MYPT1 Down-regulation by Lipopolysaccharide-SIAH1/2 E3 Ligase-Ubiquitin-Proteasomal Degradation Contributes to Colonic Obstruction of Hirschsprung Disease.

Smooth muscle contraction is evoked through multiple signaling pathways converging on myosin light chain phosphorylation., Contraction-associated proteins such as MLCK, MYPT1, RLC, RhoA, ROCK, and CPI-17 are differentially expressed in different types of smooth muscle, leading to contractile diversity.3, 4, 5 Herein we demonstrate that lipopolysaccharide (LPS) causes MYPT1 protein degradation via SIAH1/2 E3 ligases-ubiquitin-proteasomal pathway, which alters colonic contractile properties and results in obstructive phenotype.

Isolated colonic smooth muscle was treated with increasing doses of LPS ex vivo, causing significant reduction of MYPT1 protein (Supplementary Figure 1A). Compared with the proximal colon, MYPT1 protein in the distal colon was also reduced by LPS local treatment (Supplementary Figure 1B). Meanwhile, a single intraperitoneal injection of LPS (5 mg/kg) induced a significant reduction of MYPT1 within 24 hours, and the effect lasted for 5 days (Figure 1A). After injection of LPS (0.5 mg/kg) sequentially with a 24-hour interval, MYPT1 protein was maintained at a low level and recovered within 3 days after stopping the injection (Supplementary Figure 1C). Summarily, MYPT1 protein can be reduced by LPS and reversed by removing LPS.

Supplementary Figure 1

LPS treatment induced altered contractile property by decreasing expression of MYPT1 through TLR4 both. (A) In ex vivo experiment, mouse (C57BL/6) colon segments were isolated, followed by treatment of increased dose of LPS. The protein of the treated muscles was sampled for Western blot assay (n = 5) (one-way ANOVA). (B) LPS-containing cotton balls (20 μg) were inserted into distal about 1 cm from the anus for 14 days. Then MYPT1 protein was measured in the distal and proximal sections of the colons (paired t test) (n = 5). (C) C57BL/6 mice were injected every other day with LPS (0.5 mg/kg body weight) (n = 4) for 14 days. Colonic smooth muscles were sampled at indicated time points and subjected to MYPT1 protein measurement with Western blot. Amount of loaded protein was normalized by using β-actin. (D) Quantitative polymerase chain reaction showing relative mRNA expression of MYPT1 in smooth muscle treated with LPS (n = 3) (one-way ANOVA). (E) Colonic smooth muscle from C57BL/6 mice was treated with MG132 (a proteasome inhibitor) (50 μm) and LPS by increased dose for 24 hours and harvested for Western blot analysis (n = 4) (one-way ANOVA). (F) Colonic smooth muscle from C3H/HeJ mice was treated with LPS by increased dose for 24 hours (n = 3) (one-way ANOVA). (G and H) After same treatment as (B), contractility of proximal and distal colonic smooth muscles was measured with KCl stimulation. Quantitation of percentage force with sustain to the maximum force in distal and proximal colon with C57/B6 (G) and Mypt1 (H) mice, respectively (n = 8) (t test). Bars represent mean values ± standard error of the mean. *P < .05; **P < .01; ***P < .001. CTR, control.

Figure 1

MYPT1 was down-regulated by LPS-SIAH1/2 E3 ligase-ubiquitin-proteasomal degradation, leading to colonic obstruction. (A) Western blot showed MYPT1 expression after LPS intraperitoneal injection (n = 4–6) (t test). (B and C) Colonic smooth muscle from C57BL/6 mice was treated with or without LPS (0.1 mg/mL) and MG132 (50 μmol/L) for 24 hours and harvested for detection of constitutive expression of MYPT1, SIAH1, and SIAH2 by immunoblotting analysis (C); lysate was collected for immunoprecipitation with MYPT1, MYPT1-ubiquitin, SIAH2, and SIAH2 (B) (n = 3) (one-way analysis of variance [ANOVA]). (D) Position of feces in the colon of C57BL/6 mice after local treatment with LPS or phosphate-buffered saline. Bars represent the mean values ± standard error of the mean. *P < .05; **P < .01; ***P < .001.

We found MYPT1 mRNA level in colonic smooth muscle comparable with or without LPS (Supplementary Figure 1D). On treatment with MG132, MYPT1 protein was not affected by LPS (Supplementary Figure 1E). In pull-down by anti-MYPT1 antibody, the ubiquitinated MYPT1 level was increased by LPS (Figure 1B). Pulled-down MYPT1 protein complex contained E3 ligases (SIAH1 and SIAH2) that have a binding motif with MYPT1. SIAH1 and SIAH2 levels were elevated by LPS or LPS plus MG132 (Figure 1B). Furthermore, C3H/HeJ mice with a mutant TLR4 showed no apparent reduction of MYPT1 by LPS (Supplementary Figure 1F). Therefore, LPS/TLR4 enhances MYPT1 degradation through the SIAH1/2 E3 ligases-ubiquitin-proteasomal pathway.

LPS local treatment in the distal colon caused 45% of mice to have packed feces in the colon lumen (Figure 1D), and circular smooth muscles showed an enhanced sustained phase of KCl-evoked contraction (Supplementary Figure 1G). These phenotypes did not appear in MYPT1SMKO mice with the same treatment (Supplementary Figure 1H). Therefore, MYPT1 reduction by LPS causes an obstruction-like phenotype by altering colonic contraction.

To investigate the role of MYTP1 in Hirschsprung disease (HD), we collected fresh colons from HD patients: circular (Cir-) and longitudinal (Long-) muscle strips from dilated (D) and narrow (N) segments. LPS concentration was significantly higher in Cir-N than in Cir-D and Long-D and slightly higher than in Long-N (Figure 2A). The LPS average concentration in narrowed segments was higher than in the dilated (Figure 2B). Meanwhile, compared with Cir-D, MYPT1 protein in Cir-N was decreased (Figure 2C), and PP1cδ was accordingly down-regulated (Figure 2C). In Long-N and Long-D, the contractile proteins were comparable (Supplementary Figure 2A). Moreover, Cir-N muscles produced a strong contractile response with a large, robust, and sustained tension by KCl, whereas Cir-D, Long-N, and Long-D displayed a phasic contraction (Figure 2D). H1152 and GF109203X inhibitors could not relax the KCl-contracted muscle, whereas sodium nitroprusside (SNP) and nifedipine could in Cir-N (Supplementary Figure 2B–E). On KCl stimulation, Cir-N smooth muscle from HD patients exhibited significantly higher regulatory light chain (RLC) phosphorylation than Cir-D at the sustained phase (Figure 2E), in accordance with its maintained force tension.

Figure 2

Cir-N showed high LPS concentration, MYPT1 deficiency, tonic-like contraction, and high RLC phosphorylation. (A and B) LPS was measured by limulus test in Cir-D, Cir-N, Long-D, and Long-N, respectively, (A) and in the whole layers of distal and longitudinal smooth muscle (B). (C) Western blots showed the protein levels in Cir-N (left panel). Quantification of proteins in the narrow segment shown as the percentage of those in the dilated segment (right panel) (pair t test). MYPT1, n = 51; PP1c, n = 34; ROCK2, n = 29; PKC, n = 31; RLC, n = 19; RhoA, n = 15. (D) Contraction of circular (left panel) and longitudinal (right panel) smooth muscle evoked by KCl. Cir-, n = 37; Long-, n = 20 (t test). (E) The pRLC level was expressed as percentage of total RLC stimulated by KCl (n = 8) (t test). Bars represent the mean values ± standard error of the mean. *P < .05; **P < .01; ***P < .001.

Supplementary Figure 2

Relaxant effects of ROCK, PKC, L-type calcium channel inhibitors, and SNP on KCl-evoked contraction of Cir-N from HD and colon from MYPT1 (A) Western blots showing relative amounts of proteins in Long- smooth muscles (left panel). Quantification of protein levels in the narrow segment as percentage of those in the dilated segment (right panel) (paired t test). MYPT1, n = 51; PP1c, n = 34; ROCK2, n = 29; PKC, n = 31; RLC, n = 19; RhoA, n = 15. (B–E) Representative tracings of Cir-N pre-contracted using 87 mmol/L KCl, followed by exposure to 0.3 μmol/L H1152 (B), 5 μmol/L GF109203X (C), 100 μmol/L SNP (D), or 3 μmol/L nifedipine (E). Relative ratios of relaxed force during the sustained phase, which were expressed as percent of force of contraction after addition of KCl at same time point as addition of the vehicle (force % = (Fvehicle – Finhibitor)/Fvehicle, F = force) in the left panel. Bars represent mean values ± standard error of the mean; n = 3. *P < .05; ***P < .001 (t test).

We generated MYPT1 smooth muscle–specific knockout mice, Mypt1; SMA Cre-, Mypt1; SMA-Cre+ and Mypt1; and SMA-Cre+7. On KCl stimulation, the colonic circular muscle from homozygotes showed enhanced initial and sustained tension, whereas the vehicle from heterozygotes displayed a modest enhancement (Supplementary Figure 3A). Meanwhile, H1152 and GF109203 could not relax the mutant muscle, but SNP and nifedipine could (Supplementary Figure 2B–E). The number of ganglia and ganglionic cells in the mutant colon was significantly reduced (Supplementary Figure 3F). Collectively, MYPT1-deficient colon showed similar phenotypes including altered contractile properties and ganglia to Cir-N from HD patients, although the MYPT1SMKO mice showed no colonic obstruction, comparable bowel motility, transit velocity, and eating/defecation activity. Because circular smooth muscle layer constricts the bowel lumen and longitudinal muscle dilates the lumen and propels feces,, the disruption of this coordination might cause colonic obstruction.

Supplementary Figure 3

Colon from MYPT1 (A) Representative tracings of circular smooth muscle of distal colon treated with 87 mmol/L KCl from Mypt; SMA Cre-, Mypt1; SMA Cre+, and Mypt1; SMA Cre+ mice. Quantification of force responses of circular smooth muscle to treatment with KCl (one-way ANOVA) (n = 6). (B–E) Representative tracings of circular distal colon from MYPT1SMKO mice pre-contracted using 87 mmol/L KCl, followed by exposure to 0.3 μmol/L H1152 (B), 5 μmol/L GF109203X (C), 100 μmol/L SNP (D), or 3 μmol/L nifedipine (E). Relative ratios of the relaxed force during the sustained phase, which were expressed as percent of force of contraction after addition of KCl at the same time point as addition of the vehicle (force % = (Fvehicle – Finhibitor)/Fvehicle, F = force) in left panel. (F) H&E staining of colons showed significant reduction in number of ganglionic aggregates and total ganglionic cells in Mypt; SMA Cre- and Mypt1; SMA Cre+ mice. Quantitation of the ganglion and the total neuron cells count normalized by colon area (t test) (n = 6). Asterisk indicates a ganglion. Bars represent mean values ± standard error of the mean. **P < .01; ***P < .001; #P < .05; ##P < .01.

We also established an Ednrb line (Supplementary Figure 4A–D), an HD model with colonic obstruction and few ganglions. The high concentration of LPS, MYPT1 deficiency, and altered contractile property were also confirmed (Supplementary Figure 4E–G).

Supplementary Figure 4

Colonic phenotype of (A) Schematic representation of Ednrb knockout strategy by CRISP-Cas9 technology. (B and C) DNA sequencing isolated from chimeric (+/-) and wild-type (+/+) mice tail presented the deleted segment. (D) Western blot analysis of EDNRB protein expression in the colon from Ednrb+/+ and Ednrb-/- mice. (E) Limulus test showing concentration of LPS in Ednrb+/+ and Ednrb-/- mice (n = 6). (F) Western blots showing decreased expression of MYPT1 in Ednrb-/- mice (n = 4). (G) Representative tracings of responses of circular smooth muscle from colon of Ednrb+/+ (left panel) and Ednrb-/- (middle panel) mice evoked by 87 mmol/L KCl. Quantification of force tension of circular smooth muscle from HD colons that were evoked by KCl (right panel) (n = 6). Bars represent mean values ± standard error of the mean. *P < .05; **P < .01; ***P < .01 (t test). bp, base pairs.

In summary, normal intestinal function relies on multiple factors such as smooth muscle and enteric nervous system. MYPT1 is a primary regulator of smooth muscle contraction. LPS can degrade MYPT1 in colonic circular smooth muscle, thereby altering the contractile property and leading to colonic obstruction. Removing local LPS or targeting the SIAH/E3-mediated protein degradation pathway might be a useful strategy to treat colonic obstruction in HD.