PURPOSE: We assessed for mutations in a large number of oncogenes and tumor suppressor genes in primary uveal melanomas using a high-throughput profiling system. METHODS: DNA was extracted and purified from 134 tissue samples from fresh-frozen tissues (n = 87) or formalin-fixed, paraffin-embedded tissues (n = 47) from 124 large uveal melanomas that underwent primary treatment by enucleation. DNA was subjected to whole genome amplification and MALDI-TOF mass spectrometry-based mutation profiling (>1000 mutations tested across 120 oncogenes and tumor suppressor genes) using the OncoMap3 platform. All candidate mutations, as well as commonly occurring mutations in GNAQ and GNA11, were validated using homogeneous mass extension (hME) technology. RESULTS: Of 123 samples, 97 (79%, representing 89 unique tumors) were amplified successfully, passed all quality control steps, and were assayed with the OncoMap platform. A total of 58 mutation calls was made for 49 different mutations across 26 different genes in 34/98 (35%) samples. Of 91 tumors that underwent hME validation, 83 (91%) harbored mutations in the GNAQ (47%) or GNA11 (44%) genes, while hME validation revealed two tumors with mutations in EGFR. These additional mutations occurred in tumors that also had mutations in GNAQ or GNA11. CONCLUSIONS: The vast majority of primary large uveal melanomas harbor mutually-exclusive mutations in GNAQ or GNA11, but very rarely have the oncogenic mutations that are reported commonly in other cancers. When present, these other mutations were found in conjunction with GNAQ/GNA11 mutations, suggesting that these other mutations likely are not the primary drivers of oncogenesis in uveal melanoma.
The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M(1)AchR, M(2)AchR, and M(3)AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca(2+)](i) in a concentration-dependent manner. EGF-stimulated increase in [Ca(2+)](i) was blocked by inhibitors of the EGF receptor and removal of extracellular Ca(2+). Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M(1)AchR, M(2)AchR, and M(3)AchRs. Cch-stimulated increase in [Ca(2+)](i) was blocked by inhibitors for the M(1)AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca(2+)](i) and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca(2+). This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca(2+)](i), and activate ERK 1/2 leading to an increase in mucin secretion.
PURPOSE: The purpose of this study was to determine the Ca(2+)-dependent cellular signaling pathways used by histamine to stimulate conjunctival goblet cell secretion. METHODS: Cultured rat goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured by loading cultured cells with the Ca(2+) sensitive dye fura-2. The level of [Ca(2+)](i) was measured using fluorescence microscopy. Extracellular regulated kinase (ERK) 2 was depleted using small interfering RNA (siRNA). RESULTS: Histamine-stimulated conjunctival goblet cell secretion of high molecular weight glycoproteins was blocked by removal of extracellular Ca(2+) and depletion of ERK2 by siRNA. Histamine increase in [Ca(2+)](i) was desensitized by repeated addition of agonist and blocked by a phospholipase C antagonist. Histamine at higher doses increased [Ca(2+)](i) by stimulating influx of extracellular Ca(2+), but at a lower dose released Ca(2+) from intracellular stores. Activation of each histamine receptor subtype (H(1)-H(4)) increased [Ca(2+)](i) and histamine stimulation was blocked by antagonists of each receptor subtype. The H(2) receptor subtype increase in [Ca(2+)](i) was cAMP dependent. CONCLUSIONS: We conclude that histamine activates phospholipase C to release intracellular Ca(2+) that induces the influx of extracellular Ca(2+) and activates ERK1/2 to stimulate conjunctival goblet cell mucous secretion, and that activation of all four histamine receptor subtypes can increase [Ca(2+)](i).
Regulation of cGMP synthesis by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) in rod and cone photoreceptors by calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) is one of the key molecular mechanisms affecting the response to light and is involved in congenital retinal diseases. The objective of this study was to identify the physiological sequence of events underlying RetGC activation in vivo, by studying the electrophysiological and biochemical properties of mouse rods in a new genetic model lacking GCAP1. The GCAP1(-/-) retinas expressed normal levels of RetGC isozymes and other phototransduction proteins, with the exception of GCAP2, whose expression was elevated in a compensatory fashion. RetGC activity in GCAP1(-/-) retinas became more sensitive to Ca(2+) and slightly increased. The bright flash response in electroretinogram (ERG) recordings recovered quickly in GCAP1(-/-), as well as in RetGC1(-/-)GCAP1(-/-), and RetGC2(-/-)GCAP1(-/-) hybrid rods, indicating that GCAP2 activates both RetGC isozymes in vivo. Individual GCAP1(-/-) rod responses varied in size and shape, likely reflecting variable endogenous GCAP2 levels between different cells, but single-photon response (SPR) amplitude and time-to-peak were typically increased, while recovery kinetics remained faster than in wild type. Recovery from bright flashes in GCAP1(-/-) was prominently biphasic, because rare, aberrant SPRs producing the slower tail component were magnified. These data provide strong physiological evidence that rod photoresponse recovery is shaped by the sequential recruitment of RetGC isozyme activation by GCAPs according to the different GCAP sensitivities for Ca(2+) and specificities toward RetGC isozymes. GCAP1 is the 'first-response' sensor protein that stimulates RetGC1 early in the response and thus limits the SPR amplitude, followed by activation of GCAP2 that adds stimulation of both RetGC1 and RetGC2 to speed-up photoreceptor recovery.
Inflammatory cytokines and growth factors drive angiogenesis independently; however, their integrated role in pathologic and physiologic angiogenesis is not fully understood. Suppressor of cytokine signaling-3 (SOCS3) is an inducible negative feedback regulator of inflammation and growth factor signaling. In the present study, we show that SOCS3 curbs pathologic angiogenesis. Using a Cre/Lox system, we deleted SOCS3 in vessels and studied developmental and pathologic angiogenesis in murine models of oxygen-induced retinopathy and cancer. Conditional loss of SOCS3 leads to increased pathologic neovascularization, resulting in pronounced retinopathy and increased tumor size. In contrast, physiologic vascularization is not regulated by SOCS3. In vitro, SOCS3 knockdown increases proliferation and sprouting of endothelial cells costimulated with IGF-1 and TNFα via reduced feedback inhibition of the STAT3 and mTOR pathways. These results identify SOCS3 as a pivotal endogenous feedback inhibitor of pathologic angiogenesis and a potential therapeutic target acting at the converging crossroads of growth factor- and cytokine-induced vessel growth.