Role of reduced H3K9 methylation in the activation of <i>Kitl/KITLG</i> transcription by IGF1 and GSK3i.

<p><i>A</i>-<i>B</i>, Reduced occupancy of the <i>Kitl</i> core promoter by the repressive H3K9me2 (<i>A</i>) and H3K9me3 (<i>B</i>) histone modifications in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. <i>C</i>-<i>D</i>, Probing the role of HP1 isoforms in transcriptional repression of <i>KITLG</i> in LX-2 cells by siRNA- (CBX5: HP1α; 25 nM, 72 h; <i>C</i>) or shRNA-mediated knock-down (CBX1: HP1β; CBX3: HP1γ; 30 µg plasmid, 72 h; <i>D</i>). Note activation of <i>KITLG</i> expression by shRNA-mediated knock-down of HP1γ (n=3/group). <i>E</i>-<i>F</i>, Stimulation of <i>KITLG</i> expression by the G9A/GLP H3K9me1/2 HKMT inhibitor BIX-01294 in LX-2 cells (<i>E</i>) and GIST-T1 cells (<i>F</i>) (n=3/group). <i>G</i>, Stimulation of <i>KITLG</i> expression by the H3K9 HKMT inhibitor chaetocin in LX-2 cells (n=3/group). Drugs were applied for 24 h following 24-h serum deprivation. See further details in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone-0076822-g005" target="_blank">Figure 5</a>.</p

IGF1 and GSK3i stimulate <i>Kitl/KITLG</i> transcription.

<p>Results obtained in murine and human smooth muscle cells (<i>A</i>-<i>E</i>), human LX-2 stellate cells (<i>F</i>-<i>G</i>) and human GIST-T1 cells (<i>H</i>-<i>I</i>) are shown. <i>A</i>, Hoffman modulation contrast image of primary human gastric smooth muscle cells. <i>B</i>, <i>KITLG</i> mRNA (total: soluble+membrane-bound) was readily detectable in primary human smooth muscle cells (passage 3) maintained with Smooth Muscle Growth Medium-2 containing insulin, hFGF-B, hEGF and 5% FBS (Lonza) but not in 24-h growth factor- and serum-deficient basal medium. <i>C</i>, Both IGF1 (100 ng/mL) and the GSK3α/β inhibitor SB415286 (30 µM) stimulated <i>Kitl</i> expression in murine gastric <i>tunica </i><i>muscularis</i> organotypic cultures (n=3/group). <i>D</i>-<i>E</i>, SB415286 stimulated endogenous <i>Kitl</i> expression in murine primary gastric smooth muscle cells (<i>D</i>; n=3/group) and <i>KITLG</i> transcriptional activity in the same cell type transfected with a <i>KITLG</i> promoter (-2120 bp to +407 bp)-pGL3 luciferase construct (<i>E</i>; n=3/group). IGF1 (100 ng/mL; n=3/group) and SB415286 (30 µM; n=6/group) also increased endogenous <i>KITLG</i> mRNA expression in LX-2 (<i>F</i>) and GIST-T1 cells (<i>H</i>) and stimulated <i>KITLG</i> promoter activity in a time-dependent fashion (LX-2: n=6-9/group; <i>G</i>; GIST-T1: n=3-11/group; <i>I</i>). Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other (<i>P</i><0.05 by post-hoc multiple comparisons).</p

Role of histone acetylation in the activation of <i>Kitl/KITLG</i> transcription by IGF1 and GSK3i.

<p><i>A</i>, Increased occupancy of the <i>Kitl</i> core promoter region by H4ac in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. Only SB415286 increased occupancy by H3K9ac (<i>B</i>). Representatives of two independent experiments, each performed in triplicates, are shown. <i>C</i>, Dose-dependent stimulation of <i>KITLG</i> expression in LX-2 cells by 24-h treatment with the class I-II HDAC inhibitor SAHA (n=3/group). <i>D</i>, Dose-dependent inhibition of the SB415286-induced stimulation of <i>KITLG</i> expression by the p300/PCAF HAT inhibitor garcinol (24 h) in LX-2 cells (n=3/group). Drugs were applied following 24-h serum deprivation. Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other (<i>P</i><0.05 by post-hoc multiple comparisons).</p

Contribution of KITLG-activated KIT signaling to baseline proliferation of GIST and LX-2 cells.

<p><i>A</i>, KIT Y721 phosphorylation was activated by exogenous rhKITLG (100 ng/mL for 10 min following 2 h serum deprivation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#B13" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#B35" target="_blank">35</a>]) in GIST-T1 cells containing a heterozygous activating <i>KIT</i> mutation (n=4/group) and in GIST-T1-5R cells containing an additional, imatinib-resistant <i>KIT</i> mutation (n=6/group), but not in GIST882 cells lacking a WT <i>KIT</i> allele (n=5/group). <i>B</i>, Culturing with anti-human KITLG neutralizing antibody for 4 days inhibited baseline proliferation of GIST-T1 cells (<i>P</i><0.001; n=3; regression and 95% confidence band are shown as solid and dashed lines, respectively) and GIST-T1-5R cells (<i>P</i><0.001; n=6). The effect of KITLG immunoneutralization on GIST-T1 cells was blocked by preabsorbing the antibody with 10-fold molar excess of rhKITLG (see open symbols in the left panel, second row; n=3/group). KITLG immunoneutralization did not inhibit the proliferation of GIST882 or GIST48B cells lacking a WT <i>KIT</i> allele (note that GIST48B cells also express very low to undetectable levels of KIT protein, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone.0076822.s001" target="_blank">Figure S1E</a>) and of LX-2 cells lacking KIT protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone.0076822.s001" target="_blank">Figure S1E</a>) (n=3/cell line). <i>C</i>, Inhibition of the proliferation of GIST-T1 and GIST-T1-5R cells by siRNA-mediated knock-down of <i>KITLG</i> (n=4/cell line/group; <i>P</i>_GIST-T1: day 2: 0.008, days 4, 6 and 8: <0.001; <i>P</i>_GIST-T1-5R: day 2: <0.02, day 4: 0.004, days 6 and 8: <0.001).</p

Role of trithorax- and polycomb-mediated histone modifications in IGF1- and GSK3i-induced activation of <i>Kitl/KITLG</i> transcription.

<p><i>A</i>, Increased occupancy of the <i>Kitl</i> core promoter by the trithorax group-mediated, activating H3K4me2 histone modification in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. <i>B</i>-<i>C</i>, Reduced occupancy of the <i>Kitl</i> core promoter by the PRC2-mediated, repressive H3K27me3 histone modification (<i>B</i>) and by the PRC2 histone methyltransferase EZH2 (<i>C</i>) in response to rhIGF1 and SB415286 in the same tissues. <i>D</i>-<i>F</i>, Stimulation of <i>KITLG</i> expression by the indirect histone methyltransferase inhibitor Adox in murine gastric smooth muscles (<i>D</i>), LX-2 cells (<i>E</i>) and GIST-T1 cells (<i>F</i>) (n=3/group). Adox was applied for 72 h following 24-h serum deprivation. See further details in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone-0076822-g005" target="_blank">Figure 5</a>.</p

Size distribution of the polyGT repeat lengths.

<p>a) The size distribution of the polyGT repeat length in type 2 diabetic controls is significantly longer than the allele length in non-diabetic controls. The fractional frequency distribution of the lengths of polyGT repeat in <i>HMOX1</i> from non-diabetic control subjects with no GI motility disorders (n = 170) compared to subjects with type 2 diabetes and no GI motility disorders (n = 84) are shown (P < 0.05 Dunn’s test). b) Fractional frequency distribution of the lengths of polyGT repeat in <i>HMOX1</i> from subjects with type 2 diabetes and no GI motility disorders (n = 84) compared to subjects with type 2 diabetes and gastroparesis (n = 72, P = NS, Dunn’s test). c) Fractional frequency distribution of the lengths of polyGT repeat in <i>HMOX1</i> from subjects with type 1 diabetes and no GI motility disorders (n = 84) compared to subjects with type 1 diabetes and gastroparesis (n = 99, P = NS, Dunn’s test). Allele length was determined by PCR amplification of genomic DNA from blood using the ABI 3730 platform and capillary electrophoresis and using primers flanking the GT repeat region.</p

Data on gastroparetic subjects that were studied.

<p>Data on gastroparetic subjects that were studied.</p

Relationship between polyGT allele length and nausea sub-score.

<p>In patients with diabetic gastroparesis, the relationship between polyGT allele length and nausea sub-score on the GCSI questionnaire fit to a slope that was significantly different from zero (P < 0.006 by linear regression, dotted lines are 95% confidence intervals) and b) subjects with one or two long alleles (open circles in Fig 4a) had significantly higher nausea sub-scores than subjects with zero long alleles (closed circles in Fig 4a, P = 0.022, Mann Whitney test). Whiskers are the medians with the interquartile ranges for the nausea sub-score.</p