We used GH4C1 cells as a model to study the effects of the chemokine stromal cell-derived factor 1 (SDF1) in pituitary functions. In these cells, SDF1␣ induced proliferation and growth hormone secretion, suggesting a possible regulatory role for this chemokine at pituitary level. We evaluated the intracellular signaling involved in these effects: SDF1␣ increased cytosolic [Ca 2ϩ ] and activated Pyk2, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and large-conductance Ca 2ϩ -activated K ϩ channels (BK Ca ) channels. To correlate these intracellular effectors with the proliferative and secretory effects, we inhibited their activity using BAPTA-AM (Ca 2ϩ chelator), 2Ј-amino-3Ј-methoxyflavone (PD98059; a mitogen-activated protein kinase kinase inhibitor), salicylate (Pyk2 inhibitor), and tetraethyl ammonium (K ϩ channel blocker). All of these compounds reverted SDF1␣-induced proliferation, suggesting the involvement of multiple intracellular pathways. Conversely, only BAPTA-AM reverted growth hormone secretion. To identify a possible cross-talk and a molecular ordering among these pathways, we tested these antagonists on SDF1␣-dependent activation of ERK1/2, Pyk2, and BK Ca channels. From these experiments, we observed that the inhibition of [Ca 2ϩ ] i increase or BK Ca channel activity did not affect ERK1/2 activation by SDF1␣; Pyk2 activation was purely Ca 2ϩ -dependent, not involving ERK1/2 or BK Ca channels; and BK Ca channel activity was antagonized by Pyk2 but not by ERK1/2 inhibitors. These data suggest that an SDF1␣-dependent increase of [Ca 2ϩ ] i activates Pyk2, which in turn regulates BK Ca channel activity. Conversely, ERK1/2 activation is an independent phenomenon.In conclusion, we demonstrate that SDF1␣ causes both proliferation and growth hormone release from pituitary adenoma cells, suggesting that the activation of CXCR4 may represent a novel regulatory mechanism for growth hormone secretion and pituitary cell proliferation, which may contribute to pituitary adenoma development.
These data show that somatostatin and lanreotide inhibit human GH-secreting pituitary adenoma cell proliferation and hormone release in vitro, and suggest that the activation of tyrosine phosphatases may represent intracellular signals mediating the antiproliferative effects and that the inhibition of the voltage-dependent calcium channels and adenylyl cyclase activities may control GH secretion.
In this study, we analyzed the intracellular mechanisms leading to basic fibroblast growth factor (bFGF)-dependent production of NO in Chinese hamster ovary (CHO)-K1 cells and a possible physiological role for such an effect. bFGF induces NO production through the activation of the endothelial form of NO synthase (eNOS), causing a subsequent increase in the cGMP levels. In these cells, the activation of eNOS by bFGF is Ca 2ϩ -and mitogen-activated protein kinase-independent. The translocation of the enzyme from the plasma membrane, where it is located in caveolae bound to caveolin 1, to the cytosol is the crucial step for the synthesis of NO through the eNOS isoform. We demonstrate that bFGF activates a sphingomyelinase to synthesize ceramide, which, in turn, allows the dissociation of eNOS from caveolin 1 and its translocation to the cytosol in the active form, where it catalyzes the synthesis of NO. In fact, drugs interfering with sphingomyelinase activity blocked bFGF activation of eNOS, and an increase in ceramide content was detected after bFGF treatment. Moreover, in fibroblasts derived from patients with Niemann-Pick disease, in which the enzyme is genetically inactive, bFGF is unable to elicit eNOS activation. The NO produced after bFGF treatment, through the activation of guanylyl cyclase and protein kinase G, mediates a mitogenactivated protein kinase-independent cell proliferation. In conclusion, our data show that, in CHO-K1 cells, bFGF regulates the activity of eNOS through a novel intracellular pathway, involving the induction of ceramide synthesis and that the NO released participates in bFGF proliferative activity.Nitric oxide (NO) is an important intracellular and intercellular mediator involved in the modulation of many physiological processes in different tissues, including blood flow regulation, platelet aggregation, smooth muscle relaxation, apoptosis, central and peripheral neurotransmission, and different neuroendocrine responses (Moncada and Higgs, 1993;Nathan and Xie, 1994).NO is synthesized by a family of three distinctive isoforms of nitric-oxide synthase (NOS), named after the tissues in which they were originally described. Neuronal and endothelial NOS [nNOS (or NOS I) and eNOS (or NOS III), respectively] are Ca 2ϩ /calmodulin-dependent enzymes constitutively expressed not only in neuronal and endothelial cells
Nitric oxide (NO) is an intracellular and intercellular mediator involved in the modulation of many physiologic and pathologic processes including the regulation of neoangiogenesis. We analyzed the effects of basic fibroblast growth factor (bFGF) on NO production in CHO-K1 cells and the intracellular mechanisms involved. bFGF induces NO production through activation of the endothelial NO synthase (eNOS), causing a subsequent increase in cGMP levels. In most systems, eNOS activation is a Ca(2+)-calmodulin-dependent process. In CHO-K1 cells, NO production by bFGF is Ca(2+) and MAP kinase independent, because it was not reverted by pretreatment with intracellular Ca(2+) chelators or MEK inhibitors. Translocation of the eNOS from the plasma membrane, where it is bound to caveolin 1, to the cytosol is the crucial step in the synthesis of NO. We demonstrate that the cytosolic translocation of eNOS is caused by increased synthesis of ceramide dependent by the bFGF activation of sphingomyelinase. Indeed, in the presence of the sphingomyelinase inhibitors D609 or desipramine, bFGF-dependent NO production is abrogated. To support this evidence we evaluated ceramide concentration using HPLC-electrospray ionization-mass spectrometry in controls and in bFGF-treated cells: after bFGF stimulation, a substantial increase in ceramide levels was observed. These data were further confirmed by the lack of NO production in response to fibroblast growth factor in fibroblasts derived from Niemann Pick patients who genetically lack the enzyme sphingomyelinase. In conclusion, ceramide in CHO-K1 cells is responsible for a novel Ca(2+)/calmodulin-independent mechanism for eNOS activation after fibroblast growth factor stimulation.
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