CgA coding regions, putative functional domains and targets.

<p>The primary transcript, on chromosome 14, is derived from 8 exons and includes exon 1 which is untranslated but contains a signal peptide region for protein processing. In this study, PCR was performed using intron spanning primers to examine exons I-VI. Mature CgA mRNA includes 439 coding base pairs which are translated into a primary peptide of 431 amino acids. Processing of CgA following cleavage at dibasic and monobasic residues e.g. by PC1/3 and CPE results in production of a range of intermediate peptides as well biologically active peptides [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081111#B36" target="_blank">36</a>].</p

CgA silencing, processing enzyme inhibition and functional analysis of CgA peptides in SI-NEN cell lines.

<p>After successfully silencing CgA in H-STS cells (<i>data not shown</i>), proliferation was significantly decreased (<b>4A</b>, *<i>p</i><0.05). Secretion of CgA (<i>p</i><0.01) and 5-HT (<b>4B</b>, <i>*p</i><0.05) was significantly reduced following CgA antisense. Inhibition of the CgA processing enzyme prohormone convertase using Decanoyl-Arg-Val-Lys-Arg-CMK also decreased proliferation of H-STS cells (25 µM [<i>data not shown</i>] and 50 µM, <b>4D</b>, *<i>p</i><0.05). Additionally, secretion of CgA and 5-HT (<b>4E</b>, *<i>p</i><0.05) was also significantly reduced. Decreases in CgA and its fragments (Vasostatin II and Pancreastatin) after treatment with the prohormone convertase inhibitor were confirmed with western blot (<b>4C</b> and <b>F</b>). Chromostatin (<20 kDa) was too small to appear on this WB. The fragments Vasostatin I and II significantly stimulated proliferation (up to 60%, *<i>p</i><0.02) in both metastatic cell lines (L-STS and H-STS, <b>4H</b> and <b>I</b>, square) but had no effects on the primary tumor cell lines. Chromostatin inhibited the well-differentiated localized NEN cell line proliferation (P-STS, <b>4G</b>, square, ~50%, *<i>p</i><0.05) but not proliferation of the less well-differentiated cell line, KRJ-I. Mean±SEM; <i>n</i>=6, CON: control, KD: knockdown, SCR: scrambled, P: P-STS, K:KRJ-1, L: L-STS, H: H-STS. 5-HT: Serotonin.</p

CgA processing enzyme prohormone convertase expression and the effect of tumor growth on CgA and processing.

<div><p><i>PCSK1</i> mRNA expression was increased in SI-NEN metastases (METS) and primaries with metastasis (MET PRIM) compared to normal mucosa (NML, *<i>p</i><0.05) and normal EC cells (EC, ^#<i>p</i><0.05) (<b>3A</b>, Kruskal-Wallis <i>p</i>=0.0003). Western blot analysis confirmed that protein levels of prohormone convertase 1-3 were elevated in metastases compared to normal mucosa (<b>3C</b>,*<i>p</i><0.05). </p> <p>CgA mRNA (<b>3B</b>, 6 exons) and protein (3D) were elevated at the plateau growth phase (day 7) compared to logarithmic growth (day 2) in H-STS cells (*<i>p</i><0.05). PC1-3 proteins were decreased at day 7 (3D), which can be discussed as one reason for the elevation of total intracellular CgA at this time point. </p> <p>Mean±SEM. PCSK1: prohormone convertase 1. </p></div

Chromogranin A expression in normal mucosa, EC cells, small intestinal NENs (SI-NENs) and primary and metastatic SI-NEN cell lines.

<p>CgA mRNA expression in normal human mucosa (NML), EC cell preparations (EC), localized NENs (PRIM), primaries with metastasis (MET PRIM) and liver metastases (METS) demonstrated that all NENs expressed higher CgA levels (Kruskal-Wallis <i>p</i><0.0001) compared to normal mucosa (*<i>p</i><0.001) or normal EC cells (^#<i>p</i><0.001) (<b>a</b>). Levels of CgA protein expression, measured by ELISA, showed a similar pattern (<b>2C</b>, Kruskal-Wallis <i>p</i><0.0001) and was increased in PRIM (*<i>p</i><0.01), MET PRIM (*<i>p</i><0.001) and METS (*<i>p</i><0.05) compared to normal mucosa. CgA western blot in normal mucosa, normal EC cells and SI-NENs identified a mature CgA band of 75-80 kDa in all NENs but not in normal mucosa or EC cells (<b>2E</b>). Fragment sizes included peptides ranging in size from ~30-60kDa, consistent with CgA processing intermediates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081111#B36" target="_blank">36</a>]. In cell lines, CgA mRNA was expressed in higher levels in the two primary cell lines in comparison to metastatic cell lines (<b>2B</b>, Kruskal-Wallis <i>p</i><0.0001), particularly in P-STS CgA was over-expressed compared to H-STS (* <i>p</i><0.001) and L-STS cells (^#<i>p</i><0.01). In KRJ-1 CgA was also elevated in comparison to H-STS cells (*<i>p</i><0.01, <b>2B</b>). Protein level (ELISA) followed similar pattern (Kruskal-Wallis <i>p</i>=0.0273, *<i>p</i><0.05, <b>2D</b>). Using western blot, total CgA (75-80 kDa) was identified in all cell lines, highest in the primary cell lines KRJ-1 and P-STS (2F). Band sizes consistent with CgA processing were evident and exhibited different patterns of expression consistent with alterations in translational modifications (2F). No external receptor was identified for CgA, but Cy5-labeled immunofluorescence (IF) was identified within KRJ-I and H-STS cells. We interpret this dot-like signal to reflect intracellular uptake of this CgA peptide (<b>2G</b>). </p

Effect of Vasostatin I and Chromostatin on AKT phosphorylation in metastatic and localized NEN cell lines.

<p>Vasostatin I stimulated AKT phosphorylation in the liver metastasis (H-STS) (CASE ELISA: 50%, *<i>p</i><0.04, western blot: 25%) and could be completely reversed by pre-incubation with RAD001 (<b>5A/C</b>, ^#<i>p</i><0.01). AKT antisense reversed vasostatin-mediated proliferation (BrdU uptake) (<b>5E</b>). In contrast, chromostatin, inhibited AKT signaling in the primary cell line (P-STS) (<b>5B/D</b>, ~25%, *<i>p</i><0.05). AKT antisense reversed chromostatin-mediated inhibition of proliferation (BrdU uptake) (5F). Mean±SD; AS = antisense, CON: control, SCR: scrambled, V-I: vasostatin I, R: RAD001, CST: chromostatin.</p

Analysis of 5-HT release and signaling pathways in ADORA2B-deficient rat EC cells.

<p><b>5A)</b> A 12 hr antisense approach inhibited ADORA2B transcript (real-time PCR) and protein expression (membrane-bound, flow cytometry) in isolated rat EC cells. Membrane expression was reduced to an estimated 5% of non-targeted cells. <b>5B)</b> In non-targeted cells, 5-HT release was elevated by hypoxia and inhibited by curcumin. Adenosine signaling responses were similar to human EC cells. Antisense reduced hypoxic-mediated responses (compared to controls) and ameliorated cell responses to NECA and MRS1754. <b>5C)</b> Western blot identified decreased pMAPK (0.56±0.09), pCREB (0.75±0.11) and pTPH-1 (0.63±0.07) in antisense treated cells confirming down-regulation of these pathways with loss of ADORA2B expression under hypoxic conditions. Mean±SEM, <i>n</i> = 3. *<i>p</i><0.05 vs. control, **<i>p</i><0.05 vs hypoxia, ^#<i>p</i><0.05 vs. hypoxia (in non-antisense treated cells). NS = not significant.</p

HIF-1α, adenosine signaling and HRE activation.

<p><b>3A)</b> Curcumin inhibited (022±0.04 fold) while NECA (1.24±0.06 fold) stimulated and MRS1754 (0.74±0.02) inhibited HIF-1α protein levels compared to 30 minutes hypoxia in KRJ-I cells. SCH442146 had no effect. <b>3B)</b> Transient transfection with <i>Renilla</i> luciferase-encoding constructs in KRJ-I cells. Under normoxic conditions, NECA amplified activation of luciferase (RLU) while MRS1754 inhibited this. Curcumin and SCH442416 had no effect. Hypoxic conditions activated RLU (1.81±0.12), which was inhibited by curcumin and MRS1754 and amplified by NECA. <b>3C)</b> In IBD-EC cells, RLU was elevated in normoxic conditions. This could be reduced by curcumin and MRS1754 and amplified by NECA. Mean±SEM, <i>n</i> = 3–7, *<i>p</i><0.05 vs. 30 min hypoxia or control, **<i>p</i><0.05 vs. normoxic cells, ^#<i>p</i><0.05 vs. 30 min hypoxia. NS = not significant.</p

Analysis of HIF-1α and ADORA2B in mucosa from the animal TNBS-induced colitis model with or without 5-ASA-treatment.

<p>6A) TNBS was associated with a significant elevation in HIF-1α and ADORA2B transcripts. Treatment with 5-ASA reversed these values to those similar to untreated rats. 6B) Protein expression was elevated by TNBS (HIF-1α: 1.68±0.11; ADORA2B: 1.37±0.09) and reversed by 5-ASA in mucosa. Mean±SEM, <i>n</i> = 3. *<i>p</i><0.05 versus control, **<i>p</i><0.05 versus TNBS.</p

Effect of adenosine on the hypoxia-activated 5-HT pathway in KRJ-I cells.

<p><b>2A)</b> 5-HT was increased by hypoxia between 15–120 min, with a maximal effect at 30 min (2.28±0.12 fold). Curcumin and MRS1754 inhibited while NECA augmented secretion at all time points up to 120 mins. SCH442146 had no significant effect. <b>2B)</b> Total TPH-1 protein levels were unchanged after 30 min of hypoxia and after NECA or MRS1754 stimulation. Phosphorylated TPH-1 was significantly increased under hypoxia (1.80±0.26), was amplified by NECA (2.66±0.28) and reduced by MRS1754 to baseline. <b>2C)</b> VMAT-1 protein levels were significantly increased (2.83±0.31) by NECA during hypoxia and reduced by MRS1754 (2±0.1). <b>2D)</b> Chromogranin A protein levels did not change significantly after hypoxia or with the addition of NECA or MRS 1754. Mean±SEM, <i>n</i> = 3–8, *<i>p</i><0.05 vs. control, **<i>p</i><0.05 vs. hypoxia. NS = not significant.</p

<i>HIF-1α</i> transcripts and protein in normal mucosa, IBD mucosa, isolated normal and IBD-EC cells.

<p><b>1A)</b> Transcript of HIF-1α was significantly elevated in IBD-associated conditions (mucosa: 3.4±0.63 fold, cells: 2.4±0.41). <b>1B)</b> Protein levels were significantly elevated in IBD-associated conditions (mucosa: 12.2±3.4, cells: 2.6±0.36). <b>1C)</b> Immunohistochemical staining of HIF-1α and Chromogranin A (white arrows) in normal mucosa and Crohn’s mucosa identified co-staining (yellow arrows) predominantly in IBD mucosa. <b>1D</b>) Quantitation identified significantly more enteroendocrine cells to be HIF-1α positive in IBD mucosa. Mean±SEM, <i>n</i> = 4–7, *<i>p</i> = 0.03 vs. normal mucosa, ^#<i>p</i> = 0.04 vs. normal EC cells,^##<i>p</i><0.001 vs. normal EC cells. DAPI – nuclei (blue), FITC-CgA (green), Cy5-HIF-1α (red), co-localization (yellow). N–M = normal mucosa, IBD-M = IBD mucosa, N–C = normal EC cells, IBD-C = IBD EC cells.</p