrix during cell spreading is via p125 focal adhesion kinase (FAK) (39). FAK has been postulated to play a critical role in the cellular response to the extracellular matrix and in cell morphology and motility (40). We previously demonstrated that hGH stimulates the association of JAK2 and FAK and the subsequent JAK2-dependent activation of FAK and two of its substrates, namely paxillin and tensin (15). We also observed the subsequent formation of a multiprotein signaling complex centered around p130Cas and CrkII (16). Components of this complex have been identified and in addition to p130Cas and CrkII include c-Src, c-Fyn, IRS-1, c-Cbl, Nck, paxillin, tensin, the p85 regulatory subunit of PI-3 kinase, C3G, SHC, Grb-2, and Sos-1 (16). Many of the molecules reported in this complex are involved in the regulation of cellular morphology. Some examples include the following. 1) Formation of a p130Cas-CrkII complex has been demonstrated to be sufficient for cell migration (17), and concordantly, p130 Cas-deficient fibroblasts exhibit significant defects in cell movement (41). 2) The hGH-stimulated reorganization of the actin cytoskeleton is PI-3 kinase dependent (13), and we demonstrated that CrkII-dependent cytoskeletal reorganization is due to enhanced PI-3 kinase activity (Goh, E. L. K., T. Zhu, and P. E. Lobie, manuscript in preparation). 3) c-Src kinase activity is required for hepatocyte growth factor-induced cellular motility (42), and c-Src bound through its SH2 domain to phosphorylated FAK facilitates c-Src SH3 domain interactions with p130Cas, thereby promoting the formation of a ternary complex of FAK, c-Src, and p130Cas (43). 4) c-Cbl is associated with c-Cbl-associated protein (CAP), which is involved in the formation of stress fibers and focal adhesion complexes (44) and also with the p85 regulatory subunit of PI-3 kinase (45). We observed that hGH also uses c-Cbl to increase PI-3 kinase activity (Goh, E. L. K., and P. E. Lobie, unpublished observations), and c-Cbl has also been demonstrated to be required for PI-3 kinase-dependent macrophage spreading (46). 5) The Drosophila Nck homolog, DOCK, is involved in cell migration events (47). Other molecules, which are not contained in the multiprotein signaling complex but are also stimulated by hGH, such as p38 mitogen-activated protein kinase (48, 49), have been reported to be involved in actin reorganization (49, 50) and cell spreading (51). Further studies are therefore required to determine which pathway downstream of JAK2 is required for the autocrine hGH enhancement of mammary carcinoma cell spreading.

We observed a hGH-dependent increase in the spreading of human mammary carcinoma cells in vitro. It is therefore plausible to expect that hGH in vivo may promote the metastasis of human mammary carcinoma. Indeed, the formation of numerous branched filipodia in cells, as observed here for MCF-hGH cells, is associated with increased cell locomotion and metastases (52, 53, 54). Interestingly, however, hGH has actually been demonstrated to inhibit the pulmonary metastasis of prostate cancer in tumor-bearing animals (55, 56). It is likely that hGH may exert differential cell type-specific effects on cell motility or metastatic behavior. In addition, the metastatic potential of a tumor is positively correlated with size (57). In the prostate cancer model (55, 56), hGH did not increase tumor size, whereas we previously reported (11) that autocrine hGH stimulates the hyperproliferation of mammary carcinoma cells, and hGH exposure could therefore be expected to result in increased tumor volume in vivo. Exogenously administered hGH has been reported to result in an increase in mammary glandular size in primate models (58). Thus, any potential involvement of hGH in mammary carcinoma metastasis may simply be due to a hGH-dependent increase in tumor size. Interestingly, the serum level of a related cytokine, IL-6, has been positively correlated with the metastatic behavior of breast cancer in Japanese women (59). Several genes have now been identified that correlate the invasion and metastasis of breast cancer (60). One such gene product is ß-catenin, whose expression is negatively correlated with tumor cell dissemination (61, 62). In a screen of close to 600 genes (Mertani, H. C., T. Zhu, G. Morel, K. O. Lee, and P. E. Lobie, manuscript in preparation), we found that ß-catenin is one of approximately 30 genes up-regulated by the autocrine production of hGH in mammary carcinoma cells. Autocrine hGH production in mammary carcinoma cells may therefore result in high proliferative activity without promotion of metastasis, as is observed in medullary carcinoma of the breast with high expression of ß-catenin (63). In vivo studies are obviously required to determine the behavior of autocrine hGH-producing mammary carcinoma cells. We have also demonstrated here that a competitive hGH receptor antagonist (hGH-G120R) completely abrogates the effect of autocrine-produced hGH on cell spreading. It therefore remains to be determined whether hGH receptor antagonists will be of clinical utility to diminish any potential metastatic progression of breast cancer stimulated by autocrine hGH. Initial studies using a nude mouse model of metastasis may prove useful to test this hypothesis (62). Previous studies have used such a model system to study the role of the fibroblast growth factors in metastasis of human mammary carcinoma (64).

In summary, we demonstrated that autocrine production of hGH in mammary carcinoma cells resulted in an enhanced rate of cell spreading that is dependent on the kinase activity of JAK2. Of pivotal importance is to determine whether the in vitro changes in cell proliferation (11) and morphology (this study) stimulated by autocrine production of hGH in mammary carcinoma cells translates into in vivo behavioral changes in the cell that negatively impact on the clinical prognosis.


   Footnotes
 
1 This work was supported by the National Science and Technology Board of Singapore (to P.E.L.) and a National Medical Research Council grant, Singapore (to K.O.L.). Back

Received September 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mol JA, Garderen EV, Selman PJ, Wolfswinkel J, Rijnberk A, Rutteman GR 1995 Growth hormone mRNA in mammary gland tumors of dogs and cats. J Clin Invest 95:2028–2034
  2. Mol JA, Henzen-Logman SC, Hageman P, Misdorp W, Blankenstein MA, Rijnberk A 1995 Expression of the gene encoding growth hormone in the human mammary gland. J Clin Endocrinol Metab 80:3094–3096[Abstract/Free Full Text]
  3. Lincoln DT, Waters MJ, Breiphol W, Sinowatz F, Lobie PE 1990 Growth hormone receptor expression in the proliferating rat mammary gland. Acta Histochem 40:47–49
  4. Jammes H, Gaye P, Belair L, Djiane J 1991 Identification and characterization of growth hormone receptor mRNA in the mammary gland. Mol Cell Endocrinol 75:27–35[CrossRef][Medline]
  5. IIkbahar Y, Wu K, Thodarson G, Talamentes F 1995 Expression and distribution of mRNAs for GH receptor and GHBP in mice during pregnancy. Endocrinology 136:386–392[Abstract]
  6. Glimm DR, Baracos VE, Kennely JJ 1990 Molecular evidence for the presence of growth hormone receptors in the bovine mammary gland. J Endocrinol 126:R5–R8
  7. Sobrier ML, Duquesnoy P, Duriez B, Amselem S, Goossens M 1993 Expression and binding properties of two isoforms of the human growth hormone receptor. FEBS Lett 319:16–20[CrossRef][Medline]
  8. Mertani HC, Garcia-Caballero T, Lambert A, Gerard F, Palayer C, Boutin JM, Vonderhaar BK, Waters MJ, Lobie PE, Morel G 1998 Cellular expression of growth hormone and prolactin receptors in human breast disorders. Int J Cancer 79:202–211[CrossRef][Medline]
  9. Plaut K, Ikeda M, Vonderhaar BK 1993 Role of growth hormone and insulin-like growth factor-I in mammary development. Endocrinology 133:1843–1848[Abstract]
  10. Feldman M, Ruan W, Cunningham BC, Wells JA, Kleinberg DL 1993 Evidence that the growth hormone receptor mediates differentiation and development of the mammary gland. Endocrinology 13:1602–1608
  11. Kaulsay KK, Mertani HC, Tornell J, Morel G, Lee KO, Lobie PE 1999 Autocrine stimulation of human mammary carcinoma cell proliferation by human growth hormone. Exp Cell Res 250:35–50[CrossRef][Medline]
  12. Verhasselt B, Van Damme J, van Larebeke N, Put W, Bracke M, De Potter C, Mareel M 1992 Interleukin-1 is a motility factor for human breast carcinoma cells in vitro: additive effect with interleukin-6. Eur J Cell Biol 59:449–457[Medline]
  13. Goh ELK, Pircher TJ, Wood TJJ, Norstedt G, Graichen R, Lobie PE 1997 Growth hormone-induced reorganization of the actin cytoskeleton is not required for STAT5 (signal transducer and activator of transcription-5)-mediated transcription. Endocrinology 138:3207–3215[Abstract/Free Full Text]
  14. Goh ELK, Pircher TJJ, Lobie PE 1998 Growth hormone promotion of tubulin polymerization stabilizes the microtubule network and protects against colchicine-induced apoptosis. Endocrinology 139:4364–4372[Abstract/Free Full Text]
  15. Zhu T, Goh ELK, Lobie PE 1998 Growth hormone stimulates the tyrosine phosphorylation and association of p125 focal adhesion kinase (FAK) with JAK2. J Biol Chem 273:10682–10689[Abstract/Free Full Text]
  16. Zhu T, Goh ELK, LeRoith D, Lobie PE 1998 Growth hormone stimulates the formation of a multiprotein signaling complex involving p130Cas and Crk II. J Biol Chem 273:33864–33875[Abstract/Free Full Text]
  17. Klemke RL, Leng J, Molander R, Brooks PC, Vuori K, Cheresh DA 1998 CAS/Crk coupling serves as a "molecular switch" for induction of cell migration. J Cell Biol 140:961–972[Abstract/Free Full Text]
  18. Frank SJ, Woelsung Y, Yanming Z, Goldsmith JR, Gilliland G, Jiang J, Sakai I, Kraft AS 1995 Regions of the JAK2 tyrosine kinase required for coupling to the growth hormone receptor. J Biol Chem 270:14776–14785[Abstract/Free Full Text]
  19. Liu N, Mertani HC, Norstedt G, Tornell J, Lobie PE 1997 Mode of the autocrine/paracrine mechanism of growth hormone action. Exp Cell Res 237:196–206[CrossRef][Medline]
  20. Acheson A, Sunshine JL, Rutishauser U 1991 NCAM polysialic acid can regulate both cell-cell and cell-substrate interactions. J Cell Biol 114:143–153[Abstract/Free Full Text]
  21. Yap AS, Manley SW 1993 Contact inhibition of cell spreading: a mechanism for the maintenance of thyroid cell aggregation in vitro. Exp Cell Res 208:121–127[CrossRef][Medline]
  22. Parini P, Angelin B, Lobie PE, Norsdedt G, Rudling M 1995 Growth hormone specifically stimulates low density lipoprotein receptor expression in cultured human hepatoma cells. Endocrinology 136:3767–3773[Abstract]
  23. Fuh G, Wells JA 1995 Prolactin receptor antagonists that inhibit the growth of breast cancer cell lines. J Biol Chem 270:13133–13137[Abstract/Free Full Text]
  24. Safiejko-Mroczka B, Bell PB, Jr 1998 Distribution of cytoskeletal proteins in neomycin-induced protrusions of human fibroblasts. Exp Cell Res 242:495–514[CrossRef][Medline]
  25. Guan JL, Shalloway D 1992 Regulation of focal adhesion kinase-associated protein tyrosine by both cellular adhesion and oncogenic transformation. Nature 358:690–692[CrossRef][Medline]
  26. Burridge K, Turner CE, Romer LH 1992 Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to the extracellular matrix: a role in cytoskeletal assembly. J Cell Biol 119:893–902[Abstract/Free Full Text]
  27. Argetsinger LS, Campbell GS, Yang X, Witthuhn BA, Silvennoinen O, Ihle JN, Carter-Su C 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74:237–244[CrossRef][Medline]
  28. Lobie PE 1999 Signal transduction through the growth hormone receptor. In: Bengtsson BA (ed) Growth Hormone. Kluwer, Boston, pp 17–35
  29. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia, Lapidot Z, Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, Roifman CM 1996 Inhibition of acute lymphoblastic leukemia by a Jak-2 inhibitor. Nature 379:645–648[CrossRef][Medline]
  30. Harris KW, Hu XJ, Schultz S, Arcasoy MO, Forget BG, Clare N 1998 The distal cytoplasmic domain of the erythropoietin receptor induces granulocytic differentiation in 32D cells. Blood 92:1219–1224[Abstract/Free Full Text]
  31. Hu Y, Ding Y, Wang Y, Zhang H, Gu Z, Zhang H, Zhu Y 1992 Effect of human growth hormone on the proliferation of human fetal islet cells in vitro. Chin Med J 105:721–725[Medline]
  32. Calduch-Giner JA, Sitja-Bobadilla A, Alvarez-Pellitero P, Perez -Sanchez J 1997 Growth hormone as an in vitro phagocyte-activating factor in the gilthead sea bream. Cell Tissue Res 287:535–540[CrossRef][Medline]
  33. Goffin V, Bernichtein S, Carriere O, Bennett WF, Kopchick JJ, Kelly PA 1999 The human growth hormone antagonist B2036 does not interact with the prolactin receptor. Endocrinology 140:3853–3856[Abstract/Free Full Text]
  34. VanderKuur J, Wang X, Zhang L, Campbell G, Allevato G, Billestrup N, Norstedt G, Carter-Su C 1994 Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase. J Biol Chem 269:21709–21717[Abstract/Free Full Text]
  35. Yap AS, Keast JR, Manley SW 1994 Thyroid cell spreading and focal adhesion formation depend upon protein tyrosine phosphorylation and actin microfilaments. Exp Cell Res 210:306–314[CrossRef][Medline]
  36. Rui L, Mathews LS, Hotta K, Gustafson TA, Carter-Su C 1997 Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling. Mol Cell Biol 17:6633–6644[Abstract]
  37. Rui L, Carter-Su C 1999 Identification of SH2-Bbeta as a potent cytoplasmic activator of the tyrosine kinase Janus kinase 2. Proc Natl Acad Sci USA 96:7172–7177[Abstract/Free Full Text]
  38. Herrington J, Rui L, Carter-Su C SH2-Bß is required for growth hormone-induced regulation of the actin cytoskeleton. 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, p 387 (Abstract)
  39. Cary LA, Guan JL 1999 Focal adhesion kinase in integrin-mediated signaling. Front Biosci 4:D102–113
  40. Ilic D, Furuta Y, Yamamoto T 1997 Focal adhesion kinase: at the crossroads of signal transduction. J Cell Sci 110:401–407[Abstract]
  41. Honda H, Nakamoto T, Sakai R, Hirai H 1999 p130(Cas), an assembling molecule of actin filaments, promotes cell movement, cell migration, and cell spreading in fibroblasts. Biochem Biophys Res Commun 262:25–30[CrossRef][Medline]
  42. Rahimi N, Hung W, Tremblay E, Saulnier R, Elliott B 1998 c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells. J Biol Chem 273:33714–33721[Abstract/Free Full Text]
  43. Schlaepfer DD, Hunter T 1998 Integrin signaling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol 8:151–157[CrossRef][Medline]
  44. Ribon V, Herrera R, Kay BK, Saltiel AR 1998 A role for CAP, a novel, multifunctional Src homology 3 domain-containing protein in formation of actin stress fibers and focal adhesions. J Biol Chem 273:4073–4080[Abstract/Free Full Text]
  45. Liu YC, Altman A 1998 Cbl: complex formation and functional implications. Cell Signal 10:377–385[CrossRef][Medline]
  46. Meng F, Lowell CA 1998 A ß1 integrin signaling pathway involving Src- family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration. EMBO J 17:4391–4403[CrossRef][Medline]
  47. Garrity PA, Rao Y, Salecker I, McGlade J, Pawson T, Zipursky SL 1996 Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein. Cell 85:639–650[CrossRef][Medline]
  48. Hodge C, Liao J, Stofega M, Guan K, Carter-Su C, Schwartz J 1998 Growth hormone stimulates phosphorylation and activation of elk-1 and expression of c-fos, egr-1, and junB through activation of extracellular signal-regulated kinases 1 and 2. J Biol Chem 273:31327–31336[Abstract/Free Full Text]
  49. Zhu T, Lobie PE 2000 Janus kinase 2 dependent activation of p38 mitogen-activated protein kinase by growth hormone: resultant transcriptional activation of ATF-2 and CHOP, cytoskeletal organization and mitogenesis. J Biol Chem 275:2103–2114[Abstract/Free Full Text]
  50. Matsumoto T, Yokote K, Tamura K, Takemoto M, Ueno H, Saito Y, Mori S 1999 Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration. J Biol Chem 274:13954–13960[Abstract/Free Full Text]
  51. Ogura M, Kitamura M 1998 Oxidant stress incites spreading of macrophages via extracellular signal-regulated kinases and p38 mitogen-activated protein kinase. J Immunol 161:3569–3574[Abstract/Free Full Text]
  52. Schillaci R, Luparello C, Minafra S 1989 Type I and I-trimer collagens as substrates for breast carcinoma cells in culture. Effect on growth rate, morphological appearance and actin organization. Eur J Cell Biol 48:135–141[Medline]
  53. Luparello C, Schillaci R, Pucci-Minafra I, Minafra S 1990 Adhesion, growth and cytoskeletal characteristics of 8701-BC breast carcinoma cells cultured in the presence of type V collagen. Eur J Cancer 26:231–240
  54. Li C, McCarthy JB, Furcht LT, Fields GB 1997 An all-D amino acid peptide model of {alpha}1(IV)531–543 from type IV collagen binds the {alpha}3ß1 integrin and mediates tumor cell adhesion, spreading, and motility. Biochemistry 36:15404–15410[CrossRef][Medline]
  55. Torosian MH, Donoway RB 1991 Growth hormone inhibits tumor metastasis. Cancer 67:2280–2283[CrossRef][Medline]
  56. Torosian MH 1993 Growth hormone and prostate cancer growth and metastasis in tumor-bearing animals. J Pediatr Endocrinol 6:93–97[Medline]
  57. Makhlouf HR, Burke AP, Sobin LH 1999 Carcinoid tumors of the ampulla of Vater: a comparison with duodenal carcinoid tumors. Cancer 85:1241–1249