Brain-derived neurotrophic factor stimulates growth of pituitary melanotrope cells in an autocrine way

Brain-derived neurotrophic factor (BDNF) is expressed in the mammalian pituitary gland, in both the anterior and intermediate lobes, where its functional significance is unknown. Melanotrope cells in the intermediate pituitary lobe of the amphibian Xenopus laevis also produce BDNF, which co-exists in secretory granules with α-melanophore-stimulating hormone (α-MSH), a peptide that causes pigment dispersion in dermal melanophores during adaptation of the toad to a dark background. Xenopus melanotropes are highly plastic, undergoing very strong growth to support the high biosynthesis and release of α-MSH in black-adapted animals. In this study we have tested our hypothesis that this enhanced growth of the melanotrope is maintained by autocrine release of BDNF. Furthermore, since the extracellular-regulated kinase (ERK) pathway is a major component of BDNF signaling in neuronal plasticity, we investigated its involvement in melanotrope cell growth. For these purposes melanotropes were treated for 3 days in vitro, with either an anti-BDNF serum or a recombinant tropomyosin-receptor kinase B (TrkB) receptor fragment to eliminate released BDNF, or with the ERK inhibitor U0126. We also applied a novel inhibitor of the TrkB receptor, cyclotraxin-B, to test this receptor’s involvement in melanotrope cell growth regulation. All treatments markedly reduced melanotrope cell growth. Therefore, we conclude that autocrine release of BDNF and subsequent TrkB-dependent ERK-mediated signaling is important for melanotrope cell growth during its physiologically induced activation.     

Expression and characterization of the extracellular Ca(2+)-sensing receptor in melanotrope cells of Xenopus laevis

The extracellular Ca(2+)-sensing receptor (CaR) is expressed in many different organs in various species, ranging from mammals to fish. In some of these organs, this G protein-coupled receptor is involved in the control of systemic Ca(2+) homeostasis, whereas in other organs its role is unclear (e.g. in the pituitary gland). We have characterized the CaR in the neuroendocrine melanotrope cell of the intermediate pituitary lobe of the South African clawed toad Xenopus laevis. First, the presence of CaR mRNA was demonstrated by RT-PCR and in situ hybridization. Then it was shown that activation of the CaR by an elevated extracellular Ca(2+) concentration and different CaR-activators, including L-phenylalanine and spermine, stimulates both Ca(2+) oscillations and secretion from the melanotrope. Furthermore, it was revealed that activation of the receptor stimulates Ca(2+) oscillations through opening of voltage-operated Ca(2+) channels in the plasma membrane of the melanotropes. Finally, it was shown that the CaR activator L-phenylalanine could induce the biosynthesis of proopiomelanocortin in the intermediate lobe. Thus, in this study it is demonstrated that the CaR is present and functional in a defined cell type of the pituitary gland, the amphibian melanotrope cell.     

Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells

The calcium-sensing receptor (CaR) activation has recently been shown to modulate the ERK1 and ERK2 cascade in different cell lines. The present study investigated this pathway in human normal and tumoral parathyroid cells. In cells from normal parathyroids and almost all hyperplasia increasing extracellular calcium concentrations (Ca(o)(2+)) induced a significant activation of ERK1 and -2, the percent stimulation over basal activity (at 0.5 mM Ca(o)(2+)) being 545 +/- 140 and 800 +/- 205 in normal cells and 290 +/- 71 and 350 +/- 73 in hyperplasia at 1 and 2 mM Ca(o)(2+), respectively. This effect was mediated by CaR because it was mimicked by the receptor agonist gadolinium and neomycin. Basal and Ca(o)(2+)-stimulated ERK1 and -2 activity was nearly abolished by the PKC inhibitor calphostin C, and PKA changes did not affect ERK1 and -2 activity. PI3K blockade by wortmannin, known to prevent G protein betagamma subunit effect on ERK1 and -2, induced a 30% reduction of the Ca(o)(2+)-stimulated ERK1 and -2 activity. Adenomatous cells showed high PKC-dependent ERK1 and -2 activity in resting conditions that was unresponsive to high Ca(o)(2+). A role of MAPK on PTH secretion was suggested by the finding that PD98059, a specific MEK inhibitor, abolished the inhibitory effect of 1.5 mM Ca(o)(2+) on PTH release from normal parathyroid cells. In conclusion, these data first demonstrate that CaR activation, through the PKC pathway and, to a lesser extent, PI3K, increases ERK1 and -2 activity in normal parathyroid cells and this cascade seems to be involved in the modulation of PTH secretion by Ca(o)(2+). Interestingly, this signaling pathway is disrupted in parathyroid tumors.     

Activation of the extracellular calcium-sensing receptor initiates insulin secretion from human islets of Langerhans: involvement of protein kinases

The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca(2+) homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca(2+). CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulin-dependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the beta-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between beta-cells.     

Analysis of the melanotrope cell neuroendocrine interface in two amphibian species, Rana ridibunda and Xenopus laevis: a celebration of 35 years of collaborative research

This review gives an overview of the functioning of the hypothalamo-hypophyseal neuroendocrine interface in the pituitary neurointermediate lobe, as it relates to melanotrope cell function in two amphibian species, Rana ridibunda and Xenopus laevis. It primarily but not exclusively concerns the work of two collaborating laboratories, the Laboratory for Molecular and Cellular Neuroendocrinology (University of Rouen, France) and the Department of Cellular Animal Physiology (Radboud University Nijmegen, The Netherlands). In the course of this review it will become apparent that Rana and Xenopus have, for the most part, developed the same or similar strategies to regulate the release of α-melanophore-stimulating hormone (α-MSH). The review concludes by highlighting the molecular and cellular mechanisms utilized by thyrotropin-releasing hormone (TRH) to activate Rana melanotrope cells and the function of autocrine brain-derived neurotrophic factor (BDNF) in the regulation of Xenopus melanotrope cell function.     

Calcium-sensing receptor induces proliferation through p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase but not extracellularly regulated kinase in a model of humoral hypercalcemia of malignancy

Using H-500 rat Leydig cancer cells as a model of humoral hypercalcemia of malignancy (HHM), we previously showed that high Ca(2+) induces PTH-related peptide (PTHrP) secretion via the calcium-sensing receptor (CaR) and mitogen- and stress-activated kinases, e.g. MAPK kinase 1 (MEK1), p38 MAPK, and stress-activated protein kinase 1/c-Jun N-terminal kinase. Because cellular proliferation is a hallmark of malignancy, we studied the role of the CaR in regulating the proliferation of H-500 cells. Elevated Ca(2+) has a mitogenic effect on these cells that is mediated by the CaR, because the calcimimetic NPS R-467 also induced proliferation. Inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 MAPK but not MEK1 abolished the mitogenic effect. Activation of PI3K by elevated Ca(2+) was documented by phosphorylation of its downstream kinase, protein kinase B. Because protein kinase B activation promotes cell survival, we speculated that elevated Ca(2+) might protect H-500 cells against apoptosis. Using terminal uridine deoxynucleotidyl nick end labeling staining, we demonstrated that high Ca(2+) (7.5 mM) and NPS R-467 indeed protect cells against apoptosis induced by serum withdrawal compared with low Ca(2+) (0.5 mM). Because the CaR induces PTHrP secretion, it is possible that the mitogenic and antiapoptotic effects of elevated Ca(2+) could be indirect and mediated via PTHrP. However, blocking the type 1 PTH receptor with PTH (7-34) peptide did not alter either high Ca(2+)-induced proliferation or protection against apoptosis. Taken together, our data show that activation of PI3K and p38 MAPK but not of MEK1/ERK by the CaR promotes proliferation of H-500 cells as well as affords protection against apoptosis. These effects are likely direct without the involvement of PTHrP in an autocrine mode.     

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

Aims/hypothesis Proinsulin C-peptide is generally believed to be inert without any appreciable biological functions. However, it has been shown to modulate a variety of cellular processes important in the pathophysiology of diabetic complications. We therefore investigated the ability of C-peptide to stimulate intracellular signalling pathways in kidney proximal tubular cells, the altered activation of which may possibly be related to the development of diabetic nephropathy.Methods Extracellular signal-regulated kinase (ERK) and Akt phosphorylation were evaluated by western blotting. ERK activity was measured by in vitro kinase assay. Intracellular Ca²⁺ was evaluated by confocal imaging. The membrane and cytosol-associated fractions of protein kinase C (PKC) isoforms were evaluated by western blotting. Proliferation was assessed by thymidine incorporation assay.Results Using the opossum proximal tubular kidney cell line as a model, we demonstrated that at high picomolar to low nanomolar concentrations, C-peptide stimulates extracellular signal-regulated mitogen-activated kinase (3.3±0.1-fold over basal at 3 minutes) and phosphatidylinositol 3-kinase (4.1±0.05-fold over basal at 5 minutes). ERK activation was attenuated by pre-treatment with a PKC inhibitor and abolished by pertussis toxin. Elevations of intracellular [Ca²⁺] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC-. Pre-treatment with pertussis toxin abolished PKC-. C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pre-treatments. None of these effects is reproduced by scrambled C-peptide.Conclusions/interpretation This study provides evidence that C-peptide, within physiological concentration ranges, stimulates many signalling pathways in opossum kidney cells.Abbreviations Akt/PKB protein kinase B - DTT dithiothreitol - ERK extracellular signal-regulated kinase - GPCR G-protein coupled receptor - MAPK mitogen-activated protein kinase - OK opossum kidney - PI3-K phosphoinositide 3-kinase - PKC protein kinase C - PMA phorbol myristate acetate - PMSF phenylmethansulfonylfluoride - PTC proximal tubular cells - PTX pertussis toxin     

beta-Adrenergic stimulation of rat cardiac fibroblasts promotes protein synthesis via the activation of phosphatidylinositol 3-kinase.

beta -adrenergic agonists stimulate neonatal rat cardiac fibroblast growth, albeit the identity of the signaling event(s) remains equivocal. Isoproterenol (ISO) treatment increased intracellular cyclic AMP levels; however, cyclic AMP-elevating agents had no effect on protein synthesis. The tyrosine kinase inhibitor tyrphostin A25, and the inhibition of ras processing by the farnesyltransferase inhibitor BMS-191563 attenuated ISO-stimulated protein synthesis. Concomitant with increased protein synthesis, ISO stimulated extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) activity. The MEK1/2 inhibitor PD098059 abrogated ISO-stimulated ERK activity, albeit the increase in protein synthesis was unaffected. By contrast, LY294002 inhibited both ISO-stimulated PI3-K activity, and protein synthesis. ISO treatment did not increase the expression of transforming growth factor-beta(1)(TGF-beta(1)) mRNA, whereas a significant decrease in the steady-state mRNA level of TGF- beta(3)was observed. This latter effect was mimicked by cyclic AMP-elevating agents. Angiotensin II (AII) activation of the AT(1)receptor increased protein synthesis, but in contrast to ISO, the growth response was not inhibited by either tyrphostin A25 or BMS-191563, and was associated with the concomitant expression of both TGF-beta(1)and TGF-beta(3)mRNAs. Analogous to ISO, AII treatment increased ERK and PI3-K activity, and PI3-K was required for protein synthesis. These findings are the first to highlight the activation of PI3-K by a Gs(alpha)-coupled receptor, and its essential role in beta -adrenergic as well as AT(1)receptor-mediated protein synthesis in neonatal rat cardiac fibroblasts. However, despite the conserved role of PI3-K, additional disparate signaling pathways are recruited by ISO and AII, which may differentially influence fibroblast phenotype.     

Upregulation of urokinase-type plasminogen activator and inhibitor and gelatinase expression via 3 mitogen-activated protein kinases and PI3K pathways during the early development of osteoarthritis

OBJECTIVE: To examine whether upregulation of urokinase-type plasminogen activator (u-PA), PA inhibitor-1 (PAI-1), and gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] in early knee osteoarthritis (OA) of humans occurs through 3 major mitogen-activated protein kinases (MAPK): extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase signaling pathways, and the phosphatidylinositol 3-kinase (PI3K) signaling pathway. METHODS: Enzyme linked immunosorbent assay and gelatin zymography were used to investigate the effects of ERK 1/2 inhibitor U0126, JNK and p38 inhibitor SB203580, and PI3K inhibitor LY294002 on the secretion of u-PA, PAI-1, MMP-2, and MMP-9 in early osteoarthritic tissue cultures, with or without interleukin 1alpha (IL-1alpha) and lipopolysaccharide (LPS) induction. RESULTS: Our findings were: (1) latent and active forms of MMP-9 secretion in synovial and some meniscal cultures were inhibited significantly by U0126, SB203580, and LY294002; (2) latent and active forms of MMP-2 secretion were also inhibited significantly by U0126 and LY294002, but not by SB203580, except for active MMP-2 in synovial cultures; (3) a similar observation was seen in IL-1alpha- and LPS-treated cultures; and (4) U0126, SB203580, and LY294002 significantly decreased u-PA and PAI-1 levels in all cultures in the presence or absence of IL-1alpha and LPS. CONCLUSION: MAPK ERK, JNK, and p38 signaling pathways and the PI3K signaling pathway are involved in upregulation of u-PA, PAI-1, and gelatinase expression during early development of knee OA. Thus, blocking PA/plasmin and gelatinase expression by novel physiologic and pharmacological inhibitors could be an important therapeutic or preventive approach for early OA.     

A role for heterotrimeric GTP-binding proteins and ERK1/2 in insulin-mediated, nitric-oxide-dependent, cyclic GMP production in human umbilical vein endothelial cells

Aims/hypothesis Insulin is known to stimulate endothelial nitric oxide synthesis, although much remains unknown about the intracellular mechanisms involved. This study aims to examine, in human endothelial cells, the specific contribution of heterotrimeric G_i proteins and extracellular signal-regulated protein kinases 1/2 (ERK1/2) in insulin signalling upstream of nitric-oxide-dependent cyclic GMP production.Methods Human umbilical vein endothelial cells were treated with 1 nmol/l insulin in the presence or absence of inhibitors of tyrosine kinases (erbstatin), G_i proteins (pertussis toxin) or ERK1/2 (PD098059 or U0126), and nitric oxide production was examined by quantification of intracellular cyclic GMP. Activation/phosphorylation of ERK1/2 by insulin was examined by immunoblotting with specific antibodies, and direct association of the insulin receptor with G_i proteins was examined by immunoprecipitation.Results Treatment of cells with a physiological concentration of insulin (1 nmol/l) for 5 min increased nitric-oxide-dependent cyclic GMP accumulation by 3.3-fold, and this was significantly inhibited by erbstatin. Insulin-stimulated cyclic GMP production was significantly reduced by pertussis toxin and by the inhibitors of ERK1/2, PD098059 and U0126. Immunoblotting indicated that insulin stimulated the phosphorylation of ERK1/2 after 5 min and 1 h, and that this was completely abolished by pertussis toxin, but insensitive to the nitric oxide synthase inhibitor l-NAME. No direct interaction of the insulin receptor with could be demonstrated by immunoprecipitation.Conclusions/interpretation This study demonstrates, for the first time, that nitric oxide production induced by physiologically relevant concentrations of insulin, is mediated by the post-receptor activation of a pertussis-sensitive GTP-binding protein and subsequent downstream activation of the ERK1/2 cascade.     

ERK-regulated double cortin-like kinase (DCLK)-short phosphorylation and nuclear translocation stimulate POMC gene expression in endocrine melanotrope cells

We tested whether double cortin-like kinase-short (DCLK-short), a microtubule-associated Ser/Thr kinase predominantly expressed in the brain, is downstream of the ERK signaling pathway and is involved in proopiomelanocortin gene (POMC) expression in endocrine pituitary melanotrope cells of Xenopus laevis. Melanotropes form a well-established model to study physiological aspects of neuroendocrine plasticity. The amphibian X. laevis adapts its skin color to the background light intensity by the release of α-MSH from the melanotrope cell. In frogs on a white background, melanotropes are inactive but they are activated during adaptation to a black background. Our results show that melanotrope activation is associated with an increase in DCLK-short mRNA and with phosphorylation of DCLK-short at serine at position 30 (Ser-30). Upon cell activation phosphorylated Ser-30-DCLK-short was translocated from the cytoplasm into the nucleus, and the ERK blocker U0126 inhibited this process. The mutation of Ser-30 to alanine also inhibited the translocation and reduced POMC expression, whereas overexpression stimulated POMC expression. This is the first demonstration of DCLK-short in a native endocrine cell. We conclude that DCLK-short is physiologically regulated at both the level of its gene expression and protein phosphorylation and that the kinase is effectively regulating POMC gene expression upon its ERK-mediated phosphorylation.     

Meat hydrolysate and essential amino acid-induced glucagon-like peptide-1 secretion, in the human NCI-H716 enteroendocrine cell line, is regulated by extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinases

Glucagon-like peptide-1 (GLP-1) is a potent insulin secretagogue released from L-cells in the intestine. Meat hydrolysate (MH) is a powerful activator of GLP-1 secretion in the human enteroendocrine NCI-H716 cell line, but the mechanisms involved in nutrient-stimulated GLP-1 secretion are poorly understood. The objective of this study was to characterize the intracellular signalling pathways regulating MH- and amino acid-induced GLP-1 secretion. Individually, the pharmacological inhibitors, SB203580 (inhibitor of p38 mitogen-activated protein kinase (MAPK)), wortmannin (inhibitor of phosphatidyl inositol 3-kinase) and U0126 (inhibitor of mitogen activated or extracellular signal-regulated protein kinase (MEK1/2) upstream of extracellular signal-regulated kinase (ERK)1/2) all inhibited MH-induced GLP-1 secretion. Further examination of the MAPK pathway showed that MH increased the phosphorylation of ERK1/2, but not p38 or c-Jun N-terminal kinase over 2-15 min. Incubation with SB203580 resulted in a decrease in phosphorylated p38 MAPK and a concomitant increase in the phosphorylation of ERK1/2. Phosphorylation of ERK1/2 was augmented by co-incubation of MH with SB203580. Inhibitors of protein kinase A and protein kinase C did not inhibit MH-induced GLP-1 secretion. In contrast to non-essential amino acids, essential amino acids (EAAs) increased GLP-1 secretion and similar to MH, activated ERK1/2. However, they also activated p38-suggesting type of protein may affect GLP-1 secretion. In conclusion, there appears to be a crosstalk between p38 and ERK1/2 MAPK in the human enteroendocrine cell with the activation of ERK1/2 common to both MH and EAA. Understanding the cellular pathways involved in nutrient-stimulated GLP-1 secretion has important implications for the design of new treatments aimed at increasing endogenous GLP-1 release in type-2 diabetes and obesity.     

The effects of adiponectin and leptin on human endothelial cell proliferation: a live-cell study

The effect of adiponectin and leptin on the proliferation of the human microvascular endothelial cell line (HMEC-1) was studied in the absence or presence of fetal bovine serum (FBS). The participation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt (PI-3K/Akt) pathways in this effect were evaluated. We studied the effect of both adipokines on the motility, mitosis, proliferation and cell death processes of HMEC-1 cells using live-cell imaging techniques. Adiponectin but not leptin further increased the proliferative effect induced by FBS on HMEC-1. This effect seems to be the consequence of an increase in the mitotic index in adiponectin-treated cells when compared to untreated ones. The presence of either the mitogen-activated protein kinase (MAPK) inhibitor (PD98059), or PI-3K inhibitor (LY294002), reduced the effect of adiponectin in a dose-dependent manner. Neither adipokine was able to affect HMEC-1 proliferation in FBS-free conditions. Duration of mitosis, cell motility and the cell death process were similar in all conditions. These data suggest that adiponectin and leptin exert different effects on endothelial cell function. Adiponectin was able to potentiate proliferation of HMEC-1. This effect involves the activation of both PI3-K/Akt and ERK/MAPK pathways. However, it seems to exert minimal effects on HMEC-1 function in the case of leptin.     

Decreased expression of caveolin-1 and altered regulation of mitogen-activated protein kinase in cultured bovine parathyroid cells and human parathyroid adenomas

Caveolins are key components of caveolae membranes. The calcium-sensing receptor (CaR) resides within caveolin-rich membrane domains in bovine parathyroid (PT) cells. Recent studies reported reduced CaR expression, and abnormal calcium-sensing in PT tumors. To examine this altered CaR signaling, we investigated ERK activation after CaR stimulation in human and bovine PT cells. In freshly prepared bovine PT cells, high extracellular calcium (Ca(2+)(0)) stimulates ERK1/2 phosphorylation, and activated ERK1/2 colocalizes with caveolin-1 at the plasma membrane but fails to translocate to the nucleus, and cell proliferation is low. In cultured bovine PT cells, CaR and caveolin-1 levels are reduced; activated ERK1/2 localizes in the cell periphery at 10 min and in the perinuclear and nuclear regions at 60 min after exposure to high Ca(2+)(0), and cell proliferation is increased. In PT cells from adenomas, there are high levels of caveolin-2, variably reduced caveolin-1, and hyperactivation of ERK1/2, which colocalizes with caveolin-1 in some cells, but localizes in the cytosol and nucleus in others. Finally, caveolin-1 negative human PT cells exhibit reduced suppressibility of PTH secretion by high Ca(2+)(0). Thus, CaR and caveolin-1 colocalize in PT cells, and reduced levels of caveolin-1 could participate in the abnormal cellular function and proliferation of cultured bovine PT cells and PT adenomas.     

Glucose modifies the cross-talk between insulin and the beta-adrenergic signalling system in vascular smooth muscle cells

BACKGROUND: Abnormalities in the vascular function of insulin are observed in insulin resistance, and hyperglycaemia is one of the important factors inducing insulin resistance. OBJECTIVE: To investigate the role of glucose in the interaction of insulin and beta-adrenergic signalling systems in vascular smooth muscle cells (VSMC). METHODS: After cells were treated with D-glucose (525 mmol/l) and insulin (100 nmol/l), adenylyl cyclase activity was measured in the presence of isoproterenol, forskolin, and cholera toxin. Assays for insulin-induced activities of insulin receptor substrate (IRS)-1, phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) were performed. RESULTS: In the presence of low glucose concentrations (5 mmol/l), insulin enhanced isoproterenol-, forskolin- and cholera toxin-stimulated adenylyl cyclase activities. This stimulatory effect was abolished by PI3-K inhibitors, wortmannin, or LY294002. In contrast, in the presence of high glucose concentrations (25 mmol/l), insulin attenuated isoproterenol-stimulated activity but not cholera toxin- or forskolin-stimulated activity. Insulin-stimulated activities of IRS-1 and PI3-K, but not MAPK activity, were also attenuated in the presence of high concentrations of glucose. The MAPK kinase inhibitor, PD98059, abolished the inhibitory effect of insulin on the beta-adrenergic signalling system. Troglitazone and pioglitazone prevented this inhibitory effect of insulin by restoring IRS-1 and PI3-K activities. CONCLUSIONS: In the presence of low glucose concentrations, insulin stimulates the beta-adrenergic signalling system through the IRS-1/PI3-K pathway. However, in the presence of high glucose concentrations, the effect of insulin is switched to an inhibitory one, through the MAPK pathway. Our finding suggests that high glucose concentrations modify the cross-talk between insulin and the beta-adrenergic signalling systems in VSMC.     

Role of growth factor receptor transactivation in high glucose-induced increased levels of Gq/11α and signaling in vascular smooth muscle cells

We have recently shown that high glucose increased the expression of Gq/11α, PLCβ and mediated signaling in A10 vascular smooth muscle cells (VSMC). Since high glucose has been shown to increase growth factor receptor activation, we investigated the role of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) transactivation in high glucose-induced enhanced expression of Gq/11α and PLCβ. Pre-treatment of A10 VSMC with high glucose (26 mM) for 3 days, increased the levels of Gqα, G11α, PLCβ-1 and PLCβ-2 proteins which were restored to control levels by AG1478, an inhibitor of EGF-R, AG1295, an inhibitor of PDGF-R and PP2, an inhibitor of c-Src but not by PP3. In addition, endothelin-1 (ET-1)-stimulated production of IP₃ that was enhanced by high glucose was also restored towards control levels by AG1478, AG1295 and PP2. High glucose also increased the phosphorylation of EGF-R and PDGF-R which was abolished by AG1478, AG1295 and PP2. Furthermore, high glucose-induced enhanced levels of Gqα, G11α and PLCβ were also attenuated by PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). In addition, AG1478 and AG1295, also attenuated high glucose-induced enhanced phosphorylation of ERK1/2 and AKT. Furthermore, high glucose augmented the phosphorylation of c-Src which was attenuated by antioxidant, DPI. These results suggest that oxidative stress through the activation of c-Src and resultant transactivation of growth factor receptor contributes to the high glucose-induced enhanced expression of Gq/11α/PLC and -mediated cell signaling through MAPK/PI3K pathway.     

Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and uPA via MAPK pathways

Purpose

Sphingosine kinase (SphK) 1 is an oncogenic enzyme promoting transformation, proliferation, and survival of a number of human tumor cells. However, its effect on colon cancer cell behavior has not been fully clarified.

Methods

SphK1 plasmid or SphK1 shRNA transfection and N,N-dimethylsphingosine (DMS) was used to regulate the expression and activity of SphK1 in colon cancer line LOVO. Cell proliferation, apoptosis, invasion, and protein expression were detected by MTT, flow cytometry, transwell chambers model, and western blot. The levels of metalloproteinases-2/9 (MMP-2/9) and urokinase plasminogen activator (uPA) were detected by ELISA.

Results

Overexpression of SphK1 after plasmid transfection markedly enhanced LOVO cell viability and invasiveness and reduced cell apoptosis. In contrast, inhibition of SphK1 by DMS and shRNA significantly suppressed cell viability and invasiveness but promoted cell apoptosis. SphK1 increased the constitutive expression of extracellular signal-regulated kinase1/2 (ERK1/2) but reduced the constitutive expression of p38 mitogen-activated protein kinase (MAPK). Blocking ERK1/2 pathway inhibited the biological effects induced by overexpression of SphK1. Blocking p38 MAPK pathway reversed the effects of DMS and SphK1 shRNA. Moreover, SphK1 was required for the production of MMP-2/9 and uPA in tumor cells, which was suppressed by ERK1/2 inhibitor U0126, but enhanced by the p38 MAPK inhibitor SB203580.

Conclusions

SphK1 enhances colon cancer cell proliferation and invasiveness, meanwhile suppressing cell apoptosis. SphK1 promoting the secretion of MMP-2/9 and uPA via activation of ERK1/2 and suppression of p38 MAPK pathways maybe the molecular mechanisms for its regulation of the malignant behavior of colon cancer cell.