EGF-mediated phosphorylation of extracellular signal-regulated kinases in osteoblastic cells

Epidermal growth factor (EGF) induces a rapid increase in the phosphorylation of extracellular signal-regulated kinases (ERKs) in the human osteosarcoma osteoblastic cell line G292 and in primary cultures of rat osteoblastic cells. This phosphorylation is transient and time-dependent. Maximal stimulation is attained within 1 min in G292 and within 5 min in rat osteoblastic cells. Enzymatic activity in G292 cells is also induced rapidly after EGF stimulation. Western blot analysis revealed that enhancement of the phosphorylation of ERKs in the EGF-stimulated cells is not due to an increase in ERK protein, since EGF-treatment does not lead to an increase in the absolute amount of ERKs present even after 2 days of stimulation. The pattern of expression of the ERKs observed in the two cell types differs in the apparent molecular weights observed. The most slowly migrating immunoreactive protein (～45 kDa) in normal rat osteoblastic cells is ERK1, identified by an ERK1-selective antiserum. The same antiserum reacts only weakly with one of the ERK proteins (44 kDa) blotted from the human osteosarcoma cell line G292. Phorbol 12-myristate 13-acetate (PMA) is also capable of inducing ERK phosphorylation, albeit to a lesser degree. The combination of PMA and EGF does not produce a greater response than EGF alone. The role of protein kinase C (PKC) in the EGF-stimulated ERK signaling pathway was further examined by inhibition of PKC with the staurosporine analog, CGP41251, and by down-regulation of PKC via chronic treatment with PMA. Chronic PMA treatment results in a partial inhibition of the EGF-mediated phosphorylation. CGP41251 completely abolishes the increased ERK activity produced by PMA, but the effect of EGF in this regard is potentiated. We conclude that PKC and EGF act through parallel pathways to stimulate ERK phosphorylation and activity. The inhibitor studies, in addition, indicate that activation of PKC may moderate the actions of the EGF pathway via a tonic inhibitory feedback. © 1995 Wiley-Liss, Inc.     

Asbestos-mediated CREB phosphorylation is regulated by protein kinase A and extracellular signal-regulated kinases 1/2

Asbestos is a ubiquitous, naturally occurring fiber that has been linked to the development of malignant and fibrotic lung diseases. Asbestos exposure leads to apoptosis, followed by compensatory proliferation, yet many of the signaling cascades coupled to these outcomes are unclear. Because CREs (Ca(2+)/cAMP-response elements) are found in the promoters of many genes important for regulation of proliferation and apoptosis, CREB (CRE binding protein) is likely to play an important role in the development of asbestos-mediated lung injury. To explore this possibility, we tested the hypotheses that asbestos exposure leads to CREB phosphorylation in lung epithelial cells and that protein kinase A (PKA) and extracellular signal-regulated kinases 1/2 (ERK1/2) are central regulators of the CREB pathway. Persistent CREB phosphorylation was observed in lung sections from mice following inhalation of crocidolite asbestos. Exposure of C10 lung epithelial cells to crocidolite asbestos led to rapid CREB phosphorylation and apoptosis that was decreased by the inhibition of PKA or ERK1/2 using the specific inhibitors H89 and U0126, respectively. Furthermore, crocidolite asbestos selectively induced a sustained increase in MAP kinase phosphatase-1 mRNA and protein. Silencing CREB protein dramatically reduced asbestos-mediated ERK1/2 phosphorylation, yet significantly increased the number of cells undergoing asbestos-induced apoptosis. These data reveal a novel and selective role for CREB in asbestos-mediated signaling through pathways regulated by PKA and ERK1/2, further providing evidence that CREB is an important regulator of apoptosis in asbestos-induced responses of lung epithelial cells.     

Stimulation of glucose transport in osteoblastic cells by parathyroid hormone and insulin-like growth factor I

Insulin and parathyroid hormone (PTH) regulate glucose metabolism in bone cells. In order to differentiate between the effects of these hormones and to compare the potency of insulin with that of insulin-like growth factor (IGF) I, we treated rat bone-derived osteoblastic (PyMS) cells for different time periods and at different concentrations with insulin, IGF I, or PTH, and measured [1-(14)C]-2-deoxy-D-glucose (2DG) uptake and incorporation of D-[U-(14)C] glucose into glycogen. 2DG uptake was Na-independent with an apparent affinity constant (K (M)) of ~2 mmol/l. Expression of the high affinity glucose transporters (GLUT), GLUT1 and GLUT3 but not of GLUT4, was found by Northern and Western analysis. Similar to the findings with primary rat osteoblasts, but distinct from those in rat fibroblasts, 2DG uptake and glycogen synthesis were increased in this cell line after exposure to low concentrations (0.1 nmol/l and above) of PTH. IGF I at low doses (0.3 nmol/l and above) or insulin at higher doses (1 nmol/l and above) stimulated 2DG uptake and [(3)H] thymidine incorporation into DNA. 2DG transport was enhanced already after 30 min of IGF I treatment whereas the effect of PTH became significant after 6 h. It is concluded that IGF I rather than insulin may be a physiological regulator of 2DG transport and glycogen synthesis in osteoblasts.     

Angiotensin II phosphorylation of extracellular signal-regulated kinases in rat anterior pituitary cells

We studied the effects of ANG II on extracellular signal-regulated kinase (ERK)1/2 phosphorylation in rat pituitary cells. ANG II increased ERK phosphorylation in a time- and concentration-dependent way. Maximum effect was obtained at 5 min at a concentration of 10-100 nM. The effect of 100 nM ANG II was blocked by the AT1 antagonist DUP-753, by the phospholipase C (PLC) inhibitor U-73122, and by the MAPK kinase (MEK) antagonist PD-98059. The ANG II-induced increase in phosphorylated (p)ERK was insensitive to pertussis toxin blockade and PKC depletion or inhibition. The effect was also abrogated by chelating intracellular calcium with BAPTA-AM or TMB-8 by depleting intracellular calcium stores with a 30-min pretreatment with EGTA and by pretreatment with herbimycin A and PP1, two c-Src tyrosine kinase inhibitors. It was attenuated by AG-1478, an inhibitor of epidermal growth factor receptor (EGFR) activation. Therefore, in the rat pituitary, the increase of pERK is a Gq- and PLC-dependent process, which involves an increase in intracellular calcium and activation of a c-Src tyrosine kinase, transactivation of the EGFR, and the activation of MEK. Finally, the response of ERK activation by ANG II is altered in hyperplastic pituitary cells, in which calcium mobilization evoked by ANG II is also modified.     

Drosophila extracellular signal-regulated kinase involves the insulin-mediated proliferation of Schneider cells

The Drosophila insulin pathway is involved in the control of the proliferation and size of the cell. The stimulation of Schneider cells with human insulin has been observed to activate Drosophila extracellular signal regulated kinase (DERK). However, the role of DERK in the regulation of proliferation is unknown. In this study, we have identified a role of DERK in the proliferation of Drosophila Schneider cells. The inhibition of DERK activity by the overexpression of DMKP-3, an ERK-specific mitogen-activated protein kinase (MAPK) phosphatase, inhibited G(1) to S phase cell cycle progression as well as bromodeoxyuridine (BrdU) incorporation, which were previously increased by human insulin. However, DMKP-3 overexpression did not significantly reduce cell size that was also enlarged by insulin treatment, which suggests the specificity of the ERK pathway in proliferation but not for cell size. G1 to S phase cell cycle progression and BrdU incorporation were also reduced by catalytically inactive DMKP-3 mutant, and they may be acquired by the trapping of DERK into cytosol. The depletion of DERK or DMKP-3 by inhibitory double-stranded RNA decreased and increased BrdU incorporation, respectively. Thus, we propose that DERK is involved in the proliferation of Schneider cells via the insulin pathway.     

The lutropin/choriogonadotropin receptor-induced phosphorylation of the extracellular signal-regulated kinases in leydig cells is mediated by a protein kinase a-dependent activation of ras

The pathways involved in activation of the ERK1/2 cascade in Leydig cells were examined in MA-10 cells expressing the recombinant human LH receptor (hLHR) and in primary cultures of rat Leydig cell precursors. In MA-10 cells expressing the recombinant hLHR, human choriogonadotropin-induced activation of ERK1/2 is effectively inhibited by overexpression of a cAMP phosphodiesterase (a manipulation that blunts the human choriogonadotropin-induced cAMP response), by addition of H89 (a selective inhibitor of protein kinase A), or by overexpression of the heat-stable protein kinase A inhibitor, but not by overexpression of an inactive mutant of this inhibitor. Stimulation of hLHR did not activate Rap1, but activated Ras in an H89-sensitive fashion. Addition of H89 to MA-10 cells that had been cotransfected with a guanosine triphosphatase-deficient mutant of Ras almost completely inhibited the hLHR-mediated activation of ERK1/2. We also show that 8-bromo-cAMP activates Ras and ERK1/2 in MA-10 cells and in primary cultures of rat Leydig cells precursors in an H89-sensitive fashion, whereas a cAMP analog 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP (8CPT-2Me-cAMP) that is selective for cAMP-dependent guanine nucleotide exchange factor has no effect. Collectively, our results show that the hLHR-induced phosphorylation of ERK1/2 in Leydig cells is mediated by a protein kinase A-dependent activation of Ras.     

Stimulation of leukemia inhibitory factor receptor degradation by extracellular signal-regulated kinase

Leukemia inhibitory factor (LIF) signals via the heterodimeric receptor complex comprising the LIF receptor alpha subunit (LIFRalpha) and the common signal transducing subunit for interleukin-6 cytokine receptors, gp130. This study demonstrates that in different cell types, the level of LIFRalpha decreases during treatment with LIF or the closely related cytokine oncostatin M (OSM). Moreover, insulin and epidermal growth factor induce a similar LIFRalpha down-regulation. The regulated loss of LIFRalpha is specific since neither gp130 nor OSM receptor beta shows a comparable change in turnover. LIFRalpha down-regulation correlates with reduced cell responsiveness to LIF. Using protein kinase inhibitors and point mutations in LIFRalpha, we demonstrate that LIFRalpha down-regulation depends on activation of extracellular signal-regulated kinase 1/2 and phosphorylation of the cytoplasmic domain of LIFRalpha at serine 185. This modification appears to promote the endosomal/lysosomal pathway of the LIFRalpha. These results suggest that extracellular signal-regulated kinase-activating factors like OSM and growth factors have the potential to lower specifically LIF responsiveness in vivo by regulating LIFRalpha half-life.     

Distinct pathways of extracellular signal-regulated kinase activation by growth factors,fibronectin and parathyroid hormone 1-34

Growth factors, hormones, and matrix proteins regulate osteoblast proliferation and differentiation, acting through cognate receptors. Since each of the receptors are coupled to a variety of distinct signal transduction pathways, in this report we evaluated whether there is a common convergent intermediate step that allows cross-talk among the various pathways. Since extracellular signal-regulated kinases 1 and 2 (Erk1/2) play a role in mitogenesis and differentiation processes, we evaluated the effects of various osteotrophic factors on Erk1/2 phosphorylation in osteoblasts. Osteoblasts isolated from the metaphyseal marrow (MM) and diaphyseal marrow (DM) of 4-6 week old male rat longitudinal bones were grown to confluency and Erk1/2-phosphorylation was evaluated using antibodies that recognized either the total or the phosphorylated form of the kinase. There was very little Erk1/2 phosphorylation in cells kept in suspension. Both MM and DM cells attached to fibronectin (FN), demonstrated Erk1/2 phosphorylation that persisted for at least up to 8h. Platelet-derived growth factor AB (PDGF-AB) induced a transient and robust Erk1/2 phosphorylation that was attenuated by 2h. Studies with specific inhibitors indicated that the effects of these factors were mediated by protein kinase C, by receptor tyrosine kinase, as well as by protein phosphatases. Parathyroid hormone (PTH 1-34), a bone anabolic agent however, caused a down-regulation of FN stimulated Erk1/2 phosphorylation in MM derived cells. The inhibitory effect of PTH was mediated through cAMP-dependent protein kinase A (PKA) activation. The data collectively suggest that a combination of diverse extracellular stimuli regulates Erk1/2 phosphorylation that may ultimately influence osteoblast proliferation and/or differentiation.     

Stimulation of macrophage urokinase expression by polyanions is protein kinase C-dependent and requires protein and RNA synthesis.

Highly charged polyanionic ligands of the scavenger receptor trigger macrophage secretion of urokinase-type plasminogen activator (uPA). In experiments reported here, we have investigated the intracellular and extracellular regulation of polyanion-induced macrophage plasminogen activation. Exposure of a macrophage cell line (RAW264.7) to either fucoidan or phorbol myristate acetate (PMA) stimulates the secretion of uPA, whereas calcium ionophore or dibutyryl cyclic AMP had no effect. Moreover, preincubation of macrophages with inhibitors of protein kinase C reduced (50-60%) the ability of both fucoidan and PMA to trigger the secretion of uPA, whereas aspirin and eicosatetraenoic acid had no effect. Both PMA and fucoidan treatment of RAW264.7 cells resulted in a rapid and transient increase in the steady state levels of uPA mRNA. However, in marked contrast to that observed with PMA, fucoidan-induced expression of RAW264.7 uPA activity was partially insensitive to cycloheximide and actinomycin D. In addition, fucoidan-induced uPA activity was detected in conditioned media in as little as 15 min, whereas PMA-induced uPA activity did not increase until 2 h. In addition to stimulating macrophage secretion of uPA, fucoidan bound uPA and had a small stimulatory affect on uPA activity. The binding does not interfere with the catalytic site on the B chain, or require the receptor binding or kringle domains on the A chain.     

Contribution of phospholipase D in endothelin-1-mediated extracellular signal-regulated kinase activation and proliferation in rat uterine leiomyoma cells

Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [3H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth.     

Endocytosis of ligand-human parathyroid hormone receptor 1 complexes is protein kinase C-dependent and involves beta-arrestin2. Real-time monitoring by fluorescence microscopy.

Endocytosis and intracellular trafficking of the human parathyroid hormone receptor subtype 1 (hPTH1-Rc) and its ligands was monitored independently by real-time fluorescence microscopy in stably transfected HEK-293 cells. Complexes of fluorescence-labeled parathyroid hormone (PTH)-(1-34) agonist bound to the hPTH1-Rc internalized rapidly at 37 degrees C via clathrin-coated vesicles, whereas fluorescent PTH-(7-34) antagonist-hPTH1Rc complexes did not. A functional C terminus epitope-tagged receptor (C-Tag-hPTH1-Rc) was immunolocalized to the cell membrane and, to a lesser extent, the cytoplasm. PTH and PTH-related protein agonists stimulated C-Tag-hPTH1-Rc internalization. Relocalization to the cell membrane occurred 1 h after removal of the ligand. Endocytosis of fluorescent PTH agonist-hPTH1-Rc complexes was blocked by the protein kinase C (PKC) inhibitor staurosporine but not by the specific protein kinase A inhibitor N-(2-(methylamino)ethyl)-5-isoquinoline-sulfonamide. Fluorescent PTH antagonist-hPTH1-Rc complexes were rapidly internalized after PKC activation by phorbol 12-myristate 13-acetate or thrombin, but not after stimulation of the cAMP/protein kinase A pathway by forskolin. In cells co-expressing the hPTH1-Rc and a green fluorescent protein-beta-arrestin2 fusion protein (beta-Arr2-GFP), PTH agonists stimulated beta-Arr2-GFP mobilization to the cell membrane. Subsequently, fluorescent PTH-(1-34)-hPTH1Rc complexes and beta-Arr2-GFP co-localized intracellularly. In conclusion, agonist-activated hPTH1-Rc internalization involves beta-arrestin mobilization and targeting to clathrin-coated vesicles. Our results also indicate that receptor occupancy, rather than receptor-mediated signaling, is necessary, although not sufficient, for endocytosis of the hPTH1-Rc. Activation of PKC, however, is absolutely required.     

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.     

Butyrate response factor 1 is regulated by parathyroid hormone and bone morphogenetic protein-2 in osteoblastic cells

Parathyroid hormone (PTH) exerts potent and diverse effects in bone and cartilage through activation of type 1 PTH receptors (PTH1R) capable of coupling to protein kinase A (PKA) and PKC. We have used macroarrays to identify zinc finger protein butyrate response factor-1 (BRF1) as a novel PTH regulated gene in clonal and normal osteoblasts of human and rodent origin. We further demonstrate that in human osteoblast-like OHS cells, biologically active hPTH(1-84) and hPTH(1-34) stimulate BRF1 mRNA expression in a dose- and time-dependent manner, while the amino-terminally truncated hPTH(3-84) which does not activate PTH1R has no effect. Moreover, using specific stimulators or inhibitors of PKA and PKC activity, the PTH-elicited BRF1 mRNA expression is mediated through the PKA signaling pathway. In mouse calvarial osteoblasts, BRF1 mRNA levels are upregulated by PTH(1-84) and reduced in response to bone morphogenetic protein 2 (BMP-2). Hence, our data showing that BRF1 is expressed in osteoblastic cells and regulated by PTH and BMP-2, suggest an important role for BRF1 in osteoblasts within the molecular network of PTH-dependent bone remodeling.     

Effect of parathyroid hormone and forskolin on cytoskeletal protein synthesis in cultured mouse osteoblastic cells

Parathyroid hormone (PTH) has been shown to cause transient cell shape changes in bone cells. We have examined the effects of parathyroid hormone and forskolin on the organization and expression of cytoskeletal proteins in cultured mouse endosteal osteoblastic cells. Analysis of [35S]methionine-labeled cytoskeletal proteins isolated on two-dimensional gel electrophoresis showed that PTH treatment (24 h) stimulated the de novo biosynthesis of actin, vimentin and tubulins in confluent cells, whereas forskolin had a minor effect despite a huge stimulation of cAMP production. This PTH-induced stimulation was associated with cell respreading following a mild and transitory cell retraction. PTH increased the synthesis of monomeric subunits of actin and beta-tubulins in subconfluent bone cells, whereas both monomeric and polymeric levels of beta-tubulins were increased in confluent osteoblasts. Under conditions reducing cell spreading, osteoblastic cells had initially high levels of unpolymerized subunits. In these poorly spread cells, parathyroid hormone or forskolin had no effect on the de novo synthesis of cytoskeletal proteins despite a marked elevation in intracellular cAMP levels. It is concluded that PTH affects the biosynthesis of cytoskeletal proteins in osteoblastic cells and that cAMP production does not seem to be directly involved. In addition, the effect of PTH is modulated by cell spreading and by the initial pool of cytoskeletal subunits.     

Inhibition of sphingosine kinase 1 suppresses proliferation of glioma cells under hypoxia by attenuating activity of extracellular signal-regulated kinase

Objectives: Sphingosine kinase (SphK), which is regulated by hypoxia, catalyses phosphorylation of sphingosine to produce sphingosine‐1‐phosphate, which stimulates invasiveness of gliomas. However, whether SphK is involved in proliferation of glioma cells under hypoxic conditions is not clearly understood. In this study, we have investigated the role of SphK in of proliferation glioma cells under hypoxia. Materials and methods: Effects of small interfering RNA (siRNA) on SphKs, SKI (inhibitor of SphK) and U0126 (inhibitor of ERK) on proliferation of glioma cells under hypoxia were studied using CCK‐8 assay and flow cytometry. Protein expression profiles were evaluated by Western blot analysis. Results:  SKI suppressed proliferation of glioma cells under hypoxia. Similarly, downregulation of SphKs by siRNA inhibited glioma cell proliferation, and the cell cycle was arrested in G₂/M phase when SphK1 was inhibited. In addition, inhibition of SphK1 attenuated phosphorylation of ERK in hypoxic conditions. Furthermore, U0126 markedly inhibited cell population growth and arrested cells in G₂/M as effectively as SKI. However, silencing SphK2 induced cell cycle arrest in the S phase and it showed little effect on hypoxia‐induced activation of ERK. Conclusions: SphK1 and SphK2 are involved in proliferation of glioma cells in hypoxic conditions through distinct signalling pathways. SphK1, but not SphK2, promotes cell population expansion in hypoxic conditions by activating ERK.