Differential effects of exogenous and autocrine growth hormone on LNCaP prostate cancer cell proliferation and survival

The prostate gland is regulated by multiple hormones and growth factors that may also affect prostate tumorigenesis. Growth hormone (GH) contributes to prostate development and function, but the direct effects of GH on prostate cancer cells are not well understood. The expression of endogenous GH in prostate cancer cell lines has also been observed, suggesting the potential for an effect of autocrine GH. In the present study, we measure the levels of GH and GH receptor (GHR) mRNA in multiple prostate cancer and normal prostate‐derived cell lines, and compare the effects of exogenous and autocrine GH on LNCaP prostate cancer cell proliferation and apoptosis, and the associated signal transduction pathways. We found that GHR and GH expression were higher in the prostate cancer cell lines, and that exogenous GH increased LNCaP cell proliferation, but had no effect on apoptosis. In contrast, autocrine GH overexpression reduced LNCaP cell proliferation and increased apoptosis. The distinct actions of exogenous and autocrine GH were accompanied by differences in the involvement of GHR‐associated signal transduction pathways, and were paralleled by an alteration in the subcellular localization of GHR, in which autocrine GH appeared to sequester GHR in the Golgi and endoplasmic reticulum. This alteration of GHR trafficking may underlie a distinct mode of GH‐mediated signaling associated with the effect of autocrine GH. These findings clarify the potential effects of GH on prostate cancer cell function, and indicate that the activity of autocrine GH may be distinct from that of endocrine GH in prostate cancer cells. J. Cell. Biochem. 114: 1322–1335, 2013. © 2012 Wiley Periodicals, Inc.     

Studies with a growth hormone antagonist and dual-fluorescent confocal microscopy demonstrate that the full-length human growth hormone receptor, but not the truncated isoform, is very rapidly internalized independent of Jak2-Stat5 signaling.

We have investigated trafficking of two negative regulators of growth hormone receptor (GHR) signaling: a human, truncated receptor, GHR1-279, and a GH antagonist, B2036. Fluorescent-labeled growth hormone (GH) was rapidly internalized by the full-length GHR, with >80% of the hormone internalized within 5 min of exposure to GH. In contrast, <5% of labeled GH was internalized by cells expressing truncated GHR1-279. Using another truncated receptor, GHR1-317 fused to enhanced green fluorescent protein (EGFP), we have exploited fluorescence energy transfer to monitor the trafficking of ligand-receptor complexes. The data confirmed that internalization of this truncated receptor is very inefficient. It was possible to visualize the truncated GHR1-317-EGFP packaged in the endoplasmic reticulum, its rapid movement in membrane bound vesicles to the Golgi apparatus, and subsequent transport to the cell membrane. The GH antagonist, B2036, blocked Jak2-Stat5-mediated GHR signaling but was internalized with a similar time course to native GH. The results: 1) demonstrate the rapid internalization of GH when studied under physiological conditions; 2) confirm the hypothesis that internalization of cytoplasmic domain truncated human GHRs is very inefficient, which explains their dominant negative action; and 3) show that the antagonist action of B2036 is independent of receptor internalization.     

Growth hormone receptor expression in the nucleus and cytoplasm of normal and neoplastic cells

 Growth hormone (GH) exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific receptors which trigger a phosphorylation cascade, resulting in the modulation of numerous signalling pathways dictating gene expression. A panel of five monoclonal antibodies was used in mapping the presence and somatic distribution of the GH receptor by immunohistochemistry in normal and neoplastic tissues and cultured cells of human, rat and rabbit origin. A wide distribution of the receptor was observed in many cell types. Not all cells expressing cytoplasmic GH receptors displayed nuclear immunoreactivity. In general, the relative proportion of positive cells and intensity of staining was higher in neoplastic cells than in normal tissue cells. Immunoreactivity showed subcellular localisation of the GH receptor in cell membranes and was predominantly cytoplasmic, but strong nuclear immunoreaction was also apparent in many instances. Intense immunoreactivity was also observed in the cellular Golgi area of established cell lines and cultured tissue-derived cells in exponential growth phase, indicating cells are capable of GH receptor synthesis. The presence of intracellular GH receptor, previously documented in normal tissues of mostly animal origin, is the result of endoplasmic reticulum and Golgi localisation. Heterogeneity of immunoreactivity was found in normal and neoplastic tissue with a variable range of positive cells. The nuclear localisation of immunoreactivity is the result of nuclear GH receptor/binding protein, identically to the cytosolic and plasma GH-binding protein, using a panel of five monoclonal antibodies against the GH receptor extracellular region. The expression of GH receptors, not only on small proliferating tumour cells such as lymphocytes, but also on well differentiated cells including keratinocytes, suggests that GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion, differentiation and maintenance. Accepted: 4 July 1997     

Autocrine/paracrine actions of growth hormone in human melanoma cell lines

Melanoma is the most aggressive skin cancer. Its aggressiveness is most commonly attributed to ERK pathway mutations leading to constitutive signaling. Though initial tumor regression results from targeting this pathway, resistance often emerges. Interestingly, interrogation of the NCI-60 database indicates high growth hormone receptor (GHR) expression in melanoma cell lines. To further characterize melanoma, we tested responsiveness to human growth hormone (GH). GH treatment resulted in GHR signaling and increased invasion and migration, which was inhibited by a GHR monoclonal antibody (mAb) antagonist in WM35, SK-MEL 5, SK-MEL 28 and SK-MEL 119 cell lines. We also detected GH in the conditioned medium (CM) of human melanoma cell lines. GHR, JAK2 and STAT5 were basally phosphorylated in these cell lines, consistent with autocrine/paracrine GH production. Together, our results suggest that melanomas are enriched in GHR and produce GH that acts in an autocrine/paracrine manner. We suggest that GHR may constitute a therapeutic target in melanoma.     

Spatial range of autocrine signaling: modeling and computational analysis

Autocrine loops formed by growth factors and their receptors have been identified in a large number of developmental, physiological, and pathological contexts. In general, the spatially distributed and recursive nature of autocrine signaling systems makes their experimental analysis, and often even their detection, very difficult. Here, we combine Brownian motion theory, Monte Carlo simulations, and reaction-diffusion models to analyze the spatial operation of autocrine loops. Within this modeling framework, the ability of autocrine cells to recapture the endogenous ligand and the distances traveled by autocrine ligands are explicitly related to ligand diffusion coefficients, density of surface receptors, ligand secretion rate, and rate constants of ligand binding and endocytic internalization. Applying our models to study autocrine loops in the epidermal growth factor receptor system, we find that autocrine loops can be highly localized--even at the level of a single cell. We demonstrate how the variations in molecular and cellular parameters may "tune" the spatial range of autocrine signals over several orders of magnitude: from microns to millimeters. We argue that this versatile regulation of the spatial range of autocrine signaling enables autocrine cells to perceive a broad spectrum of environmental information.     

Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase.

The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels of the cloned liver GH receptor bound to anti-phosphotyrosine antibody, suggesting that the cloned GH receptor is tyrosyl phosphorylated in vivo. GH-GH receptor complexes purified from transfected COS-7 cells using anti-GH antibody incorporated 32P when incubated with [gamma-32P]ATP, indicating association of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present at low levels in the COS-7 cells. To test whether a higher level of GH receptor phosphorylation would be observed when the GH receptor was expressed in a different cell line, GH receptor cDNAs were stably transfected into mouse L and CHO cells, which have few or no endogenous GH receptors, and RIN5-AH cells, which do express endogenous GH receptors. In vivo tyrosyl phosphorylation of the cloned GH receptor in mouse L cells and in vitro phosphorylation of the cloned GH receptor in both L and CHO cells were higher than in transfected COS-7 cells but still substantially lower than in untransfected 3T3-F442A cells. Significantly increased 32P incorporation into tyrosyl residues in GH receptors in the in vitro kinase assay was demonstrated for GH receptors isolated from the transfected RIN5-AH cells. These studies show that the cloned liver GH receptor can be tyrosyl phosphorylated when expressed in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific.     

PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma

PDGFs and their cognate tyrosine kinase alpha- and beta-receptors are involved in multiple tumor-associated processes including autocrine growth stimulation of tumor cells, stimulation of tumor angiogenesis and recruitment and regulation of tumor fibroblasts. The recent development of clinically useful PDGF antagonists, like STI571/Glivec, has increased the interest in PDGF receptors as cancer drug targets. Autocrine PDGF receptor signaling occurs in certain malignancies characterized by mutational activation of PDGF or PDGF receptors, for instance, dermatofibrosaracoma protuberans, gastrointestinal stromal tumors, and hypereosinophilic syndrome. The roles of PDGF in regulation of tumor angiogenesis and tumor fibroblasts are more general, and probably occur in most common solid tumors. Concerning tumor angiogenesis recent studies have predominantly focused on the importance of PDGF receptor signaling for tumor pericyte recruitment. PDGF receptors in the tumor stroma have also attracted attention as interesting drug targets because of their function as regulators of tumor interstitial fluid pressure, tumor transvascular transport and tumor drug uptake. In summary, the improved understanding of the role of PDGF signaling in tumor biology, and the introduction of PDGF antagonists, has set the stage for a continued development of PDGF antagonists as novel cancer drugs.     

Stochastic model of autocrine and paracrine signals in cell culture assays

Autocrine signaling systems are commonly studied under cell culture conditions. In a typical cell culture assay, a layer of liquid medium covers a random two-dimensional dispersion of cells, which secrete ligands. In a growing number of experiments, it is important to characterize the spatial range of autocrine and paracrine cell communication. Currently, the spatial distribution of diffusing signals can be analyzed only indirectly, from their effects on the intracellular signaling or physiological responses of autocrine cells. To directly characterize the spatial range of secreted ligands, we propose a stochastic model for autocrine cell cultures and analyze it using a combination of analytical and computational tools. The two main results derived within the framework of this model are 1), an expression for the fraction of autocrine trajectories, i.e., the probability for a ligand to be trapped by the same cell from which it has been secreted; and 2), an expression for the spatial distribution of trapping points of paracrine trajectories. We test these analytical results by stochastic simulations with efficient Brownian dynamics code and apply our model to analyze the spatial operation of autocrine epidermal growth factor receptor systems.     

Growth hormone action in rat insulinoma cells expressing truncated growth hormone receptors

Transfection of the insulin-producing rat islet tumor cell line RIN-5AH with a full length cDNA of the rat hepatic growth hormone (GH) receptor (GH-R1-638) augments the GH-responsive insulin synthesis in these cells. Using this functional system we analyzed the effect of COOH-terminal truncation of the GH receptor. Two mutated cDNAs encoding truncated GH receptors, GH-R1-294 and GH-R1-454, respectively, were generated by site-directed mutagenesis and transfected into the RIN cells. Both receptor mutants were expressed on the cell surface and displayed normal GH binding affinity. Whereas GH-R1-638 had a molecular mass of about 110 kDa, GH-R1-294 and GH-R1-454 showed molecular masses of 49 and 80 kDa, respectively. Cells expressing GH-R1-454 internalized GH to a similar extent as cells transfected with the full length receptor and the parent cell line, but GH-R1-294-expressing cells showed a markedly reduced capability of GH internalization. In contrast to cells transfected with GH-R1-638, none of the cell lines expressing truncated GH receptors exhibited any increase of the GH-stimulated insulin production. We conclude that domains within the COOH-terminal half of the cytoplasmic part of the GH receptor are required for transduction of the signal for GH-stimulated insulin synthesis, whereas cytoplasmic domains proximal to the transmembrane region are involved in receptor-mediated GH internalization.     

Growth factor/growth factor receptor loops in autocrine growth regulation of human prostate cancer DU145 cells

Autocrine growth factors produced by epithelial cells mediate the development and proliferation of neoplastic human prostate tissue. Various approaches have been used to down-regulate neoplastic growth of prostate cancer using natural flavonoids, soluble receptors, pseudo-ligands, monoclonal antibodies and tyrosine kinase inhibitors (tyrphostins). Selected growth factor/growth factor receptor loops (mainly TGFα/EGFR and IGFs/IGFIR) have been proposed as regulators of prostate cancer cell growth. We have previously determined that blockade of IGFIR or VEGF2R signaling pathways by tyrphostin AG1024 and SU1498 inhibits autocrine growth and viability of DU145 cells in vitro. Recently, we compared the activity of AG1024 and SU1498 with the inhibiting effect of tyrphostin A23 (a selective inhibitor of EGFR). The results described in this paper confirm that DU145 cells do not produce IGFI or EGF. In contrast, DU145 cells produce a great amount of VEGF, much more than TGFα (about 60-fold), and VEGF may be the real autocrine growth factor of the investigated cells. The results indicate that the growth of DU145 may be regulated by at least three autocrine loops: TGFα/EGFR, IGFII/IGFIR and VEGF/VEGFR2. Neither AG1024 nor SU1498 affected the production of TGFα substantially, which excludes the possibility that IGFRs or VEGFR2 inhibitors arrest the growth of these cells by inhibition of synthesis and/or secretion of TGFα. The obtained data indicate that all tree investigated tyrphostins (AG1024, SU1498 and A23) inhibit signal transmission by Akt (PKB), ERK(1/2), Src and STAT in a similar manner. A comparison of the effects of the investigated tyrphostins indicates that TGFα, IGFII and VEGF stimulate cell growth by affecting the same signaling pathway. The hypothesis was confirmed by the effect of the investigated tyrphostins on activation of EGFR. All these inhibitors decreased phosphorylation of EGFR to the same extent, and after the same time of incubation with cell culture. These results strongly suggest that stimulation of EGFR kinase is the main step in the initiation of mitogen signaling in DU145 cells, regardless of the type of ligand (TGFα, IGFs or VEGF) and their specific receptors.     

Effect of cell density on induction of growth hormone receptors by dexamethasone in primary cultured rat hepatocytes

The effect of cell density on the regulation of growth hormone (GH) receptors was studied by measuring specific binding of [125I]hGH to primary cultured hepatocytes with or without dexamethasone, which induces GH receptors. In cell cultures without dexamethasone, the cell density did not affect the level of binding of labeled GH appreciably. On the other hand, in the presence of dexamethasone, which induced an increase in the level of GH receptors on the cells, GH-binding by cultured cells at low cell density (3.3 x 10(4) cells/cm2) was about one-third of that of cells at high cell density (10(5) cells/cm2). Scatchard plot analysis showed that the cell-density dependent change in induction of GH binding, by dexamethasone was due to change in the number of binding sites without significant change in their affinity. The binding capacity of glucocorticoid receptors, measured as specific binding of [3H]dexamethasone to the hepatocytes, was not significantly affected by cell density. These results suggest that cell density modulates GH receptor induction by dexamethasone via events after glucocorticoid receptor binding.     

Rapid turnover of the platelet-derived growth factor receptor in sis-transformed cells and reversal by suramin. Implications for the mechanism of autocrine transformation

In cells transformed by either v-sis or c-sis, the majority of the newly synthesized platelet-derived growth factor (PDGF) receptors fail to reach the cell surface and are rapidly degraded. This rapid turnover (t1/2 less than 30 min) appears to result from interaction of the sis gene product with the PDGF receptor in the endoplasmic reticulum and/or Golgi apparatus during their intracellular routing from the endoplasmic reticulum to the plasma membrane or extracellular compartment. Several lines of evidence support this hypothesis. 1) Both the 160-kDa precursor and the intracellular 180-kDa mature form of the PDGF receptor possessed ligand binding activity for PDGF; 2) both the 160-kDa precursor and the 180-kDa mature form of the receptor in sis-transformed cells were found to be activated (phosphorylated); 3) protamine, a competitive inhibitor for PDGF or v-sis gene product binding to the cell-surface receptor, did not affect the rapid turnover of the PDGF receptor in sis-transformed cells; 4) suramin, an inhibitor for PDGF or v-sis gene product binding to the PDGF receptor, not only reversed the rapid turnover of the PDGF receptor in sis-transformed cells, but also increased the secretion of sis gene products; and 5) rapid turnover of the PDGF receptor was only observed in sis-transformed cells but not in cells transformed by other oncogenes. We suggest that the persistence of a mitogenic signal from cellular organelles, arising from the intracellular interaction of sis gene products with newly synthesized PDGF receptors, is the mechanism for autocrine transformation by sis.     

Subcellular trafficking of growth hormone receptor and Jak2 under ligand exposure.

In CHO cells, growth hormone stimulation induces a rapid degradation of mature and precursor forms of its receptor, but does not affect Jak2 concentration. Confocal analysis of the receptor and of specific markers for subcellular localization shows that ligand exposure induced the disappearance of cell surface receptors, while some receptors seem to be sequestered in the endoplasmic reticulum (ER) and in the Golgi apparatus. Using a tagged version of Jak2 (HA-Jak2) and double immuno-fluorescence analysis with anti-HA and anti-Stat5 antibodies, we demonstrate that ligand stimulation induces Stat5 nuclear accumulation while Jak2 remains localized in the cytoplasm. Immunoblots of nuclear extracts confirm the Jak2 nuclear exclusion.     

Phosphorylation of highly purified growth hormone receptors by a growth hormone receptor-associated tyrosine kinase

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.     

Multimeric Growth Hormone Receptor Complexes Serve as Signaling Platforms

Growth hormone (GH) signaling is required for promoting longitudinal body growth, stem cell activation, differentiation, and survival and for regulation of metabolism. Failure to adequately regulate GH signaling leads to disease: excessive GH signaling has been connected to cancer, and GH insensitivity has been reported in cachexia patients. Since its discovery in 1989, the receptor has served a pivotal role as the prototype cytokine receptor both structurally and functionally. Phosphorylation and ubiquitylation regulate the GH receptor (GHR) at the cell surface: two ubiquitin ligases (SCF^βTrCP2 and CHIP) determine the GH responsiveness of cells by controlling its endocytosis, whereas JAK2 initiates the JAK/STAT pathway. We used blue native electrophoresis to identify phosphorylated and ubiquitylated receptor intermediates. We show that GHRs occur as ∼500-kDa complexes that dimerize into active ∼900-kDa complexes upon GH binding. The dimerized complexes act as platforms for transient interaction with JAK2 and ubiquitin ligases. If GH and receptors are made in the same cell (autocrine mode), only limited numbers of ∼900-kDa complexes are formed. The experiments reveal the dynamic changes in post-translational modifications during GH-induced signaling events and show that relatively simple cytokine receptors like GHRs are able to form higher order protein complexes. Insight in the complex formation of cytokine receptors is crucially important for engineering cytokines that control ligand-induced cell responses and for generating a new class of therapeutic agents for a wide range of diseases.